The Retina and its Disorders

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THE RETINA AND ITS DISORDERS

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THE RETINA AND ITS DISORDERS EDITORS

JOSEPH C. BESHARSE Department of Cell Biology, Neurobiology and Anatomy Medical College of Wisconsin Milwaukee, WI USA DEAN BOK Department of Neurobiology and Jules Stein Eye Institute David Geffen School of Medicine at UCLA University of California Los Angeles, CA USA

Amsterdam • Boston • Heidelberg • London • New York • Oxford Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Copyright # 2011 Elsevier Ltd. All rights reserved Material in the text originally appeared in the Encyclopedia of the Eye, edited by Darlene A. Dartt, Joseph C. Beshare and Reza Dana (Elsevier Limited 2010) No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at (http://elsevier.com/locate/permissions), and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-382198-0 For information on all Academic Press publications visit our website at www.elsevierdirect.com PRINTED AND BOUND IN CHINA 11 12 13

10 9 8 7 6 5 4 3 2 1

CONTRIBUTORS

A P Adamis University of Illinois, Chicago, IL, USA J Adijanto National Eye Institute, Bethesda, MD, USA K R Alexander University of Illinois at Chicago, Chicago, IL, USA J Ambati University of Kentucky, Lexington, KY, USA B Anand-Apte Cleveland Clinic, Cleveland, OH, USA

P Bovolenta Instituto Cajal (CSIC) and CIBER de Enfermedades Raras (CIBERER), Madrid, Spain N C Brecha UCLA School of Medicine, Los Angeles, CA, USA; VAGLAHS, Los Angeles, CA, USA R Bremner University of Toronto, Toronto, ON, Canada S E Brockerhoff University of Washington, Seattle, WA, USA

D H Anderson University of California, Santa Barbara, CA, USA

N L Brown Cincinnati Children’s Research Foundation, Cincinnati, OH, USA

R E Anderson University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

J A Brzezinski, IV University of Washington, Seattle, WA, USA

A C Arman University of Southern California, Los Angeles, CA, USA V Y Arshavsky Duke University, Durham, NC, USA J D Ash University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA T J Bailey University of Notre Dame, Notre Dame, IN, USA J L Barbur City University, London, UK S Barnes Dalhousie University, Halifax, NS, Canada B-A Battelle University of Florida, St. Augustine, FL, USA M Ba¨hr University of Go¨ttingen, Go¨ttingen, Germany J C Besharse Medical College of Wisconsin, Milwaukee, WI, USA P Bex Schepens Eye Research Institute, Boston, MA, USA

B Burnside University of California, Berkeley, Berkeley, CA, USA P D Calvert SUNY Upstate Medical University, Syracuse, NY, USA J Carroll Medical College of Wisconsin, Milwaukee, WI, USA G J Chader USC School of Medicine, Los Angeles, CA, USA T Chan-Ling University of Sydney, Sydney, NSW, Australia D G Charteris Moorfields Eye Hospital, London, UK C F Chicani University of Southern California-Keck School of Medicine, Los Angeles, CA, USA T Cogliati National Institutes of Health, Bethesda, MD, USA N J Colley University of Wisconsin, Madison, WI, USA I Conte Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy

v

vi

Contributors

M F Cordeiro UCL Institute of Ophthalmology, London, UK

M B Gorin Jules Stein Eye Institute, Los Angeles, CA, USA

S W Cousins Duke Eye Center, Durham, NC, USA

M S Gregory Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA

K M Coxon UCL Institute of Ophthalmology, London, UK T W Cronin University of Maryland Baltimore County, Baltimore, MD, USA M D Crossland UCL Institute of Ophthalmology/Moorfields Eye Hospital, London, UK

L Guo UCL Institute of Ophthalmology, London, UK E V Gurevich Vanderbilt University, Nashville, TN, USA V V Gurevich Vanderbilt University, Nashville, TN, USA

J Cunha-Vaz AIBILI, Coimbra, Portugal

A Ha Ottawa Hospital Research Institute, Ottawa, ON, Canada

M Cwinn Ottawa Hospital Research Institute, Ottawa, ON, Canada

A Harris Indiana University, Indianapolis, IN, USA

P A D’Amore Schepens Eye Research Institute, Boston, MA, USA

M E Hartnett Moran Eye Center, University of Utah, Salt Lake City, UT, USA

M del Pilar Gomez Universidad Nacional de Colombia, Bogota´, Colombia J B Demb University of Michigan, Ann Arbor, MI, USA D Deretic University of New Mexico, Albuquerque, NM, USA W Drexler Medical University Vienna, Vienna, Austria J Duggan UCL Institute of Ophthalmology, London, UK R Ehrlich Indiana University, Indianapolis, IN, USA K Ford Schepens Eye Research Institute, Boston, MA, USA

S Haverkamp Max-Planck-Institute for Brain Research, Frankfurt/Main, Germany S S Hayreh University of Iowa, Iowa City, IA, USA C C Heikaus University of Washington, Seattle, WA, USA K Hein University of Go¨ttingen, Go¨ttingen, Germany D B Henson University of Manchester, Manchester, UK A A Hirano UCLA School of Medicine, Los Angeles, CA, USA

D H Foster University of Manchester, Manchester, UK

P Hiscott University of Liverpool, Liverpool, UK; Royal Liverpool University Hospital, Liverpool, UK

P J Francis Oregon Health and Sciences University, Portland, OR, USA

J G Hollyfield Cleveland Clinic, Cleveland, OH, USA

R N Frank Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI, USA F D Frentiu University of Queensland, St. Lucia, QLD, Australia Y Fu Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, UT, USA I Glybina Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI, USA

A Horsager USC School of Medicine, Los Angeles, CA, USA M S Humayun USC School of Medicine, Los Angeles, CA, USA D R Hyde University of Notre Dame, Notre Dame, IN, USA C Insinna Medical College of Wisconsin, Milwaukee, WI, USA P M Iuvone Emory University School of Medicine, Atlanta, GA, USA

Contributors K Jian Texas A&M University, College Station, TX, USA

D G McMahon Vanderbilt University, Nashville, TN, USA

L V Johnson University of California, Santa Barbara, CA, USA

B McNeill Ottawa Hospital Research Institute, Ottawa, ON, Canada

B Katz Hebrew University, Jerusalem, Israel V J Kefalov Washington University School of Medicine, Saint Louis, MO, USA M R Kesen Duke Eye Center, Durham, NC, USA C King-Smith Saint Joseph’s University, Philadelphia, PA, USA H J Klassen University of California, Irvine, Orange, CA, USA M E Kleinman University of Kentucky, Lexington, KY, USA G Y-P Ko Texas A&M University, College Station, TX, USA M L Ko Texas A&M University, College Station, TX, USA T D Lamb The Australian National University, Canberra, ACT, Australia

S Meredith Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK S S Miller National Eye Institute, Bethesda, MD, USA B Minke Hebrew University, Jerusalem, Israel J Mitchell University of Toronto, Toronto, ON, Canada T A Mu¨nch University of Tu¨bingen, Tu¨bingen, Germany J L Morgan University of Washington, Seattle, WA, USA O L Moritz University of British Columbia, Vancouver, BC, Canada A M Moss Indiana University, Indianapolis, IN, USA E Nasi Universidad Nacional de Colombia, Bogota´, Colombia

D C Lee University of British Columbia, Vancouver, BC, Canada

I Nasonkin National Institutes of Health, Bethesda, MD, USA

S Lee Yale University School of Medicine, New Haven, CT, USA

R W Nickells University of Wisconsin, Madison, WI, USA

A A Lewis University of Washington, Seattle, WA, USA

vii

S Nusinowitz UCLA School of Medicine, Los Angeles, CA, USA

R Li National Eye Institute, Bethesda, MD, USA

T H Oakley University of California, Santa Barbara, Santa Barbara, CA, USA

A Maminishkis National Eye Institute, Bethesda, MD, USA

M Pacal University of Toronto, Toronto, ON, Canada

N A Mandal University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

K Palczewski Case Western Reserve University, Cleveland, OH, USA

S C Mangel The Ohio State University College of Medicine, Columbus, OH, USA M B Manookin University of Michigan, Ann Arbor, MI, USA

R Payne University of Maryland, College Park, MD, USA L Peichl Max Planck Institute for Brain Research, Frankfurt am Main, Germany

R E Marc University of Utah, Salt Lake City, UT, USA

M E Pennesi Oregon Health and Sciences University, Portland, OR, USA

R Marco-Ferreres Instituto Cajal (CSIC) and CIBER de Enfermedades Raras (CIBERER), Madrid, Spain

D C Plachetzki University of California, Santa Barbara, Santa Barbara, CA, USA

viii

Contributors

I Provencio University of Virginia, Charlottesville, VA, USA

D M Tait Medical College of Wisconsin, Milwaukee, WI, USA

L P Pulagam Case Western Reserve University, Cleveland, OH, USA

W B Thoreson University of Nebraska Medical Center, Omaha, NE, USA

E Pyza Jagiellonian University, Krako´w, Poland Y D Ramkissoon Royal Hallamshire Hospital, Sheffield, UK I D Raymond UCLA School of Medicine, Los Angeles, CA, USA T A Reh University of Washington, Seattle, WA, USA C P Ribelayga The Ohio State University College of Medicine, Columbus, OH, USA L J Rizzolo Yale University School of Medicine, New Haven, CT, USA A A Sadun University of Southern California-Keck School of Medicine, Los Angeles, CA, USA A P Sampath University of Southern California, Los Angeles, CA, USA L Shi Texas A&M University, College Station, TX, USA

S I Tomarev National Institutes of Health, Bethesda, MD, USA G H Travis UCLA School of Medicine, Los Angeles, CA, USA B A Tucker Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA R L Ufret-Vincenty University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA S A Vinores Johns Hopkins University School of Medicine, Baltimore, MD, USA V A Wallace Ottawa Hospital Research Institute, Ottawa, ON, Canada Y Wang University of Maryland, College Park, MD, USA J Weiland USC School of Medicine, Los Angeles, CA, USA

W E Smiddy Bascom Palmer Eye Institute, Miami, FL, USA

R G Weleber Oregon Health and Sciences University, Portland, OR, USA

R G Smith University of Pennsylvania, Philadelphia, PA, USA

F S Werblin UC Berkeley, Berkeley, CA, USA

M Snead Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK

A F Wiechmann University of Oklahoma College of Medicine, Oklahoma City, OK, USA

N C Steinle University of Kentucky, Lexington, KY, USA

D R Williams University of Rochester, Rochester, NY, USA

S L Stella, Jr. UCLA School of Medicine, Los Angeles, CA, USA

D S Williams UCLA School of Medicine, Los Angeles, CA, USA

A Stockman UCL Institute of Ophthalmology, London, UK

P R Williams University of Washington, Seattle, WA, USA

E Strettoi Istituto di Neuroscienze CNR, Pisa, Italy

D Wong University of Hong Kong, Hong Kong, People’s Republic of China

E E Sutter The Smith-Kettlewell Eye Research Institute, San Francisco, CA, USA

R O L Wong University of Washington, Seattle, WA, USA

W Swardfager University of Toronto, Toronto, ON, Canada

S C Wong Moorfields Eye Hospital, London, UK

A Swaroop National Institutes of Health, Bethesda, MD, USA

S M Wu Baylor College of Medicine, Houston, TX, USA

Contributors S Yazulla Stony Brook University, Stony Brook, NY, USA

D-Q Zhang Vanderbilt University, Nashville, TN, USA

L Yin University of Rochester, Rochester, NY, USA

Z J Zhou Yale University School of Medicine, New Haven, CT, USA

M J Young Schepens Eye Research Institute, Harvard Medical School, Boston, MA, USA

M E Zuber SUNY Upstate Medical University, Syracuse, NY, USA

ix


PREFACE During our recent efforts (2008–2009) at identifying authors and subsequently editing the retina section for Elsevier’s Encyclopedia of the Eye, we were greatly impressed by the overall quality and depth of the chapters produced by our colleagues. What seemed at first to be a very large undertaking in fact became a pleasure when time had come to read and edit the chapters our colleagues had written. They had little incentive in this effort other than to expose their own research area to the broader community. In retrospect, we cannot thank them enough for what we now regard as a major service to students, postdoctoral fellows, residents, optometrists, and ophthalmologists. It was with this sentiment that we did not hesitate when we were asked to organize and reassemble their effort as a separate derivative volume for the retina community. The Retina and its Disorders provides a readily accessible and comprehensive compendium on the retina in health and disease. Coverage extends from embryology and early patterning to age-related macular degeneration, a complex trait disease that now affects about 30% of individuals over the age of 75 in industrialized countries. Included are lucid descriptions of the anatomy, physiology, cell biology, neural pathways, and pharmacology of the retina. In addition, key experts cover its vasculature as well as state-of-the-art noninvasive testing of structure and function. Comprised of 111 chapters selected from Elsevier’s Encyclopedia of the Eye, this volume provides a valuable desk reference for biomedical scientists, ophthalmologists, optometrists, and psychologists. Joseph C. Besharse Dean Bok Editors

xi


CONTENTS

Contributors

v–ix

Preface

Acuity

xi

M D Crossland

Adaptive Optics

1

L Yin and D R Williams

Alternative Visual Cycle in Mu¨ller Cells

7

G H Travis

17

Anatomically Separate Rod and Cone Signaling Pathways Anatomy and Regulation of the Optic Nerve Blood Flow Animal Models of Glaucoma Blood–Retinal Barrier

S Nusinowitz R Ehrlich, A Harris, and A M Moss

S I Tomarev

44 S A Vinores

Breakdown of the RPE Blood–Retinal Barrier

51

M E Hartnett

Circadian Metabolism in the Chick Retina Central Retinal Vein Occlusion

P M Iuvone

68 75

M R Kesen and S W Cousins

Chromatic Function of the Cones

87

D H Foster

96

The Circadian Clock in the Retina Regulates Rod and Cone Pathways Circadian Photoreception

58

S S Hayreh

Choroidal Neovascularization

28 38

J Cunha-Vaz

Breakdown of the Blood–Retinal Barrier

22

S C Mangel and C P Ribelayga

I Provencio

105 112

Circadian Regulation of Ion Channels in Photoreceptors Circadian Rhythms in the Fly’s Visual System

G Y-P Ko, K Jian, L Shi, and M L Ko

E Pyza

118 124

Color Blindness: Acquired

D M Tait and J Carroll

134

Color Blindness: Inherited

J Carroll and D M Tait

140

The Colorful Visual World of Butterflies

F D Frentiu

Cone Photoreceptor Cells: Soma and Synapse Contrast Sensitivity

148

R G Smith

156

P Bex

163

Coordinating Division and Differentiation in Retinal Development Developmental Anatomy of the Retinal and Choroidal Vasculature Development of the Retinal Vasculature Embryology and Early Patterning

R Bremner and M Pacal B Anand-Apte and J G Hollyfield

T Chan-Ling P Bovolenta, R Marco-Ferreres, and I Conte

169 179 186 198

xiii

xiv

Contents

Evolution of Opsins

T H Oakley and D C Plachetzki

Eye Field Transcription Factors

205

M E Zuber

Fish Retinomotor Movements

212

B Burnside and C King-Smith

GABA Receptors in the Retina

219

S Yazulla

228

Ganglion Cell Development: Early Steps/Fate

N L Brown

Genetic Dissection of Invertebrate Phototransduction Hereditary Vitreoretinopathies

235

B Katz and B Minke

240

S Meredith and M Snead

Histogenesis: Cell Fate: Signaling Factors

252

M Cwinn, B McNeill, A Ha, and V A Wallace

Immunobiology of Age-Related Macular Degeneration Information Processing: Amacrine Cells

R L Ufret-Vincenty

270

R E Marc

Information Processing: Bipolar Cells

263

276

S M Wu

284

Information Processing: Contrast Sensitivity

M B Manookin and J B Demb

290

Information Processing: Direction Sensitivity

Z J Zhou and S Lee

295

Information Processing: Ganglion Cells

T A Mu¨nch

Information Processing: Horizontal Cells

A A Hirano, S Barnes, S L Stella, Jr., and N C Brecha

Information Processing in the Retina

F S Werblin

Information Processing: Retinal Adaptation

Injury and Repair: Light Damage

Injury and Repair: Prostheses

A A Sadun and C F Chicani

Innate Immune System and the Eye

346

R E Marc B A Tucker, M J Young, and H J Klassen

M S Gregory

381

J L Morgan, P R Williams, and R O L Wong

389

S S Hayreh

399 P D Calvert and

B-A Battelle

R N Frank and I Glybina

Morphology of Interneurons: Bipolar Cells Morphology of Interneurons: Horizontal Cells Morphology of Interneurons: Interplexiform Cells Neuropeptides: Function Neuropeptides: Localization

412 416 426

Microvillar and Ciliary Photoreceptors in Molluskan Eyes Morphology of Interneurons: Amacrine Cells

367 374

R W Nickells

Limulus Eyes and Their Circadian Regulation

354 360

Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors V Y Arshavsky

Macular Edema

338

G J Chader, A Horsager, J Weiland, and M S Humayun

Injury and Repair: Stem Cells and Transplantation

Ischemic Optic Neuropathy

333

M E Kleinman and J Ambati

Injury and Repair: Retinal Remodeling

Intraretinal Circuit Formation

325

N A Mandal, R E Anderson, and J D Ash

Injury and Repair: Neovascularization

309 318

K R Alexander

Optic Nerve: Inherited Optic Neuropathies

IOP and Damage of ON Axons

301

E Nasi and M del Pilar Gomez

438

E Strettoi

447

S Haverkamp

452

L Peichl

461

D G McMahon and D-Q Zhang

N C Brecha, I D Raymond, and A A Hirano N C Brecha, A A Hirano, and I D Raymond

Neurotransmitters and Receptors: Dopamine Receptors

P M Iuvone

470 477 487 494

Contents

Neurotransmitters and Receptors: Melatonin Receptors

A F Wiechmann

Non-Invasive Testing Methods: Multifocal Electrophysiology Optical Coherence Tomography Optic Nerve: Optic Neuritis

E E Sutter

506 525

K Hein and M Ba¨hr

536

A P Adamis

541

D B Henson

551

Photopic, Mesopic and Scotopic Vision and Changes in Visual Performance A Stockman Photoreceptor Development: Early Steps/Fate

The Photoresponse in Squid

J C Besharse and C Insinna

610

R Payne and Y Wang

Phototransduction: Phototransduction in Cones

616

V J Kefalov

Phototransduction: Phototransduction in Rods

624

Y Fu

631

L P Pulagam and K Palczewski

Phototransduction: The Visual Cycle

637

G H Travis

Physiological Anatomy of the Retinal Vasculature

648 S S Hayreh

The Physiology of Photoreceptor Synapses and Other Ribbon Synapses Polarized-Light Vision in Land and Aquatic Animals

653 W B Thoreson

T W Cronin

Post-Golgi Trafficking and Ciliary Targeting of Rhodopsin

Primary Photoreceptor Degenerations: Terminology

676

M E Pennesi, P J Francis, and 684

M E Pennesi, P J Francis, and R G Weleber

P Hiscott and D Wong

698 708

S Yazulla

717

Retinal Degeneration through the Eye of the Fly

N J Colley

Retinal Ganglion Cell Apoptosis and Neuroprotection M F Cordeiro

726

K M Coxon, J Duggan, L Guo, and 734

J A Brzezinski, IV and T A Reh

Retinal Pigment Epithelial–Choroid Interactions

745

K Ford and P A D’Amore

Retinal Pigment Epithelium: Cytokine Modulation of Epithelial Physiology R Li, and J Adijanto

753 S S Miller, A Maminishkis,

L J Rizzolo

761 773

Retinal Vasculopathies: Diabetic Retinopathy Retinopathy of Prematurity

661 668

D Deretic

Primary Photoreceptor Degenerations: Retinitis Pigmentosa R G Weleber

Retinal Histogenesis

605

V V Gurevich and E V Gurevich

Phototransduction in Limulus Photoreceptors

Phototransduction: Rhodopsin

596

V V Gurevich and E V Gurevich

Phototransduction: Inactivation in Rods

575

589

T D Lamb

Phototransduction: Inactivation in Cones

567

582

T D Lamb

Phototransduction: Adaptation in Rods

Retinal Cannabinoids

558

J Mitchell and W Swardfager

Phototransduction: Adaptation in Cones

Proliferative Vitreoretinopathy

J L Barbur and

I Nasonkin, T Cogliati, and A Swaroop

The Photoreceptor Outer Segment as a Sensory Cilium

RPE Barrier

500

W Drexler

Pathological Retinal Angiogenesis Perimetry

xv

N C Steinle and J Ambati

M E Hartnett

Rhegmatogenous Retinal Detachment

S C Wong, Y D Ramkissoon, and D G Charteris

781 790 801

xvi

Contents

Rod and Cone Photoreceptor Cells: Inner and Outer Segments

D H Anderson and D S Williams

Rod and Cone Photoreceptor Cells: Outer Segment Membrane Renewal D H Anderson Rod Photoreceptor Cells: Soma and Synapse

D S Williams and 815

R G Smith

The Role of the Vitreous in Macular Hole Formation

819

W E Smiddy

825

Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration Secondary Photoreceptor Degenerations

Zebra Fish as a Model for Understanding Retinal Diseases S E Brockerhoff Zebra Fish–Retinal Development and Regeneration

830 836

Unique Specializations – Functional: Dynamic Range of Vision Systems A P Sampath

Index

L V Johnson

M B Gorin

Xenopus laevis as a Model for Understanding Retinal Diseases

811

A C Arman and

O L Moritz and D C Lee

841 847

A A Lewis, C C Heikaus, and

T J Bailey and D R Hyde

853 863

875

Acuity M D Crossland, UCL Institute of Ophthalmology/Moorfields Eye Hospital, London, UK ã 2010 Elsevier Ltd. All rights reserved.

Glossary Cycles per degree – The number of complete phases of a grating (e.g., the distance between the center of a white bar and the center of the next bright bar in a square-wave grating; or the distance between two adjacent areas of maximum brightness on a sine-wave grating) contained in 1 of visual angle. Minimum angle of resolution – The size of the angle subtended at the eye of the smallest feature which can be reliably identified on an optotype. Minute of arc – One-sixtieth of a degree. Optotype – A letter, symbol, or other figure presented at a controlled size to measure vision. Visual angle – The angle, which a viewed object subtends at the eye.

Detection and Resolution Acuity Visual acuity can be defined in two broad ways. Detection acuity is measured by determining the size of the smallest object which can be reliably seen (is there a circle on the first or second screen?). Detection can be elicited reliably with targets, which subtend an angle at the eye as small as 1 s of arc (1/3600 ). Even a small point of light will stimulate several photoreceptors due to the point-spread function of the eye: that is, the way in which light is diffracted through the eye’s optics (Figure 1(a)). Tests that require the identification of a target are a measurement of resolution acuity. These tests frequently involve identifying a letter or reporting an object’s orientation (what direction is this letter C facing?). Acuity for these tests depends on the separation of the target features: if they are too close, the point-spread function from each element will overlap and they will not be identified (Figure 1(b)). The smallest separation of the elements required for identification of the target (Figure 1(c)) is known as the minimum angle of resolution (MAR). For an adult observer with good vision, a typical MAR for a centrally presented, high-contrast target can be as good as 30 s of arc (1/120 ). Figure 2 shows the feature critical for the MAR for some commonly used tests of visual acuity.

Measurement of Visual Acuity Visual acuity tests have been used for millennia: the ancient Egyptians are reported to have used discrimination of the twin stars of Mizar and Alcor as a measurement of vision. The most familiar clinical test of visual acuity, the Snellen chart, was introduced in 1862, and is still widely used today. Detection acuity is often measured psychophysically by means of a temporal two-alternative forced-choice experiment (did the light appear in the first or the second interval?). Detection acuity is rarely measured clinically. In psychophysical experiments of the visual system, resolution acuity is commonly measured by asking observers to report the orientation of a grating with variable separation between each dark and light bar (Figure 2(b)). In clinical practice, gratings are rarely used, with the exception of forced-choice preferential looking tests in preverbal children. These tests consist of a uniform gray field with an isoluminant grating toward one side of the chart (Figure 3(a)). In a featureless room, the test is presented to the child and the clinician observes whether the child looks toward the grating. The finest grating toward which the child repeatedly looks is recorded as the visual acuity. For cooperative patients, optotypes are more often used to measure clinical resolution acuity. The Landolt C (Figure 2(c)) is the standard to which letter visual acuity tests are compared. This target consists of a ring of fixed width with a gap, of height equal to the stroke width, at the top, left, right, or bottom of the circle. The observer is asked to report the position of this gap. The smallest gap whose position can be reliably reported is equivalent to the MAR. The National Academy of Sciences standard for visual acuity measurement advocates the presentation of 10 optotypes, of equivalent difficulty to the Landolt C, at each acuity size. The horizontal spacing between each optotype should be at least one character width, and vertical spacing between lines should be 1–2 times the height of the larger optotypes. It suggests that the number of characters on each line should be equal, and that the size difference between consecutive lines is 0.1 log units: in other words, for each target size, the next line should be approximately 1.26 times smaller. The Snellen chart (Figure 3(b)) does not meet these recommendations: the number of letters per line and step

1

Acuity

2

Target

(a)

(b)

(c) (a)

2-D PSF

1-D PSF

(b)

Figure 1 Schematic illustration of the point-spread function of three visual targets: (a) a point target; (b) two adjacent lines, too close to be resolved; and (c) two adjacent lines, with sufficient separation to be resolved. Middle row: two-dimensional representation of the target point-spread function; bottom row: one-dimensional representation of the point-spread function; and red line indicates the sum of energy incident on the retina. PSF, point-spread function.

θ

(a) θ

(b)

θ

(c)

θ

(d)

Figure 2 Examples of the limiting feature for four commonly used resolution tasks: (a) two-point discrimination task; (b) grating; (c) Landolt C; and (d) Sloan letter E (note that white gap size is equal in width to black bar elements).

size between the lines are variable, as is the horizontal and vertical spacing on the chart. There is also a marked difference in the legibility of different letters on the Snellen chart: a W, for example, has far less separation

(c)

Figure 3 (a) A forced-choice preferential looking test consisting of a grating against an isoluminant background. Note the peephole in the center for the clinician to observe the child’s visual behavior; (b) the Snellen chart; and (c) The ETDRS chart. ETDRS, Early treatment of diabetic retinopathy study.

between the elements of the letter and is more difficult to identify than a letter L. In the 1950s, Sloan suggested the use of 10 letters with a selection of vertical, horizontal, oblique, and round strokes which are each about as legible as a Landolt C. These Sloan letters are C, D, H, K, N, O, R, S, V, and Z. Each of the Sloan letters has a stroke width of the MAR and has a total height and width of five times the MAR. The Bailey–Lovie chart, introduced before the recommendations of the National Academy of Sciences, conforms to most of these requirements, although it only has five letters per line. Further, the letters on the Bailey–Lovie chart are taller than they are wide: their height-to-width ratio is 5:4 and they are selected from the British Standards set of letters (D, E, F, H, N, P, U, V, R, and Z). The ETDRS chart (Figure 3(c)), developed for the early treatment of diabetic retinopathy study (ETDRS), is similar in design but does use the recommended 5 5 Sloan letters. A criterion of 7/10 letters being read correctly for a line to be marked as seen was suggested by the National Academy of Sciences. This threshold reduces the chance of the line being scored correctly by chance (by a blind observer) to around 1 in 9 000 000. On a chart with five letters per line, recording a visual acuity where four of the five letters are read correctly equates to a chance success rate of 1 in 46 000. There is a theoretical advantage if the observer knows there are only 10 letters which can be presented on the chart: if an observer guesses from all 26 letters rather than the ten Sloan letters, the probability of the observer getting four out of five letters correct reduces to about 1 in 100 000.

Acuity

3

Test–retest variability of the Snellen chart is around 0.3 logMAR, while the ETDRS chart has far better repeatability (test–retest variability 0.1–0.2 logMAR). Despite the many limitations of the Snellen chart, it is still widely used in clinical practice. While this is likely to be largely due to clinicians’ familiarity with the Snellen chart, there is also a perception that Snellen acuity measurement is quicker than that on the Bailey–Lovie or ETDRS charts. Various modified versions of the ETDRS chart exist: for example, a version with an altered letter set (A, B, E, H, N, O, P, T, X, and Y) has been developed for use by readers of most European languages, including those based on Cyrillic or Hellenic alphabets. For observers unable to report letters on a sight chart, other frequently used optotypes include the tumbling E chart (formerly and less politically correctly known as the illiterate E chart), where a letter E is shown in each of four rotations; the HOTV chart, where only these four letters are used; symbols such as the Lea or Kay pictures; and simple shapes, such as the Cardiff card.

In much of Europe, the Snellen fraction is reduced into a decimal fraction. A further confusion with the Snellen system is that in countries not using the metric system, distances are expressed in feet rather than meters, with 20/20 being exactly equivalent to 6/6 but with a test distance of 20 ft rather than 6 m. Although Snellen recommended adoption of the metric system in 1875 and, in 1980, the US National Academy of Sciences favored adoption of a standard defined in meters, given the imminent adoption of the metric system, the feet system is still widely used in the USA, and among lay people in the UK. The accepted standard for expressing visual acuity in clinical research, and increasingly in clinical practice, is to use the base 10 logarithm of the MAR (logMAR), such that 0.0 logMAR is equivalent to 6/6 or 20/20, and 1.0 logMAR is the same as 6/60 or 20/200. Table 1 gives approximately equivalent values in MAR, cycles per degree, Snellen fractions in meters and feet, decimal acuity, and logMAR for a range of visual acuities.

Reporting Visual Acuity

Optical and Neural Limits on Visual Acuity

Clinicians have traditionally used Snellen fractions to record visual acuity, where the numerator is the test distance and the denominator the target size. The target size is expressed, counterintuitively, as the distance from which the target has an MAR of 1 min of arc. Therefore, a visual acuity of 6/6 indicates that from 6 m, letters with MAR 1-min arc are correctly identified, while a visual acuity of 3/36 indicates that from 3 m, the targets identified have a MAR of 1 min of arc when viewed from 36 m. The reciprocal of the Snellen fraction gives the visual acuity in MAR: so a visual acuity of 3/36 indicates a MAR of 12 min of arc.

Visual acuity is limited by many factors: the optics and refraction of the eye; the clarity of the optical media; the spacing and function of the retinal photoreceptors; the ratio of retinal ganglion cells to photoreceptors; and the resolution of the primary visual cortex and higher areas of visual processing. Each diopter of myopia reduces visual acuity: a –1.00DS myope will typically have uncorrected visual acuity of around 0.5 logMAR (6/18; 20/60) and a two-diopter myope will have vision of around 0.8 logMAR on a distance test. Hypermetropia can often be relieved by accommodation in young people, but each diopter of hypermetropia

Table 1

Visual acuity conversion table

a

MAR (min)

Cycles/ degree

Snellen (metric)

Snellen (feet)

Decimal

Log MAR

60 20 10 6.3 4 3.2 2 1.6 1.3 1 0.83 0.67 0.5 0.33

0.5 1.5 3 4.7 7.5 9.4 15 18.8 23 30 36 44 60 91

1/60 3/60 6/60 6/36 6/24 6/18 6/12 6/9 6/7.5 6/6 6/5 6/4 6/3 6/2

20/1200 20/400 20/200 20/120 20/80 20/60 20/40 20/30 20/25 20/20 b 20/17 b 20/13 20/10 20/7

0.017 0.05 0.1 0.17 0.25 0.33 0.5 0.67 0.8 1 1.2 1.5 2 3

1.8 1.3 1 0.8 0.6 0.5 0.3 0.2 0.1 0 0.1 0.2 0.3 0.4

a

Each row contains approximately equivalent values of visual acuity. Log MAR values have been rounded to 1 decimal place. On US Snellen charts, these lines are 20/16 and 20/12 respectively.

b

Acuity

beyond the accommodative ability of the eye will reduce visual acuity by a similar amount to an equivalent degree of myopia. Astigmatism, particularly where the meridia of astigmatism are oblique, will also reduce uncorrected vision significantly. Other aberrations of the eye beyond defocus and astigmatism further limit visual acuity. Retinal image quality can be improved by viewing monochromatic stimuli (to reduce chromatic aberration) and by using a deformable mirror to correct coma, trefoil, and other higher-order aberrations of the eye. Under these ideal conditions, Williams and colleagues have shown that subjects are able to resolve gratings of up to 55 cycles per degree, equivalent to a visual acuity of approximately –0.30 logMAR (6/3; 20/10). Assuming that an image is perfectly focused on the retina, the next limit on visual resolution is the spacing of the retinal photoreceptors. In order to detect a grating, alternate black and white bars must fall on adjacent photoreceptors. This theoretical limit of vision, known as the Nyquist limit, is equivalent to a grating with light to dark separation of 1/√D, where D is the center-to-center separation of two photoreceptors. In the fovea, D is approximately 3 mm, equivalent to a visual angle of approximately 55 cycles per degree – almost identical to the value found by Williams. This confirms that in people with good vision, all of the limits on visual acuity are precortical. Amblyopia, where vision is reduced despite the absence of any eye disease, is dealt with elsewhere in the encyclopedia.

Visual Acuity across the Retina Nonfoveal vision is limited by many elements. First, the eye’s optics are not optimized for viewing off the visual axis, and peripheral vision is subject to greater aberration than central vision. Second, the size of photoreceptors increases and their density falls with increasing eccentricity. The number of photoreceptors per retinal ganglion cell also increases, from less than one photoreceptor per ganglion cell in the fovea to more than 20 photoreceptors per ganglion cell in the far periphery. The volume of visual cortex devoted to noncentral retina is also proportionally lower. It is unsurprising, therefore, that visual acuity falls quickly with increasing distance from the fovea (Figure 4). This is one reason for the severely reduced visual acuity of people with central vision loss from diseases such as age-related macular disease.

Visual Acuity over Life Over the first year of life, visual acuity assessed by a preferential looking test appears to be reasonably stable

250 Letter visual acuity (min arc)

4

200

150

100

50

0

0

10

20

30 40 Eccentricity (⬚)

50

60

Figure 4 Letter visual acuity measured in peripheral vision as a function of degrees of eccentricity. Data from Anstis, S. M. (1974). Letter: A chart demonstrating variations in acuity with retinal position. Vision Research 14(7): 589–592.

at around 6 min of arc. Between a child’s first and third birthday, visual acuity improves exponentially to reach 1 min of arc. A further small improvement in resolution ability to approximately 0.75 min of arc is achieved by age 5 years. In the absence of eye disease, this value remains relatively constant until the sixth decade. In a populationbased study of nearly 5000 older adults, Klein found a decrease in visual acuity to a mean value of approximately 2 min of arc in those aged over 75 years. Of course, this reflects the age-related nature of many diseases which affect visual acuity, such as cataract, glaucoma, diabetic retinopathy, and age-related macular degeneration. Figure 5 plots data from the studies of Mayer and Klein. Visual Standards In most countries, there is a visual-acuity requirement for car drivers. While the level and measurement technique varies between countries, the acuity limit is usually approximately 0.3 logMAR. Commercial airline pilots are required to have a binocular visual acuity of 0.0 logMAR. Best corrected binocular visual acuity of 1.0 logMAR or poorer is used as a definition of low vision or partial sight in many countries, with acuity of worse than 1.3 logMAR being described as severe sight impairment.

Hyperacuity Some visual tasks can be performed with a far greater degree of precision than would be suggested by the MAR. Alignment tasks such as Vernier discrimination (where the offset of one line with respect to another is detected, Figure 6(a)) can be performed with misalignment of less

Acuity 6

Visual acuity (min arc)

5 4 3 2 1 0

1

10 Age (years)

100

Figure 5 Variation in visual acuity over life. From Mayer, D. L. and Dobson, V. (1982). Visual acuity development in infants and young children, as assessed by operant preferential looking. Data from Vision Research 22(9): 1141–1151 and Klein, R., Klein, B. E., Linton, K. L., and De Mets, D. L. (1991). The beaver dam eye study: Visual acuity. Ophthalmology 98(8): 1310–1315.

5

must be for it to be seen. If a target moves with velocity of 40 s1, the MAR is increased to about 2 min of arc, while at 80 s1, acuity is about 3 min of arc. In peripheral vision, slow image motion (less than 10 s1) slightly improves visual acuity for peripherally presented targets, perhaps because it breaks the phenomenon of Troxler fading. Target motion at the retina can be induced by target movement, by eye motion, or by head motion. Many eye diseases, particularly those of the macula, are associated with poor fixation stability of the eye. This poor eye stability increases retinal image motion, and is significantly associated with poorer visual function. Small degrees of head motion do not significantly decrease visual acuity under normal conditions, but have a marked deleterious effect for subjects viewing through telescopic spectacles. Therefore, subjects with macular disease who have poor fixation stability and who view through telescopic low-vision aids have a marked impairment in their dynamic visual acuity.

See also: Chromatic Function of the Cones; Contrast Sensitivity; Photopic, Mesopic and Scotopic Vision and Changes in Visual Performance.

Further Reading

(b)

(a) Figure 6 Examples of hyperacuity tasks. Misalignment of the lower element is easily visible. (a) Vernier alignment; (b) dot alignment: the offset of the middle dot with respect to the upper and lower dot is easily discerned.

than 5 s of arc – considerably less than the center-tocenter spacing of a foveal photoreceptor. This is thought to be due to interpolation of the inputs of two or more adjacent neural elements.

Dynamic Visual Acuity Throughout this article, visual acuity has been discussed for static targets. If the target is moved, central visual acuity decreases: the faster the target moves, the larger it

Anstis, S. M. (1974). Letter: A chart demonstrating variations in acuity with retinal position. Vision Research 14(7): 589–592. Bailey, I. L. and Lovie, J. E. (1976). New design principles for visual acuity letter charts. American Journal of Optometry and Physiological Optics 53: 740–745. Bennett, A. G. and Rabbetts, R. B. (eds.) (1989). Visual acuity and contrast sensitivity. In: Clinical Visual Optics, pp. 23–72. Oxford: Butterworth-Heinemann. Brown, B. (1972). Resolution thresholds for moving targets at the fovea and in the peripheral retina. Vision Research 12(2): 293–304. Committee on vision. (1980). Recommended standard procedures for the clinical measurement and specification of visual acuity. Report of working group 39. Advances in Ophthalmology ¼ Fortschritte der Augenheilkunde ¼ Progres en Ophtalmologie 41: 103–148. Assembly of Behavioral and Social Sciences, National Research Council, National Academy of Sciences, Washington, DC Crossland, M. D., Culham, L. E., and Rubin, G. S. (2004). Fixation stability and reading speed in patients with newly developed macular disease. Ophthalmic and Physiological Optics 24: 327–333. Demer, J. L. and Amjadi, F. (1993). Dynamic visual acuity of normal subjects during vertical optotype and head motion. Investigative Ophthalmology and Visual Science 34(6): 1894–1906. Klein, R., Klein, B. E., Linton, K. L., and De Mets, D. L. (1991). The beaver dam eye study: Visual acuity. Ophthalmology 98(8): 1310–1315. Liang, J., Williams, D. R., and Miller, D. T. (1997). Supernormal vision and high-resolution retinal imaging through adaptive optics. Journal of the Optical Society of America. A, Optics, Image Science, and Vision 14: 2884–2892. Mayer, D. L. and Dobson, V. (1982). Visual acuity development in infants and young children, as assessed by operant preferential looking. Vision Research 22(9): 1141–1151.

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Plainis, S., Tzatzala, P., Orphanos, Y., and Tsilimbaris, M. K. (2007). A modified ETDRS visual acuity chart for European-wide use. Optometry and Vision Science 84(7): 647–653. Rosser, D. A., Cousens, S. N., Murdoch, I. E., Fitzke, F. W., and Laidlaw, D. A. (2003). How sensitive to clinical change are ETDRS logMAR visual acuity measurements? Investigative Ophthalmology and Visual Science 44: 3278–3281.

Thibos, L. N., Cheney, F. E., and Walsh, D. J. (1987). Retinal limits to the detection and resolution of gratings. Journal of the Optical Society of America. A, Optics, Image Science, and Vision 4: 1524–1529. Westheimer, G. (1987). Visual acuity. In: Moses, R. A. and Hart, W. M. (eds.) Adler’s Physiology of the Eye: Clinical Application, pp. 415–428. St Louis, MO: Mosby.

Adaptive Optics L Yin and D R Williams, University of Rochester, Rochester, NY, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Deformable mirror – A mirror equipped with an array of actuators on the back surface that can warp the mirror surface by small amounts into arbitrary shapes, allowing the correction of the eye’s aberrations. Diffraction-limited resolution – The resolution of an optical system that has no aberrations, the image quality of which is reduced only by the diffraction of the light in the pupil of the system. Lipofuscin autofluorescence – Lipofuscin is composed of many molecules that are by-products of the visual or retinoid cycle. These accumulate in the retinal pigment epithelium with aging and can be visualized in the living eye because they fluoresce when exposed to short wavelength light. Point-spread function (PSF) – The light distribution in the image plane of an optical system such as the eye, formed from light from a point source outside the eye, such as a very distant star. Retinal densitometry – A method to measure the density of photopigment in the living eye. Shack–Hartmann wavefront sensor – A device capable of measuring the optical defects of an optical system such as the human eye. The output of a wavefront sensor can be used to control the shape of a deformable mirror in an adaptive optics system. Stiles and Crawford effect – The fact that the eye is far more sensitive to light entering through a point near the center of the pupil than the pupil margin even though the irradiance at the retina is very little dependent on entry point in the pupil. This effect is caused by the waveguide properties of cone photoreceptors, which are more sensitive to light falling on their optical axes (which point near the center of pupil) than obliquely incident light.

The Benefit of Adaptive Optics in Vision Science The history of ophthalmoscopy after its invention by Helmholtz until today is marked by efforts to extract the most information possible from the light reflected from the retina. Over the last two decades, there has been a

concerted effort to improve the resolution of the imaging process in all three spatial dimensions. The development of optical coherence tomography (OCT) improved the resolution in the axial dimension, and has allowed the routine imaging of individual layers of cells in the retina. More recently, the introduction of adaptive optics (AO) has improved the resolution of fundus cameras in both transverse dimensions. The transverse resolution of the conventional fundus camera is limited not by the camera itself but by the optics of the human eye. The sources of image blur in the eye’s optics include diffraction, aberrations, and scatter. Diffraction is the image blur that results from the wave nature of light as it passes through the eye’s pupil. Blurring by diffraction is not inevitable; there are exciting techniques on the horizon that may eventually overcome the fundamental resolution limit set by diffraction, but no one has yet demonstrated this in the eye. A hypothetical eye that suffered only from diffraction would allow a resolution no smaller than about 1.4 mm when imaging wavelengths of light in the middle of the visible spectrum (550 nm). This is smaller than the smallest cells in the retina, so that if one could make the natural human eye limited only by diffraction, cellular and even subcellular features that are invisible in conventional fundus imaging could be seen. As shown in Figure 1 (upper panels), in a diffraction-limited eye, the larger the pupil, the smaller the image of a single point of light and therefore the better the resolution. The monochromatic and chromatic aberrations of all eyes further blur the retinal image. The monochromatic aberrations of the human eye alone are greater than those of even a mediocre man-made optical system. As shown in Figure 1 (lower panels), increasing the pupil reduces the effect of diffraction, but exacerbates the effect of aberrations, with the best trade-off between the two occurring for pupil sizes around 3 mm. Light scatter, the third cause of loss in retinal image quality, is relatively unimportant in young eyes but it can greatly reduce retinal image contrast in older eyes, especially those with cataract. Not only is the retinal image blurred by the optical factors mentioned above, it is also exceedingly dim. The amount of light that can be delivered to the retina is limited for safety reasons and the retinal reflectance is low: 10–3–10–5 across the spectrum. This would be less of a problem if it were easy to integrate light over long exposures, but the eye is always moving; even an eye with excellent fixation moves about 20 mm root mean square (rms) velocity. Eye motion artifacts are all the more troublesome in instruments with high magnification that are designed to look at cellular

7

8

Adaptive Optics 6 mm

Typical human eye

Diffraction-limited eye

2 mm

Figure 1 The point-spread function (PSF) for a diffraction-limited eye and a normal eye at two different pupil diameters. The PSF corresponds to the light distribution on the retina produced by a point source of light infinitely distant from the eye. For the hypothetical diffraction-limited eye, the PSF diameter decreases in inverse proportion to the pupil diameter such that large pupils produce the best image quality. However, in the typical human eye, aberrations increase with increasing pupil size, eliminating the benefit of escaping diffraction at the largest pupils. The goal of AO is to correct the aberrations to produce the PSF of a diffraction-limited eye with a large pupil. Adapted from Roorda, A. Garcia, C. A., Martin, J. A., et al. (2006). What can adaptive optics do for a scanning laser ophthalmoscope? Bulletin de la Socie´te´ Belge d’Ophthalmologie 302: 231–244, Figure 1, with permission from Bulletin of the Belgian Societies of Ophthalmology (Copyright 2006).

structures that are often far smaller than 20 mm. Despite all these formidable limitations, it is possible to design fundus cameras that address all of them with varying degrees of success, making microscopic resolution of the living retina possible as described below.

Correcting the Eye’s Monochromatic Aberration It is possible to overcome the eye’s monochromatic aberrations with AO, a two-step process in which the eye’s wave aberration is measured and corrected, usually in real time. Figure 2 describes the principle of AO for imaging the eye. The monochromatic aberration of the eye is measured with a wavefront sensor. The measured aberration data are used to control a wavefront compensation device, usually a deformable mirror that corrects the wave aberration. Ideally, it would completely remove all the monochromatic aberrations, leaving diffraction and scatter as the only remaining sources of image blur. It usually takes several iterations of the measurement and correction loop to achieve the best correction, at which point it is possible to obtain a retinal image that is almost

Light from retina

Aberrated wavefront

Beamsplitter

Control system

Corrected wavefront

Adaptive mirror

Wavefront sensor

High-resolution retinal image

Figure 2 Principle of adaptive optics. The system contains two key parts: the wavefront sensor and wavefront corrector. The wavefront sensor, usually of the Shack–Hartmann type, measures the monochromatic aberration of the eye. It uses a 2-D array of lenslets conjugate with the eye’s pupil to break the light from an infrared point source imaged on the retina into several hundred individual beams. Each beam is imaged on a CCD array. Its displacement on the CCD from where it would have landed had the eye been aberration-free indicates the slope of the wave aberration at that lenslet’s location in the pupil. Information from all the lenslets is combined to compute the overall wave aberrations of the eye. These data are used to control a wavefront compensation device that corrects the wave aberration. The most commonly used device is a continuous surface deformable mirror. This mirror has a flexible surface overlying an array of actuators that can push or pull on the mirror surface locally. If the mirror surface is shaped so as to mimic the shape of the wave aberration but with half the amplitude of the wave aberration, the wavefront reflecting from the surface will be perfectly flat, and the monochromatic aberrations of the eye will have been corrected. Adapted from Carroll, J. Gray, D. C., Roorda, A., Williams, D. R. (2005). Recent advances in retinal imaging with adaptive optics. Optics and Photonics News 16: 36–42, Figure 1, with permission from Optical Society of America (Copyright 2005).

completely aberration free in eyes with normal amounts of aberrations. The rate of measurement and correction required to keep up with the temporal variations in the eye’s wave aberration is relatively slow, at least compared with applications of AO in astronomy. Heidi Hofer has shown that measuring and correcting the wave aberration at 30 Hz or so is adequate to track the most important changes in the wave aberration, which are caused by microfluctuations in accommodation that have a temporal bandwidth of only a few Hertz. With complete wavefront correction, the point-spread function (PSF) is very

Adaptive Optics

(a)

(b)

9

(c)

Figure 3 Adaptive optics and motion correction greatly improve the resolution of images of human cone mosaic. (a) Single frame of the reflectance image of the cone mosaic of a typical human eye at 1 degree of eccentricity imaged with all the aberrations of the eye, or (b) After the monochromatic aberrations of the eye were corrected with AO. (c) The summed frames of many images of the same cone mosaic with aberration and eye motion corrected with AO. The frames were registered before summing to correct for eye motion between frames, which increases the SNR over that obtained with single frames. The individual cones at this eccentricity are approximately 5 mm in diameter.

compact, approaching the light distribution produced by diffraction alone. Typically, one can achieve as much as an order of magnitude reduction in the rms wavefront error of a normal eye with this method. This provides a substantial improvement in image quality as shown in Figure 3.

Vision Correction with AO One of the convenient features of AO correction is that the correction required to focus light onto the retina is the same as the correction required to image the retina at high resolution outside the eye. Many investigators have capitalized on the advantages of AO for vision correction as well as for retinal imaging. Correcting the higher order monochromatic aberrations (i.e., those other than defocus and astigmatism corrected by spectacles) produces a modest improvement in visual acuity and contrast sensitivity in the normal eye. The improvements can be dramatic in eyes with large amounts of higher-order aberrations such as those that suffer from keratoconus. The demonstration of these improvements in spatial vision with a deformable mirror has stimulated improvements in the control of laser ablation in refractive surgery as well as the fabrication of customized contact lenses that can correct higher-order aberrations. AO continues to be a valuable tool not only for correcting aberrations but also for generating specific patterns of aberrations so that their effects on vision can be studied conveniently. For example, it is possible to explore the design of contact lenses for presbyopes that increase the depth of field of the eye without the need to fabricate optical elements for each pattern one wishes to evaluate.

Retinal Imaging with AO In retinal imaging, AO can be combined with almost any other imaging technology. David Williams’ laboratory at

the University of Rochester first demonstrated the value of a closed-loop AO system for retinal imaging, incorporating AO into a flood-illuminated system that acquired single snapshots of the retina with a resolution adequate to resolve cone photoreceptors near the fovea. Austin Roorda, then at the University of Houston, demonstrated that AO could also improve the resolution of the scanning laser ophthalmoscope (SLO). SLOs are potentially confocal devices and AO offers improvements in both axial and transverse resolutions. Moreover, AO improves the focus of the light on the confocal pinhole in front of detector, which increases the available signal. The AOSLO has a high lateral resolution of less than about 2 mm. The axial resolution of better than 60 mm, though poor by OCT standards, is nonetheless adequate for some optical sectioning of the retina. AO has also improved the transverse resolution of OCT, allowing a resolution of less than 3 mm in all three spatial dimensions in the retina. AO systems can also be combined with other imaging modalities such as phase contrast microscopy, polarization imaging, or fluorescence microscopy. Compensating for Eye Motion In many cases, the signals acquired in a single video frame of an AOSLO are weak enough to warrant frame averaging to increase the signal-to-noise ratio (SNR) of the image (Figure 4(a)). Eye motion between frames, which is substantial in the high-magnification images of AO systems, must be corrected before frame averaging can be achieved. One correction method is to register successive frames with normalized cross-correlation, the benefit of which is shown in Figure 3(c). However, it is not uncommon for single frames, such as the one shown in Figure 4(a), to have inadequate SNR for this method. In the case, illustrated here of autofluorescence imaging by Jessica Morgan at the University of Rochester, there was typically less than

Adaptive Optics

10

one photon for every 5 pixels in each frame. As shown in Figure 4(b), this problem can be solved by simultaneously recording infrared reflectance images of the cone mosaic at the same retinal location. Eye motion between frames can be reliably recovered from reflectance images of cone mosaic with an accuracy of one-fifth of the diameter of a foveal cone, and this information can be used to register the low SNR images as shown in Figure 4(b). Eye movements can also produce warping artifacts within each frame. Austin Roorda and Scott Stevenson have developed methods in which the relative locations of local retinal features are compared across frames to compute and correct the eye movement warping within each frame as well as translation between frames. Figure 5 shows

an AOSLO image motion before and after removing distortions from eye movements. Image motion after correction is reduced to a standard deviation of only 7 arcsec. David Arathorn at Montana State University, working in collaboration with Austin Roorda, has a fast software algorithm that can stabilize the retinal image in real time. This has very exciting applications for delivering light stimuli to single photoreceptors in both psychophysical and electrophysiological studies. Software registration approaches alone cannot address all the problems created by eye movements, particularly in AO instruments designed for routine clinical use where eye movements are much larger than the size of a single frame. In that case, successive frames do not share common features and cross-correlation cannot be

10 ⬚ superior

(a)

(b)

(c)

Figure 4 Dual registration improves the transverse resolution of autofluorescence images of the RPE mosaic. (a) Single frame of autofluorescence image of primate RPE mosaic. Because of the low SNR and photon density, the image does not have any apparent spatial structure. (b) 1000 frames of autofluorescence images of the same RPE mosaic shown in (a) summed with eye motion corrected using the dual-registration technique. The eye motion was calculated from reflectance images of the cone mosaic obtained simultaneously with the dim autofluorescence images, providing the translations necessary to register the autofluorescence images. The summed image reveals single cells in the RPE mosaic. (c) Autofluorescence image of human RPE mosaic at retinal eccentricity of 10 degrees. Bright regions in the images correspond to the accumulation of lipofuscin within the RPE cells. Dark regions correspond to the nuclei of RPE cells. Scale ¼ 50 mm. Adapted from Morgan, J. I. Dubra, A., Wolfe, R., et al. (2009). In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic. Investigative Ophthalmology and Visual Science 50: 1350–1359, Figure 5, with permission from Association for Research in Vision and Ophthalmology (Copyright 2009).

4 Movement (arcmin)

3 2 1 0 −1

hor. vert.

−2

unstabilized stabilized

−3 −4 0

2

4

6

8

10

Time (s) Figure 5 Eye motion in the AOSLO system before and after eye motion correction. Eye motion of a human subject within 10 s duration is captured at 480 Hz through AOSLO imaging (dotted red and blue lines: horizontal and vertical eye movements). After offline correction, the eye motion in the AOSLO images is reduced to flat lines (in red and blue: horizontal and vertical eye movement), with a standard deviation of 7 arcsec. This compares favorably with the most accurate methods to track the eye, having an accuracy that is roughly one-fourth of the diameter of the smallest foveal cones. Courtesy of A Roorda.

Adaptive Optics

used. Dan Ferguson and Dan Hammer at Physical Sciences Incorporated have developed a hardware eye tracking system specifically for AO retinal imaging that complements the software approaches described above.

11

1.25⬚

Imaging Cones The ability to image cones at high resolution with AO opened a crucial window to examine both normal and abnormal processes in the retina. AO has made possible the first measurements of the antenna properties of single cones in the living human eye. Cone photoreceptors concentrate the image-forming light that passes through the pupil in the photopigment of the outer segment while simultaneously excluding stray light from sources that do not contribute to a sharp retinal image. This beneficial effect of the waveguide nature of cones gives rise to the psychophysical effect known as the Stiles and Crawford effect, in which the sensitivity of the eye declines dramatically for light beams that enter the margin instead of the center of the pupil. Austin Roorda and David Williams at the University of Rochester showed that nearby cones are remarkably well aligned with each other optically so that the angular tuning of a large group of cones is similar to the tuning function for a single cone. Single cones imaged with AO in the living human eye show a striking variability in reflectance on a time scale of hours or days. The cause of this variability is unknown but could be related to the process of disc shedding in the cone outer segments. Don Miller at Indiana University has shown that, especially when the incoming light is highly coherent, there can be dramatic changes on very short time scales of 5–10 ms. These changes depend on the history of the cone’s light exposure and provide an optical method to monitor to the response of cones to light. Kate Grieve and Austin Roorda have also reported increases in infrared reflectance following exposure to light that may provide an alternative method to monitor functional activity in single cones in the retina. The Cone Mosaic and Color Vision The first major scientific application of AO in the eye, undertaken at the University of Rochester, was to determine the organization of the human trichromatic cone mosaic. By combining AO retinal imaging with retinal densitometry, individual cones can be characterized by their sensitivity to long (L), middle (M), or short (S) wavelength light, according to the cone opsin it contains, as the example shown in Figure 6. Experiments using this method have shown that mosaic of L and M cones in the human are essentially randomly organized. It has been known for some time from indirect methods that the relative numbers of L and M cones varies greatly from eye to eye. AO revealed just how large this variation can be even across normal subjects where a variation in the order of 40-fold in L to M cone

Figure 6 Cone mosaic of a human subject at a retinal eccentricity of 1.25 degree with normal color vision. Individual cones in each mosaic were categorized as L, M, or S cone types, using retinal densitometry, and false colored, respectively in red, green, and blue. Courtesy of O. Masuda.

ratio has been observed. The human S cones are arranged randomly near the fovea but with slight tendency toward regular distribution, a tendency that is more pronounced in the macaque monkey. The development of AO for the eye has also made it possible to study color vision in living human eyes in novel ways because it is now possible to deliver tiny flashes of light that are smaller than single cones. Heidi Hofer showed that near-threshold AO-delivered flashes of monochromatic light at a single wavelength produce a rich variety of color percepts. Indeed, for every subject she studied, the range of color experiences was too large to be explained by a simple model in which all cones of the same class produced the same color experience upon stimulation. David Brainard at the University of Pennsylvania has successfully described the range of color experiences in Heidi Hofer’s data with a Bayesian model in which each cone feeds a specific circuit that provides the best estimate of the external stimulus given the local distribution of photon catches in the stimulated cone and its surrounding neighbors. One of the most powerful applications of AO to date involves its use to characterize the topography of the cone mosaic in eyes in which the genotype is known. Until recently, it has been difficult to perform these studies with standard histological methods because of the difficulty in obtaining post-mortem tissue from eyes with specific and often rare genetic anomalies. Joe Carroll has shown that the cone mosaics of single gene dichromats appear completely normal despite the fact that they have two instead of the usual three cone photopigments (Figure 7(b)). On the contrary, dichromats with a specific polymorphism in one of the genes coding for a particular cone photopigment have a cone mosaic with numerous gaps corresponding to a spatially random loss of one class of cones (Figure 7(c); also see Figure 8 for another example).

12

Adaptive Optics

(b)

(a)

(c)

Figure 7 Pathological change of human cone mosaic in red or green dichromat. (a) Cone mosaic from a normal subject at approximately 1 degree of retinal eccentricity. (b) and (c) Cone mosaics at a similar eccentricity of two human subjects having red–green color-vision deficiency. In contrast to the normal cone mosaic in (a), the cone mosaic in (b) did not contain L cones, but appeared to have normal cone density. The cone mosaic in (c), did not contain M cones, and had reduced cone density with patches of functional loss of M cones. Scale ¼ 50 mm for all panels. Reproduced from Carroll, J. Gray, D. C., Roorda, A., Williams, D. R. (2005). Recent advances in retinal imaging with adaptive optics. Optics and Photonics News 16: 36–42, Figure 3, with permission from Optical Society of America (Copyright 2005).

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Figure 8 Pathological change of human cone mosaic in rod monochromat. (a) Cone mosaic from a normal subject at approximately 4 degrees of retinal eccentricity. (b) Possible rod mosaic of one subject who was a congenital achromat. The reason why the photoreceptors in (b) were believed to be rods is that their density and size match with the anatomical characteristic of the rod mosaic at this eccentricity, and differ greatly from the normal cones in (a). Scale ¼ 50 mm for all panels. Reproduced from Carroll, J., Gray, D. C., Roorda, A., Williams, D. R. (2005). Recent advances in retinal imaging with adaptive optics. Optics and Photonics News 16: 36–42, Figure 3, with permission from Optical Society of America (Copyright 2005).

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Figure 9 Fluorescence AOSLO images of primate retinal ganglion cells in vivo. (a–c) Fluorescence AOSLO imaging revealed the morphology of retinal ganglion cells labeled with fluorophore (rhodamine dextran) in living monkey eye. The transverse resolution of the images is fine enough to resolve the individual dendrites. The fluorophore was introduced into the ganglion cells through retrograde labeling through injections in the lateral geniculate nucleus (LGN). Scale ¼ 50 mm for all panels. (a,c) Reproduced from Gray, D. C., Wolfe, R., Gee, B. P., et al. (2008). In vivo imaging of the fine structure of rhodamine-labeled macaque retinal ganglion cells. Investigative Ophthalmology and Visual Science 49: 467–473, Figures 1 and 5, with permission from Association for Research in Vision and Ophthalmology (Copyright 2008).

Adaptive Optics

Imaging Retinal Pigment Epithelium The retinal pigment epithelium (RPE) lies immediately behind the photoreceptors and plays several critical roles in maintaining their function. RPE cell damage is implicated in many retinal degenerative diseases such as agerelated macular degeneration, retinitis pigmentosa, and Stargardt’s disease. The ability to image these cells in the living retina and to track changes in them over time may prove valuable for understanding both normal RPE function and retinal disease. In AOSLO reflectance imaging, the more reflective photoreceptor mosaic normally obscures RPE cells. Occasionally, RPE cells can be seen in patients

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with retinal degenerative diseases, such as cone–rod dystrophy, where the overlying photoreceptors are absent. However, it has recently become possible to image the RPE mosaic in living human eyes in which the photoreceptor layer is intact, by taking advantage of the autofluorescence properties of lipofuscin in the RPE as shown in Figure 4(c). Statistical characterization of the RPE mosaic, for example, packaging arrangement and cell density across eccentricity, in both normal subject and patients may eventually prove to be valuable for the clinical diagnosis of earlier stages of retinal degenerative disease. It may ultimately prove possible to use AO to image subcellular structures in RPE cells

(b)

Figure 10 Fluorescence AOSLO images of rat retinal ganglion cells in vivo. (a) Fluorescence AOSLO image of rat retina with ganglion cell expressing EGFP. Scale ¼ 50 mm. Image was taken at a large field of view (FOV). Gene encoding EGFP was delivered to ganglion cells through an AAV2 viral vector administrated intravitreally. The ganglion cell indicated by the white arrow was shown in (a) at higher magnification. Image at this view reveals the dendritic morphology of the cell. Such images could provide basis for morphological classification in vivo. Scale ¼ 20 mm.

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Figure 11 AOSLO images of retinal vasculature. (a) Retinal vasculature in the macular region in primate eye imaged by fluorescence AOSLO in combination with fluorescence angiography. Scale ¼ 150 mm. (b) Direction of blood flow and leukocyte velocity within the capillaries of the macular region in human eye calculated from reflectance AOSLO images. Movement of discrete leukocytes was detected through an image-processing algorithm. The velocities of the leukocytes labeled on the images were in mm s–1. Scale ¼ 1 degree. (a) Reproduced from Gray, D. C., Merigan, W., Wolfing, J. I. et al. (2006). In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells. Optics Express 14(16): 7144–7158, Figure 6, with permission from Optical Society of America (Copyright 2006). (b) Adapted from Roorda, A., Garcia, C. A., Martin, J. A., et al. (2006). What can adaptive optics do for a scanning laser ophthalmoscope? Bulletin de la Socie´te´ Belge d’Ophthalmologie 302: 231–244, Figure 6, with permission from Bulletin of the Belgian Societies of Ophthalmology (Copyright 2006).

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Adaptive Optics

and/or to monitor changes in RPE cells over time. Jessica Morgan has already shown that it is possible to use AO to track the time course of lipofuscin autofluoresence bleaching and RPE damage following exposure to bright light. Imaging Retinal Ganglion Cells The retinal image is conveyed to the brain through an array of 17 or more parallel ganglion cell pathways in the primate. We know remarkably little about the functional significance of many of these morphologically distinct ganglion cell classes. One of the major impediments to learning more about them is that the less numerous cells are difficult to target with a microelectrode that is capable of recording from only one single cell at a time. The development of optical methods to record from many ganglion cells

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simultaneously in the intact living eye could greatly accelerate our understanding of ganglion cell function. There are numerous technical hurdles to overcome before this is feasible, but AO retinal imaging has now solved an important one: it is now possible to image ganglion cells along with some of their subcellular structure in vivo in nonhuman primate retina. This capability may also eventually prove useful in the study of diseases such as glaucoma, in which blindness results from the death of ganglion cells. New ways to image ganglion cells at a microscopic spatial scale may lead to earlier diagnosis and delivery of therapy. Dan Gray, working in the AO group at the University of Rochester, imaged the dendritic morphology of macaque ganglion cells retrogradely labeled with fluorescent dye (rhodamine dextran) in vivo, with a transverse resolution of 1.6 mm (characterized as full width at half maximum;

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(f) Figure 12 Structural change of cone mosaic of patients having retinal degenerative diseases. (a) and (c) Cone mosaics of a patient having cone–rod dystrophy compared, respectively with (b) and (d) cone mosaics of normal subjects at matched retinal eccentricity. For the patient, severe cone loss was apparent in (c), while in (a), cones still form a continuous mosaic, but the cone density is much decreased and cone size is much larger. Scale ¼ 25 mm for panels (a–d). (e) and (f) Foveal cone mosaics of patients having disease caused by mitochondrial DNA (mtDNA) mutation. The two patients were affected by the disease at different severity. Cone mosaic of the less-affected patient in (f) showed normal cone spacing, while the cone mosaic of the more-affected patient in (e) showed increased cone size and cone spacing. The preferred fixation point of each subject is visualized as dark dots in (e) and (f). Scale ¼ 1 degree for both panels. (a–d), Reproduced from Wolfing J.I., Chung, M., Carroll, J., et al. (2006). High-resolution retinal imaging of cone-rod dystrophy. Ophthalmology 113: 1014–1019, Figure 4, with permission from the American Academy of Ophthalmology, Elsevier Ltd (2006). (e,f) Adapted from Yoon, M.K., Roorda, A., Zhang Y., et al. (2009). Adaptive optics scanning laser ophthalmoscopy images in a family with the mitochondrial DNA T8993C mutation. Investigative Ophthalmology and Visual Science 50: 1838–1847, Figure 4, with permission from Association for Research in Vision and Ophthalmology (Copyright 2009).

Adaptive Optics

Figure 9). Although in some cases, they were able to distinguish ganglion cell bodies at different depths in the retina, which is a valuable way to distinguish different classes, the depth resolution is modest, about 115 mm. Viral vector (e.g., AAV2)-mediated-gene delivery is another promising approach for delivering fluorophores (e.g., EGFP) to single cells. It requires a simpler intravitreal rather than an intracranial injection and can produce fluorescence that is essentially permanent in each cell that is transduced by the virus. Melissa Geng and Jason Porter at University of Rochester in collaboration with John Flannery at UC Berkeley combined this method with fluorescence AOSLO in the living rat eye (Figure 10). Another possibility is to use transgenics to create an animal whose cells express a fluorophore. Charles Lin and colleagues used this method to image microglia cells in living mouse eye. Work is underway to extend the viral vector method to nonhuman primate and to achieve expression of activity-dependent fluorophores such as G-CaMP (a green flurescent protein calcium probe) in single ganglion cells. These methods may ultimately allow AO retinal imaging to monitor the functional responses of ganglion cells. Imaging Retinal Vasculature Many retinal diseases, such as diabetic retinopathy or wet macular degeneration, have important effects on the retinal

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vascular structure and blood flow. AO combined with fluorescein angiography allows imaging of even the fine structure of the retinal capillary bed (Figure 11(a)). Austin Roorda, then at the University of Houston, showed that AO retinal imaging could image leukocyte motion in the smallest retinal capillaries without the need for fluorescein (Figure 11(b)). Steve Burns at University of Indiana has even succeeded in imaging the flow of erythrocytes by tracking movement of single erythrocytes in successive images of a single scan line superimposed on an oriented parallel to a small vessel.

Imaging Retinal Disease The use of an AO to image retinal disease is one of the most exciting and also one of the most challenging applications. AO is at its best when the pupil is completely dilated and the ocular media are clear and scatter free. The challenge of imaging retinal disease lies in the fact that patients often have small pupils, poor fixation, and/or large amounts of light scatter. Despite these limitations, progress has been made in the deployment of AO in the clinical arena. For many retinal degenerative diseases, the structural changes of the cone mosaic and the RPE mosaic of patients have been impressively documented in vivo. The diseases affecting cones include not only those relevant

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8⬚ TVF

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Figure 13 Correspondence between structural changes in the cone mosaic and functional measurement of visual sensitivity. Measured visual field sensitivity of a patient having rod–cone dystrophy is shown in the upper middle panel. The rest of the panels show the cone mosaics from retinal regions corresponding to locations in the visual field map. In those cone mosaic images, the darker regions indicate areas where cones are not reflecting light as in normal retina. From these images, it is not possible to determine if these dark regions represent loss of cones or structurally altered cones, hence became less effective in waveguiding and reflecting light. The extent of these dark regions strongly correlated with the level of visual sensitivity. The more extensive the dark regions (i.e., reduced cone density) were the lower the visual function (e.g., the comparison between the left and right panels). A strong positive relationship was found between the cone density and the level of visual sensitivity in patients with various types of retinal dystrophy. Adapted from Choi, S.S., Doble, N., Hardy, J.L., et al. (2006). In vivo imaging of the photoreceptor mosaic in retinal dystrophies and correlations with visual function. Investigative Ophthalmology and Visual Science 47: 2080–2092, Figure 5, with permission from Association for Research in Vision and Ophthalmology (Copyright 2006).

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Adaptive Optics

to color blindness mentioned previously, but also more common ones such as retinitis pigmentosa and photoreceptor dystrophy. As per the examples shown in Figure 12, cone loss can occur as a patchy, random dropout of cones that otherwise appear normal. In other cases, presumably when the mosaic has had the opportunity to remodel, the mosaic completely tiles the retina but the cones are larger and their density is reduced. Because the structure of the cone mosaic can be documented in living eyes, the specific structure in individual eyes can be correlated with the genotype of the patient, and also with functional measurements such as Humphrey visual field sensitivity, multifocal ERG, and contrast sensitivity (Figure 13). Retinal degenerative diseases affect many other retinal structures, besides photoreceptors and RPE. As described in this article, AO imaging has enabled the accumulation of normative data of many retinal structures in both human and nonhuman primates, as well as data from animal disease models. These data will advance the clinical investigation of retinal degenerative disease. For example, the structure of lamina cribrosa was imaged in both normal primate eye and eye with experimentally induced glaucoma. Ultimately, AO imaging may play a role in the diagnosis of glaucoma and other retinal diseases in clinical practice. See also: Color Blindness: Acquired; Color Blindness: Inherited; Optical Coherence Tomography.

Further Reading Arathorn, D. W., Yang, Q., Vogel, C. R., et al. (2007). Retinally stabilized cone-targeted stimulus delivery. Optics Express 15: 13731–13744. Artal, P., Chen, L., Fernandez, E. J., et al. (2004). Neural compensation for the eye’s optical aberrations. Journal of Vision 4: 281–287. Biss, D. P., Sumorok, D., Burns, S. A., et al. (2007). In vivo fluorescent imaging of the mouse retina using adaptive optics. Optics Letters 32: 659–661. Carroll, J., Neitz, M., Hofer, H., Neitz, J., and Williams, D. R. (2004). Functional photoreceptor loss revealed with adaptive optics: An alternate cause of color blindness. Proceedings of the National Academy of Sciences of the United States of America 101: 8461–8466. Carroll, J., Gray, D. C., Roorda, A., and Williams, D. R. (2005). Recent advances in retinal imaging with adaptive optics. Optics and Photonics News 16: 36–42.

Chen, L., Artal, P., Gutierrez, D., and Williams, D. R. (2007). Neural compensation for the best aberration correction. Journal of Vision 7(9): 1–9. Geng Y., Greenberg K. P., Wolfe R., Gray D. C., et al. (2009). In vivo imaging of microscopic structures in the rat retina. Investigative Ophthalmology and Visual Science 50: 5872–5879. Gray, D. C., Wolfe, R., Gee, B. P., et al. (2008). In vivo imaging of the fine structure of rhodamine-labeled macaque retinal ganglion cells. Investigative Ophthalmology and Visual Science 49: 467–473. Hofer, H., Artal, P., Singer, B., Aragon, J. L., and Williams, D. R. (2001). Dynamics of the eye’s wave aberration. Journal of Optical Society of America A, Optics, Image Science, and Vision 18: 497–506. Hofer, H., Carroll, J., Neitz, J., Neitz, M., and Williams, D. R. (2005). Organization of the human trichromatic cone mosaic. Journal of Neuroscience 25: 9669–9679. Hofer, H., Singer, B., and Williams, D. R. (2005). Different sensations from cones with the same photopigment. Journal of Vision 5: 444–454. Liang, J., Williams, D. R., and Miller, D. T. (1997). Supernormal vision and high-resolution retinal imaging through adaptive optics. Journal of Optical Society of America A, Optics, Image Science, and Vision 14: 2884–2892. Martin, J. A. and Roorda, A. (2005). Direct and noninvasive assessment of parafoveal capillary leukocyte velocity. Ophthalmology 112: 2219–2224. Morgan, J. I., Dubra, A., Wolfe, R., Merigan, W. H., and Williams, D. R. (2009). In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic. Investigative Ophthalmology and Visual Science 50: 1350–1359. Morgan, J. I., Hunter, J. J., Masella, B., et al. (2008). Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium. Investigative Ophthalmology and Visual Science 49: 3715–3729. Porter, J., Guirao, A., Cox, I. G., and Williams, D. R. (2001). Monochromatic aberrations of the human eye in a large population. Journal of Optical Society of America A, Optics, Image Science, and Vision 18: 1793–1803. Porter, J., Queener, H., Lin, J., Thorn, K., and Awwal, A. A. S. (2006). Adaptive Optics for Vision Science: Principles, Practices, Design and Applications. Hoboken, NJ: Wiley-Interscience. Roorda, A. and Williams, D. R. (1999). The arrangement of the three cone classes in the living human eye. Nature 397: 520–522. Thibos, L. N., Hong, X., Bradley, A., and Cheng, X. (2002). Statistical variation of aberration structure and image quality in a normal population of healthy eyes. Journal of Optical Society of America A, Optics, Image Science, and Vision 19: 2329–2348. Yoon, G. Y. and Williams, D. R. (2002). Visual performance after correcting the monochromatic and chromatic aberrations of the eye. Journal of Optical Society of America A, Optics, Image Science, and Vision 19: 266–275. Zhong, Z., Petrig, B. L., Qi, X., and Burns, S. A. (2008). In vivo measurement of erythrocyte velocity and retinal blood flow using adaptive optics scanning laser ophthalmoscopy. Optics Express 16: 12746–12756.

Alternative Visual Cycle in Mu¨ller Cells G H Travis, UCLA School of Medicine, Los Angeles, CA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Mu¨ller cell – A glial cell in the vertebrate retina that spans from the vitreal surface to the external limiting membrane, with apical processes that extend into the interphotoreceptor matrix. These cells perform multiple functions in the retina including the processing of visual retinoids. Opsin visual pigment – A member of the G-proteincoupled receptor superfamily that functions as the light receptor in rods and cones. These pigments represent a complex between an opsin protein and a visual chromophore (see below). Outer segment – An elongated light-sensitive structure attached to the connecting cilium of rod and cone photoreceptors. The rod outer segment in humans comprises a stack of approximately 1000 membranous disks. These disks are loaded with rhodopsin or cone opsin visual pigments. Rpe65 – An abundant, membrane-associated protein in retinal pigment epithelium cells that functions as a retinoid isomerase. In particular, Rpe65 catalyzes the conversion of an all-trans-retinyl ester to 11-cis-retinol and a free fatty acid. Visual chromophore – The light-absorbing molecular species in an opsin protein. The most common visual chromophore in vertebrates is 11-cis-retinaldehyde, which isomerizes to all-trans-retinaldehyde upon absorption of a photon.

Vision in vertebrates is provided by two types of photoreceptor cells, rods and cones. Rods mediate vision in dim light while cones mediate high-resolution color vision in bright light. Approximately 95% of photoreceptors in the human retina are rods. Nonetheless, cones are far more important for vision in humans. With the advent of artificial lighting, humans spend much of the time under conditions where the rod photoresponse is saturated and vision is mediated by cones. Both rods and cones contain a lightsensitive structure called the outer segment (OS) comprising a stack of densely packed membranous disks. These disks are packed with rhodopsin or cone-opsin visual pigment. The light-absorbing chromophore in most opsin pigments is 11-cis-retinaldehyde (11-cis-RAL). Absorption of a photon induces its isomerization to all-trans-retinaldehyde (all-transRAL), which activates the opsin pigment and stimulates the visual transduction cascade. After a brief period of activation,

the pigment dissociates to yield free all-trans-RAL and apoopsin, which is no longer light sensitive. The pigment is regenerated by recombination of apo-opsin with another 11-cis-RAL. To sustain light sensitivity, all-trans-RAL released by bleached opsin pigments is converted back to 11-cis-RAL by a multi-step enzyme pathway called the visual cycle (Figure 1).

Visual Cycle in Retinal Pigment Epithelium Cells All but the first step of the visual cycle takes place within cells of the retinal pigment epithelium (RPE), an epithelial monolayer adjacent to photoreceptor OS. In brief, alltrans-RAL is reduced by all-trans-retinol dehydrogenase (all-trans-RDH) in rod and cone OS to all-trans-retinol (all-trans-ROL) or vitamin A. The all-trans-ROL is released by the OS into the extracellular space or interphotoreceptor matrix (IPM), where it is bound to interphotoreceptor retinoid-binding protein (IRBP). The all-trans-ROL is taken up by apical processes of RPE cells, where it binds to cellular retinol binding protein type-1 (CRBP1). HoloCRBP1 is the substrate for lecithin:retinol acyl transferase (LRAT), which transfers a fatty acid from phosphatidylcholine in internal membranes to the all-trans-ROL. The resulting fatty-acyl ester of all-trans-ROL (all-trans-RE) is the substrate for Rpe65-isomerase. Rpe65 catalyzes hydrolysis of the carboxylate ester and utilizes the energy released for isomerization of all-trans-ROL to 11-cis-retinol (11-cis-ROL). The final catalytic step in the visual cycle is oxidation of 11-cis-ROL, by one of several 11-cis-ROL dehydrogenases (11-cis-RDH) to 11-cis-RAL. Both 11-cisROL and 11-cis-RAL are bound to cellular retinaldehyde binding protein (CRALBP) in the RPE. The 11-cis-RAL is released by the apical RPE to the IPM, where it binds IRBP. IRBP carries the 11-cis-RAL to the photoreceptor OS, where it is taken up and recombines with apo-opsin to reform a rhodopsin or cone-opsin pigment.

A Role for Mu¨ller Cells in Visual Pigment Regeneration Evidence suggests that Mu¨ller glial cells in the retina also participate in the processing of visual retinoids. Mu¨ller cells span nearly the full thickness of the retina. The apical microvilli of Mu¨ller cells extend into the IPM and hence are well situated to exchange visual retinoids

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+ 11-cis-RAL CH=NH− (rhodopsin) hv ApoMetarhodopsin II opsin

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Figure 1 Visual cycle for rhodopsin regeneration. Absorption of a photon (hv) by a rhodopsin pigment molecule induces isomerization of 11-cis-RAL to all-trans-RAL, which converts the pigment to its active metarhodopsin II state. After deactivation, the pigment dissociates to yield apo-opsin and free all-trans-RAL. The all-trans-RAL is reduced to all-trans-ROL by one or more all-trans-RDHs that use NADPH as a co-factor. The all-trans-ROL is released by the OS to the IPM and is taken up by the apical RPE where it is esterified by LRAT to yield an all-trans-RE. The all-trans-RE is isomerized and hydrolyzed by Rpe65 to yield 11-cis-ROL. The 11-cis-ROL is oxidized by one or more 11-cis-RDHs to yield 11-cis-RAL chromophore. The 11-cis-RAL is released by the RPE into the IPM where it binds to IRBP. Finally, the 11-cis-RAL is delivered to the OS where it re-combines with apo-opsin to form a new visual pigment.

with rod and cone OS, similar to the apical microvilli of RPE cells. Importantly, Mu¨ller cells contain several proteins involved in the processing of visual retinoids. These include (1) CRALBP, (2) RPE-retinal guaninenucleotide-binding protein (G-protein) coupled receptor (RGR-opsin), a non-photoreceptor opsin that effects light-dependent regulation of the visual cycle in RPE cells, (3) CRBP1, and (4) retinol dehydrogenases types 10 and 11. Mu¨ller cells neither express Rpe65 nor LRAT, which are both present in RPE cells. Therefore, Mu¨ller cells do not simply duplicate the function of RPE cells in the regeneration of visual chromophore.

A Second Source of Chromophore for Cones Several lines of evidence suggest that an alternate source of chromophore precursor is available to cones but not rods. When frog retinas were separated from the RPE, cone opsins, but not rhodopsin, regenerated spontaneously after a photobleach. After photobleaching, isolated salamander cones recovered sensitivity with the addition of either 11-cis-ROL or 11-cis-RAL, while isolated rods only recovered sensitivity with the addition of 11-cisRAL. Mu¨ller cells in primary culture were shown to take up all-trans-ROL and synthesize 11-cis-ROL, which they secreted into the medium. Salamander cones were shown to dark-adapt and regenerate visual chromophore in isolated retinas separate from the RPE. The intrinsic capacity of cones to recover sensitivity and regenerate

visual chromophore in salamander retinas was lost after exposure to a selective Mu¨ller-cell toxin (a-aminoadipic acid), suggesting that Mu¨ller cells play a role in these processes.

Functional Differences between Rods and Cones Although morphologically similar, rods and cones differ greatly in sensitivity, dynamic range, and speed of the photoresponse. Rods are single-photon detectors and show saturation of the photoresponse under relatively dim background illumination, producing 500 or more photoisomerizations per second. At saturation, all cyclic guanosine monophosphate (cGMP)-gated cation channels are closed and the rod no longer responds to light. Cones, on the other hand, are 100-fold less sensitive than rods but still exhibit a photoresponse to a light flash under bright background illuminations producing up to 106 photoisomerizations per second. The response kinetics of cones is also several-fold faster than rods. These disparities reflect differences in the properties of rhodopsin and the cone-opsin pigments. Rhodopsin is exceedingly quiet, with a spontaneous activation rate in the dark of one thermal isomerization every 2000 years. Cone opsins are much noisier than rhodopsin for two reasons. First, the spontaneous isomerization rate of cone opsin is 10 000fold higher than of rhodopsin. Second, 11-cis-RAL spontaneously dissociates from cone-opsins. A dark-adapted


red cone contains approximately 10% apo-cone-opsin due to spontaneous dissociation of chromophore. In contrast, 11-cis-RAL combines almost irreversibly with apo-rhodopsin in rods. Apo-opsin, which results from dissociation of 11-cis-RAL, activates the transduction cascade, producing the phenomenon of dark light. These effects contribute to the noisy background of cones and explain their much lower sensitivity than rods. Although the rod response is saturated in bright light, photon capture by rhodopsin continues unabated. Thus, in a rod-dominant retina under daylight conditions, the vast majority of photoisomerization events contributes nothing to useful vision. Under these circumstances, cones must compete with rods for the limited supply of 11-cis-RAL. The reversible binding of 11-cis-RAL to apocone-opsins and its irreversible binding to apo-rhodopsin confers a tendency of rods to steal chromophore from cones. This tendency aggravates the competition between rods and cones for limited chromophore.

An Alternate Retinoid Isomerase Activity in Cone-Dominant Retinas Studies on the biochemistry of visual pigment regeneration in cone-dominant retinas suggest a mechanism whereby cones may escape competition from rods for visual chromophore. Experiments were performed on separated retinas and RPE from mice and cattle, as model roddominant species, and from chickens and ground squirrels, as model cone-dominant species. Measurement of endogenous retinoids showed that the rod-dominant species contain high levels of all-trans-retinyl esters (REs) in the RPE but no detectable REs in the retina. Cone-dominant species contain REs in both the RPE and retina, with predominantly 11-cis-REs in the retina. The activities of retinoidprocessing enzymes were measured by incubating total homogenates or microsomes prepared from RPE or retinas with all-trans-ROL, then assaying for synthesis of new retinoids by high-performance liquid chromatography (HPLC). Incubation of RPE homogenates with all-transROL resulted in rapid synthesis of all-trans-REs, due to the activity of LRAT. Only after accumulation of all-trans-REs was 11-cis-ROL synthesis detected, due to the activity of Rpe65. This profile of retinoid synthesis was observed with RPE homogenates from both rod- and cone-dominant species. A strikingly different profile was observed when retina homogenates from cone-dominant chicken or ground squirrels were incubated with all-trans-ROL under similar conditions. Addition of all-trans-ROL resulted in rapid synthesis of 11-cis-REs and 11-cis-ROL, with negligible initial synthesis of all-trans-REs. Addition of palmitoyl coenzyme A (palm CoA) to the assay mixture increased synthesis of 11-cis-REs, again with minimal initial synthesis

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of all-trans-REs. Synthesis of 11-cis-ROL and 11-cis-REs from all-trans-ROL without prior formation of all-transREs suggests that the mechanism of retinoid isomerization is different in retina versus RPE. Instead of converting an all-trans-RE to 11-cis-ROL and a free fatty acid, as catalyzed by Rpe65 in RPE cells, cone-dominant retinas appear to catalyze the direct conversion of all-trans-ROL to 11-cisROL. This energetically unfavorable reaction appears to be driven by mass action through secondary esterification of the 11-cis-ROL product. Here, the energy of isomerization is indirectly supplied by hydrolysis of the thio-ester in palm CoA versus hydrolysis of the carboxyl ester in phosphatidylcholine, as catalyzed by Rpe65. The isomerase machinery in retinas may therefore involve two catalytic activities, as depicted in Figure 2. One is an isomerase that catalyzes the passive interconversion of all-trans-ROL and 11-cisROL. This enzyme has not yet been identified. The second is a palm-CoA-dependent RE synthase. Several proteins with acyl CoA:retinol acyltransferase (ARAT) activity have been described. At least one of these, diacylglycerol acyltransferase type-1 (DGAT1), is expressed in the retina.

11-cis-ROL Dehydrogenase Activity in Cones but Not in Rods As discussed above, cones can regenerate visual pigments and restore light sensitivity with the addition of either 11-cis-RAL or 11-cis-ROL, while rods can only regenerate rhodopsin pigment and recover sensitivity with addition of 11-cis-RAL. Since 11-cis-RAL is the visual chromophore for both cone and rod pigments, these observations suggest that cones, but not rods, express an 11-cis-RDH activity that oxidizes 11-cis-ROL to 11-cis-RAL. Robust nicotinamide adenine dinucleotide phosphate (NADPH)dependent 11-cis-RDH activity has been detected in cone Palm CoA ARAT

RetinolAll-trans-ROL

11-cis-ROL

Isomerase-2

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Ester synthase

Isomerosynthase complex

HS-CoA

Figure 2 Proposed isomerase-2 complex in Mu¨ller cells. The isomerase in Mu¨ller cells appears to catalyze direct conversion of all-trans-ROL to 11-cis-ROL. This energetically unfavorable reaction appears to be driven by mass action through secondary esterification of 11-cis-ROL to an 11-cis-RE. This synthase uses palm CoA as an acyl donor, and hence is a type of ARAT. The isomerase-2/ARAT complex has been named isomerosynthase to denote its activities. Neither isomerase-2 nor its ARAT partner has been identified to date.

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but not rod in photoreceptors. Reduction of all-trans-RAL to all-trans-ROL in bleached photoreceptors requires NADPH, and hence is an energy-consuming process. Oxidation of an 11-cis-ROL to 11-cis-RAL generates an NADPH reducing-equivalent. Since reduction of all-trans-RAL and oxidation of 11-cis-ROL occurs with 1:1 stoichiometry in a cone that is utilizing 11-cis-ROL as a chromophore precursor, the presence of reciprocal NADP+/NADPH-specific dehydrogenases in cones affords a self-renewing supply of dinucleotide substrate at no energy cost to the cell. Eliminating the need for energydependent synthesis of NADPH may remove the metabolic bottleneck of all-trans-RAL-reduction in cones at high rates of photoisomerization.

An Alternate Visual Cycle that Mediates Pigment Regeneration in Cones The observations outlined above can be explained by the hypothesized alternate visual cycle shown in Figure 3. According to this model, all-trans-ROL-isomerase (isomerase-2) and the 11-cis-ROL-specific ARAT are in Mu¨ller cells, which also express the 11-cis-ROL and 11-cis-RAL binding-protein, CRALBP. The major source of substrate for isomerase-2 is all-trans-ROL released by rods and cones during light exposure. Isomerization of alltrans-ROL to 11-cis-ROL and subsequent esterification of 11-cis-ROL to 11-cis-REs are probably limited by substrate

availability. It has been shown that apo-CRALBP stimulates 11-cis-RE-hydrolase (11-cis-REH) in RPE cells. These observations suggest a regulatory mechanism for the proposed visual cycle: in the dark-adapted state, cones contain fully regenerated opsin pigment and hence do not use 11-cis-ROL. CRALBP is saturated with 11-cis-ROL and the level of 11-cis-REs are high in Mu¨ller cells. Both isomerase-2 and 11-cis-RE-synthase are inactive due to the absence of available substrate. With the onset of light and the bleaching of photopigments, cones begin to take up 11-cis-ROL. This results in desaturation of CRALBP, activation of 11-cis-REH, and mobilization of 11-cis-RE-stores in Mu¨ller cells. Rods and cones begin to release all-transROL, which permits replenishment of 11-cis-REs through activation of isomerase-2 and 11-cis-RE-synthase. This pathway becomes progressively more active with increasing light intensity, availability of all-trans-ROL substrate, and consumption of the 11-cis-ROL product by cones.

A New Role for IRBP IRBP is the major extracellular retinoid-binding protein in retinas. It has been suggested that the primary function of IRBP is to bind all-trans-ROL and 11-cis-RAL during translocation of these retinoids between OS and the RPE. However, loss of IRBP in irbp / mice has only a mild effect on rhodopsin regeneration. Might IRBP play another role? The endogenous ligands of IRBP have been studied.

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Figure 3 Proposed alternate visual cycle in Mu¨ller cells. Absorption of a photon (hv) induces 11-cis to all-trans isomerization of the retinaldehyde chromophore, resulting in activated opsin (MII) inside a cone outer segment. Decay of MII releases all-trans-RAL, which is reduced to all-trans-ROL by one or more NADPH-dependent all-trans-RDHs. The all-trans-ROL is released to the IPM where it binds IRBP and is carried to a Mu¨ller cell apical process. After uptake into the Mu¨ller cells, the all-trans-ROL is isomerized by isomerase-2 and esterified by ARAT to yield an 11-cis-RE (11-cis-RP). Mu¨ller cells also contain CRALBP, which binds 11-cis-ROL but not all-trans-retinol. 11-cis-REH is activated by apo-CRALBP to hydrolyze the 11-cis-RE, yielding 11-cis-ROL. Binding of 11-cis-ROL by CRALBP prevents its back-isomerization to all-trans-ROL. The 11-cis-ROL is released by CRALBP to IRBP in the IPM, where it is carried back to the cone OS. Within the cone, an NADP+-dependent 11-cis-RDH oxidizes the 11-cis-ROL to 11-cis-RAL, which re-combines with apo-opsin to regenerate a new, light-sensitive cone pigment. The reciprocal reduction of all-trans-RAL and oxidation of 11-cis-ROL results in a self-renewing supply of NADP-co-factor at all rates of photoisomerization and no energy cost to the cell.


In light-adapted frog and bovine retinas, IRBP contains higher levels of 11-cis-ROL than 11-cis-RAL, in addition to all-trans-ROL. Moreover, IRBP immunoreactivity was found in association with cone but not rod OS. These observations suggest that the critical function of IRBP may not be exchange of all-trans-ROL and 11-cis-RAL between rods and RPE cells, as previously assumed, but rather exchange of all-trans-ROL and 11-cis-ROL between cones and Mu¨ller cells.

Alternate Visual Cycle in Rod-Dominant Species Although clearly present in cone-dominant retinas, the evidence for an alternate visual cycle in Mu¨ller cells of roddominant species is less compelling. Endogenous 11-cis-REs and isomerase-2 catalytic activity were undetectable in homogenates from rod-dominant bovine and mouse retinas. The absence of 11-cis-retinoids in rpe65 / mice has also been put forth as further evidence against the Mu¨ller-cell pathway in this species. However, this observation is difficult to interpret since rpe65 / photoreceptors contain no visual chromophore, hence all-trans-ROL, the substrate for the alternative visual cycle, is not released following light exposure. The effective Michaelis constant (Km) of all-trans-ROL for 11-cis-RE-synthesis by chicken retina homogenates is 13.5 mM. Thus, even in chickens, the alternate visual cycle is only active under conditions that yield high concentrations of all-trans-ROL. Recently, isolated mouse retinas were photobleached and analyzed physiologically for recovery of rod and cone light sensitivity. Cone sensitivity and the cone photoresponse recovered in these retinas despite the absence of RPE cells, while rod sensitivity and the rod response were dramatically attenuated. These physiological observations argue for existence of the alternate visual cycle in mouse retinas. The role of this pathway in rod-dominant retinas should be resolved upon identification of the proteins responsible for isomerase-2 activity, and measurement of their expression levels in rod- and cone-dominant retinas. See also: Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Phototransduction: The Visual Cycle.

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Further Reading Chen, Y. and Noy, N. (1994). Retinoid specificity of interphotoreceptor retinoid-binding protein. Biochemistry 33: 10658–10665. Das, S. R., Bhardwaj, N., Kjeldbye, H., and Gouras, P. (1992). Muller cells of chicken retina synthesize 11-cis-retinol. Biochemical Journal 285: 907–913. Fain, G. L., Matthews, H. R., Cornwall, M. C., and Koutalos, Y. (2001). Adaptation in vertebrate photoreceptors. Physiological Reviews 81: 117–151. Jin, M., Li, S., Nusinowitz, S., et al. (2009). The role of interphotoreceptor retinoid-binding protein on the translocation of visual retinoids and function of cone photoreceptors. Journal of Neuroscience 29: 1486–1495. Jones, G. J., Crouch, R. K., Wiggert, B., Cornwall, M. C., and Chader, G. J. (1989). Retinoid requirements for recovery of sensitivity after visual-pigment bleaching in isolated photoreceptors. Proceedings of the National Academy of Sciences of the United States of America 86: 9606–9610. Kefalov, V. J., Estevez, M. E., Kono, M., et al. (2005). Breaking the covalent bond – a pigment property that contributes to desensitization in cones. Neuron 46: 879–890. Mata, N. L., Radu, R. A., Clemmons, R., and Travis, G. H. (2002). Isomerization and oxidation of vitamin A in cone-dominant retinas. A novel pathway for visual-pigment regeneration in daylight. Neuron 36: 69–80. Mata, N. L., Ruiz, A., Radu, R. A., Bui, T. V., and Travis, G. H. (2005). Chicken retinas contain a retinoid isomerase activity that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol. Biochemistry 44: 11715–11721. Miyazono, S., Shimauchi-Matsukawa, Y., Tachibanaki, S., and Kawamura, S. (2008). Highly efficient retinal metabolism in cones. Proceedings of the National Academy of Sciences of the United States of America 105: 16051–16056. Pugh, E. N. and Lamb, T. D. (2000). Phototransduction in vertebrate rods and cones: Molecular mechanisms of amplification, recovery and light adaptation. In: Stavenga, D. G., DeGrip, W. J., and Pugh, E. N., Jr (eds.) Handbook of Biological Physics, pp. 184–255. Amsterdam: Elsevier Science B.V. Radu, R. A., Hu, J., Peng, J., et al. (2008). Retinal pigment epitheliumretinal G protein receptor-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells. Journal of Biological Chemistry 283: 19730–19738. Saari, J. C. and Bredberg, D. L. (1987). Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina. Journal of Biological Chemistry 262: 7618–7622. Stecher, H., Gelb, M. H., Saari, J. C., and Palczewski, K. (1999). Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein. Journal of Biological Chemistry 274: 8577–8585. Wang, J. S., Estevez, M. E., Cornwall, M. C., and Kefalov, V. J. (2009). Intra-retinal visual cycle required for rapid and complete cone dark adaptation. Nature Neuroscience 12: 295–302. Yen, C. L., Monetti, M., Burri, B. J., and Farese, R. V., Jr. (2005). The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters. Journal of Lipid Research 46: 1502–1511.

Anatomically Separate Rod and Cone Signaling Pathways S Nusinowitz, UCLA School of Medicine, Los Angeles, CA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Neurotransmitter – A chemical substance that is released at synaptic connections that are used to relay, amplify, and modulate signals between cells and neurons. Photopic vision – The scientific term for color vision mediated by multiple cone photoreceptor types in bright light. In the human retina, photopic vision is tri-chromatic. Phototransduction – A process by which light is converted into electrical signals in rod and cone photoreceptor cells in the retina of the eye. Retinal circuitry – It refers to the neuronal pathways in the retina that carry information from photoreceptor cells to ganglion cells. Scotopic vision – Vision mediated by rod photoreceptors in dim light. Scotopic vision is color blind. Spatio-temporal vision – A term commonly used to describe the spatial and temporal properties of human visual processing, and the neural mechanisms which underpin them. Visual adaptation – A process by which vision adjusts to or gets used to a change in overall brightness, color, and other spatio-temporal properties, in order to maximize visual sensitivity.

Rod and Cone Photoreceptors The process of vision is initiated by the absorption of light by a photoreceptor pigment molecule. The mammalian retina contains two classes of photoreceptors, referred to as rods and cones, each distinct in their structural morphology and in their behavior in response to light. Rod photoreceptors are highly sensitive to light and are capable of responding to a single photon. In contrast, cone photoreceptors are less sensitive to light, but are exquisitely tuned to mediate color and spatio-temporal vision. There are three types of cone photoreceptors in the human retina, each with different, but overlapping, spectral sensitivities, and the interaction of the output from these photoreceptor types mediates the ability to discriminate small color differences. Based on studies of the evolution of visual pigments, it is hypothesized that rods evolved from cones. A single

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amino acid substitution has been shown to exchange the molecular properties of rod and cone visual pigments. This suggests that a single spontaneous amino acid substitution could have been one of the key steps in the divergence of rod- and cone-mediated vision. The first step in the formation of rods themselves may have been the spontaneous formation of disk membranes that were separated from the plasma membrane of the outer segment. This step is thought to have increased the light sensitivity of the photoresponse, a physiological property that is typical of rod photoreceptors. The post-receptoral retinal circuitry for rods is superimposed on pre-existing retinal circuitry mediating cone function. This feature of retinal circuitry predates the evolution of the rod photoreceptor itself and likely represents the pathway of a redundant cone photoreceptor that evolved into a rod. As described below, there are multiple sites in the retinal circuitry where rods have the opportunity to penetrate the cone system circuitry. The absorption of a photon of light by a photoreceptor pigment molecule initiates a sequence of events referred to as the phototransduction cascade. Much of what we know about the specifics of the phototransduction cascade comes from the study of the rod photoresponse. In mammalian rods, the absorption of a photon of light by rhodopsin, the visual chromophore in rods, initiates a sequence of biochemical events that ultimately leads to a decrease in the intracellular level of cyclic guanosine 3,50 -monophosphate (cGMP) and the closure of the cGMP-gated ion channels in the outer segment. The resultant closure of the cGMPgated channels in the outer-segment membrane decreases the circulating current by blocking inward current flow. This results in an hyperpolarization of the photoreceptor cell membrane and a decrease in the release of neurotransmitter at the photoreceptor synapse with second-order neurons. The biochemical steps in cone outer-segment phototransduction are thought to be similar to that which occurs in rods. In fact, there are corresponding components in rod and cone phototransduction at each step in the cascade, including rod and cone versions of visual pigment, tranducin, phosphodiesterase, arrestin, kinase, cGMP-gated channels, and Naþ/Kþ, Ca2þ exchangers.

Cone Postreceptoral Circuitry Consider first the retinal circuitry mediating cone function. Cone photoreceptor cells synapse with bipolar and horizontal cells in the outer plexiform layer of the retina

Anatomically Separate Rod and Cone Signaling Pathways

(see Figure 1). Cones release glutamate at a steady rate in darkness but this rate is slowed in a graded response that is correlated to the change in the outer-segment membrane potential after stimulation by light. The modulation of synaptic transmitter release drives signaling to secondorder bipolar cells. There are at least nine different types of cone bipolar cells in the mammalian retina that are distinguished on the basis of a number of features, including the number of

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Figure 1 Cone signaling pathway in the mammalian retina. Cone photoreceptors respond to light with a graded hperpolarization and synapse with bipolar and horizontal (not shown) cells in the outer plexiform layer (OPL) of the retina. The release of glutamate at the synaptic terminal is modulated by the change in the outer-segment membrane potential. Cone signals are delivered to ON and OFF bipolar cells (BC) which carry signals to ON and OFF ganglion cells (GC) in the inner plexiform layer (green and red channels, respectively), thereby maintaining segregated cone pathways through the retina. ON and OFF bipolar cells provide excitatory and inhibitory inputs to amacrine cells and ganglion cells and respond with a graded depolarization (sign-inverting) or hyperpolarizing (sign-conserving) response, respectively. Cones communicate laterally with other cones (and rods) via electrical couplings called gap junctions (GJ). Lateral communication is also afforded by horizontal cells in the OPL and by amacrine cells in the IPL. Abbreviations: OS – outer segment, ONL – outer nuclear layer, OPL – outer plexiform layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer, GJ – gap junction.

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cone photoreceptors contacting the bipolar cell, the spread of the dendritic terminals, and the stratification of their axon terminals in the inner plexiform layer. The precise function of each of these types of bipolar cells is not well understood but they are presumed to be tuned to process different aspects of the visual stimulus. Morphologically, bipolar cells can be subdivided into classes commonly referred to as diffuse and midget bipolar cells. Diffuse bipolar cells (of which there are at least six types) have broad dendritic spreads and make contact with 5–20 long (L-) and middle (M-) wavelength cones; the number of connections depends on where they are located in the retina. These cells are likely involved in tasks requiring broad spatial averaging and color processing but have poor spatial resolution. In contrast, midget bipolar cells have a narrow dendritic tree and can make contact with a single cone and with a single ganglion cell. This cone-mediated circuitry provides a mechanism for high spatial resolution of a scene and is thought to mediate an acuity channel. The midget bipolar cell also transmits color information by virtue of its one-on-one connections with L- and M-sensitive cones. In addition, bipolar cells that make specialized contacts with short (S-) wavelength sensitive cones have been described in the primate, rat, and mouse retina. Functionally, there are two broad classes of bipolar cells in the mammalian retina mediating the cone signaling pathway. They are commonly referred to as ON and OFF bipolar cells, because of their expression of either excitatory glutamate receptors (mGluRs) or inhibitory glutamate receptors (iGluRs). ON cone bipolar cells make contact with photoreceptors through invaginations in the cone pedicle and are flanked by a pair of horizontal cell dendrites, an arrangement commonly referred to as a triad. The triads are apposed to a presynaptic ribbon, which is the release site for neurotransmitter mediating signal transfer to second-order neurons. There are typically about 25 invaginations in a cone pedicle, but as many as 50 invaginations have been reported. A recent study has identified the protein pikachurin, a previously unknown dystroglycan-binding protein, as critical for the apposition of photoreceptor and bipolar cells dendrites at the ribbon synapse. OFF bipolar cells make contact directly at the cone pedicle base where up to 500 contacts are made with postsynaptic cells. ON bipolar cells that express mGluRs are depolarized (sign-inverting) by the light response in cone photoreceptor cells. Their dendritic processes terminate in sublamina B of the inner plexiform layer (see Figure 1). In contrast, OFF bipolar cells express iGluRs. In these cells glutamate expression is linked to Naþ influx. OFF cone bipolar cells are hyperpolarized (sign-conserving) by the light response in cones and have processes that end in sublamina A of the inner plexiform layer. The two types of bipolar cells provide excitatory and inhibitory inputs to downstream cells

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and tertiary neurons in response to light stimulation. In general, ON cone bipolar cells are excited by light that is brighter than its surround, whereas OFF cone bipolar cells are excited by light that is dimmer. This segregation of visual input is maintained in parallel signaling pathways to ON and OFF ganglion cells (see Figure 1).

Lateral Communication Networks Signal transfer through the retinal layers, particularly for the rod system, depends on lateral communication between retinal cells. There are several cell types and neural connections within retinal layers that mediate lateral transfer of signal. The major cell types include amacrine and horizontal cells but lateral transfer is also accomplished by a class of low-resistance electrical gap junctions. Gap Junctions Low-resistance electrical gap junctions are ubiquitous in the mammalian retina. They enable the intercellular, bidirectional transport of ions, metabolites, and secondorder messengers. These gap junctions are mediated by connexins of which several different types have been reported in the mammalian retina. The most abundant of these is neuronal Connexin36. Connexin36 is associated with processes in the outer and inner plexiform layers, consistent with expression in multiple cell types. In the outer plexiform layer, cone photoreceptors communicate laterally via Connexin36-mediated gap junctions between cone pedicles (see Figure 1). The gap junctions also permit signal transfer between cone pedicles and rod spherules. In the inner plexiform layer, the expression of Connexin36 co-localizes with the dendritic processes of AII-type amacrine cells. The latter gap junction mediates transfer of signal from AII amacrine cells to ON cone bipolar cells, a signaling pathway that is crucial for rod signals to infiltrate the cone postreceptoral signaling pathway. In addition, the gap junctions in the outer plexiform layer (between cone pedicles and rod spherules) allow rod signals to infiltrate the cone signaling pathway very early in visual processing. These pathways are described later in more detail. Horizontal Cells Horizontal cells are second-order, mainly inhibitory, neurons located in the outer plexiform layer. While these cells make synaptic connections with photoreceptors, they are also extensively coupled by either Connexin50 or Connexin57 gap junctions. Morphologically, three types of horizontal cells have been identified in the primate and human retina, referred to as HI, HII, and HIII. The HI-type horizontal cells have small dendritic fields (75–150 mm), but with long axons (300 mm) ending in a broad dendritic tree. HIII horizontal cells are similar to the HI horizontal cell

but have larger dendritic trees at all retinal locations. In addition, HIII cells contact many more cones than HI. HII horizontal cells are more spidery and intricate in dendritic field characteristics than either of the other types. The three types of horizontal cells in the human retina demonstrate evidence of color-specific coding. HI horizontal cells contact primarily with green- and red-sensitive cones, with a smaller number of contacts with blue-sensitive cones. HII horizontal cells contact blue-sensitive cones primarily, and HIII horizontal cells contact only greenand red-sensitive cones. Most, if not all, of the input to horizontal cells is derived from the response of cone photoreceptors to light and, depending on the type of horizontal cell, can make contact with many cones over broad retinal areas. In addition, because horizontal cells are extensively coupled via electrical gap junctions, their receptive fields can be much larger than their dendritic spreads. The connection to rod photoreceptors has traditionally been thought to occur at the axon terminal process, implying that signal transfer from cones to rods is unidirectional. However, rod inputs have been recorded in horizontal cell somata in the cat retina, presumed to be delivered via cone-rod gap junctions or direct dendritic connections. Like cone OFF bipolar cells, horizontal cells express iGluRs at their dendrites and are hyperpolarized in response to light. In the primate retina, the transmitter release from cones that drives horizontal cells and bipolar cells is also regulated by feedback from horizontal cells. This feedback loop provides a mechanism for the inhibition of signals from adjacent cone photoreceptors. The precise mechanism by which horizontal cells produce this lateral inhibition is not well understood but may occur as a result of the modulated release of the inhibitory transmitter g-aminobutyric acid (GABA) at the synaptic terminal of cones or via the modulation of the Ca2þ channels that regulate the release of glutamate. Regardless of the precise mechanism of lateral inhibition, the lateral interconnections provided by horizontal cells contribute to the formation of the antagonistic surrounds of bipolar cell receptive fields. The antagonistic center-surround interaction is thought to enhance the detection of edges but have also been implicated in the processing of color information where the center-surround configuration modulates antagonistic (or opponent) color information, and in illusory surface filling effects. In addition, the observation of rod and cone inputs at horizontal cell somata provides a mechanism for integrating light signals over broad retinal areas to ensure optimal retinal sensitivity over the entire intensity range. Amacrine Cells There are up to 30 types of amacrine cells located in the inner retina of the mammalian retina that have been


distinguished on the basis of morphological characteristics, physiological properties, and pharmacological criteria. While cells upstream from amacrine cells generate graded potentials in response to stimulation by light, the amacrine cell is the first site in the retina where action potentials are generated. Amacrine cells receive their input from bipolar cells, mediated by iGluRs at the synaptic terminals, and from ganglion cells and other types of amacrine cells. The main job of the amacrine cells is to provide a mechanism for transfer of signals from bipolar cells within and between sublamina of the inner plexiform layer, and with ganglion cells. However, amacrine cells, like horizontal cells, provide a mechanism for lateral signal communication between retinal cells, including providing a feedback loop to bipolar cells. They are assumed to play an important role in modulating activity in the antagonistic surrounds of ganglion cell receptive fields that shape higher visual functions, such as object segregation and spatio-temporal adaptation. The feedback from amacrine cells has also been implicated in switching the site of light adaptation between receptor and postreceptoral sites. The extent of lateral transfer depends on the morphology of the amacrine cell. Wide-field amacrine cells transmit lateral information across a broad expanse of the inner plexiform layer and are present in many species, including the mouse, rat, cat, rabbit, salamander, and monkey. Small-field amacrine cells mediate local interactions between different sublaminae of the IPL. The best characterized of these is the AII amacrine cell which plays an important role in mediating signal transfer through the rod-mediated neural circuitry. Unlike cone bipolar cells, rod ON polar cells do not make direct contact with ON ganglion cells. Rather rod signals are transmitted to ON ganglion cells by AII electrical coupling (gap junctions) with ON cone bipolar cells and by synaptic connections with OFF-cone bipolar cells (see below). Another amacrine cell type, the A17 amacrine cell, has also been implicated in the rod-signaling pathway of the mammalian retina. Up to 11 other small-field amacrine cells have been identified in the cat, rabbit, and mouse, but their precise function is not well understood. Ganglion Cells Signals carried through the retinal layers converge on ganglion cells, the latter responsible for carrying information to higher-order visual centers. Up to 25 different types of ganglion cells have been identified in the mammalian retina, dependent on species. These retinal ganglion cells are broadly grouped into classes based on morphological characteristics and physiological properties. In the primate retina, for example, there are at least 18 different types of retinal ganglion cells that are classified morphologically into Pa (parasol), Pb (midget), and Pg types, and physiologically into two major types:

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parasol or magnocellular (M), and midget or parvocellular (P) cells. The parvocellular cells project exclusively to the parvocellular layers of the lateral geniculate nucleus (LGN) and play a key role in central acuity. Parasol ganglion cells are motion-sensitive cells and primarily project to the magnocellular layers of the LGN. Intrinsically photosensitive retinal ganglion cells (ipRGCs) containing melanopsin as the photosenstitive pigment have also been described recently. These neurons, which receive input from both rod and cone photoreceptors, have been implicated in nonimage-forming responses to environmental light such as the pupillary light reflex and circadian entrainment. Multiple Rod Signaling Pathways Cone photoreceptors synapse directly with both ON and OFF bipolar cells, which then transmit signals in parallel pathways to ON and OFF ganglion cells, respectively (Figure 1). In contrast, rod photoreceptors, which synapse with rod ON bipolar cells, do not make direct contact with ganglion cells, but rather transmit their signals to these cells through several alternate pathways. Consider first the evidence for multiple rod pathways. The alternative signaling pathways are described later and shown in Figure 2. It is generally accepted that rod-mediated vision, like cone vision, involves multiple signaling pathways. The evidence in support of this hypothesis derives from early psychophysical experiments in humans in which critical fusion frequency (CFF; the intensity at which flicker can just be detected) was measured under conditions mediated by rods. These experiments revealed two distinct branches in the function that relates CFF and stimulus intensity. In the lower branch, over dim flash intensities, the CFF was no better than 15 Hz, and remained at this level over a broad range of stimulus intensities. However, at higher intensities, covering mesopic light levels, CFFs increased rapidly and could be as high as 28 Hz. The doublebranched CFF versus intensity response function implied the existence of at least two signaling pathways mediating rod function in the mammalian retina. This hypothesis was supported by the observation that patients with achromatopsia, a retinal abnormality in which cone function is absent, also display the same response properties. Additional support for at least dual rod signaling pathways comes from psychophysical measurements of rod flicker perception in humans. These experiments demonstrated that for 15 Hz flickering stimuli (the optimal stimulus presentation frequency for demonstrating the interaction), there is an intensity region, well above flicker detection threshold, where the perception of flicker is minimized or nulled. The perceptual nulling of flicker has been assumed to result from the mutual cancellation of signals originating from at least two signaling pathways


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Figure 2 Rod signaling pathway in the mammalian retina. In the primary rod pathway (left panel), rod photoreceptors synapse with rod ON bipolar cells, which in turn make connections with amacrine AII cells in the inner plexiform layer (yellow pathway). Signals from the AII amacrine cells infiltrate the cone pathway by exciting ON cone bipolar cells via electrical gap junctions (green pathway) and via glycinergic (sign-inverting) synapses with OFF cone bipolar cells (red pathway). Rod signaling through the secondary rod pathway (middle panel) is mediated through rod-cone gap junctions between rod spherules and cone pedicles located in the outer plexiform layer. Through the rod-cone gap junction, rod signals have access to both ON and OFF cone bipolar cells and to ON and OFF ganglion cells (green and red pathways, respectively). A third pathway for rod signal transmission (right panel), in which rod photoreceptors bypass the rod ON bipolar cell and directly excite cone OFF bipolar cells, has also been hypothesized (red pathway). Abbreviations: OS – outer segment, ONL – outer nuclear layer, OPL – outer plexiform layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer, GJ – gap junction.

having different speeds of signal transmission. The hypothesis argues that when signals are in phase, despite different speeds of transmission, they are mutually additive but, when out of phase, produce destructive interference, which contributes to the inhibition of signal strength and the flicker nulling perception. This mutual cancellation has also been demonstrated with the electroretinogram (ERG), which is a noninvasive measure of the massed response of the retina to light. It is assumed only to reflect activity of outer and middle retinal cells. As in the perceptual experiments described above, supportive evidence derives from a unique feature of the function that relates ERG signal amplitude and stimulus. A local response minimum is observed at an intensity that is well above ERG flicker detection threshold but still within the scotopic range of intensities and occurs at the same intensity where the perception of flicker in humans is also minimized. Much of the electrophysiological evidence in support of the existence of multiple signaling pathways comes from experiments in which single unit extracellular recordings are made from ganglion cells in the mouse and rabbit retina. Using pharmacological agents to disrupt different cellular connections in the retinal circuitry, combined with animal models with known genetic defects affecting these connections, electrophysiological support for multiple rod

pathways has come from what signals remain detectable at the ganglion cell level. On the basis of these types of experiments, rod photoreceptor signals are presumed to be transmitted to ganglion cells via three alternate pathways. The anatomical substrates mediating rod postreceptoral signaling in the retina seem to be well established and are assumed to be conserved across mammalian species. They are illustrated in Figure 2. In the primary rod pathway (left panel), rod photoreceptors synapse with rod ON bipolar cells, via sign-inverting glutamatergic synapses. The output from the rod ON bipolar cell is then transmitted to AII amacrine cells in the inner plexiform layer via sign-conserving glutamatergic synapses. Signals from the AII amacrine cells then converge onto the cone pathway by exciting ON cone bipolar cells via electrical gap junctions and inhibit cone OFF bipolar cells via sign-inverting glycinergic synapses (see Figure 2 for color coding). Rod signaling through the secondary rod pathway (middle panel) converges onto the cone circuitry at an even earlier stage. The secondary rod pathway is mediated through rod-cone gap junctions that exist between rod spherules and cone pedicles located in the outer plexiform layer. Through the rod-cone gap junction, rod


photoreceptors can transmit signals directly to both ON and OFF cone bipolar cells and to ON and OFF ganglion cells. The circuitry for the primary and secondary rod pathways has been shown to exist in the cat, rabbit, primate, and more recently in the mouse. A third pathway for rod signal transmission (right panel), in which rod photoreceptors bypass the rod ON bipolar cell and directly excite cone OFF bipolar cells, has also been hypothesized and supported by anatomical and physiological data. This alternative pathway has been demonstrated to exist using electrophysiological methods. In these experiments, a ganglion cell signal continues to be observed in animals without cones (thereby eliminating the rod–cone gap junction of the secondary pathway) and in which all signal transmission through the primary rod pathway is blocked with pharmacological agents. Thus, the retinal circuitry comprising the rod system offers multiple signaling routes for carrying information from rod photoreceptors to inner retinal ganglion cells. While these signaling pathways provide the rod system with multiple opportunities for system redundancy, they also subserve specialized functions related to scotopic vision. It has been suggested that signal transfer from the primary to secondary rod signaling pathway affords the rod system the capability of enhanced temporal resolution at the expense of light sensitivity. However, further work is needed to better understand the precise role of each of the rod signaling pathways in the processing of visual information.

Concluding Statements A major step in forming our perceptions of the visual world is accomplished in the retina, where information from rod and cone photoreceptors is filtered, processed, and channeled through multiple parallel signaling pathways. In addition to the different spectral sensitivities of rod and cone photoreceptors and the intensity range over which they operate, the different types of bipolar cells, amacrine cells, and horizontal cells are presumed to be tuned to capture or enhance specific attributes of a visual scene – color processing, brightness contrast, temporal processing, signal enhancement and integration, and adaptation mechanisms. Ultimately, these processed and filtered signals from the retina are transmitted to higher-order visual centers, such as the lateral geniculate nucleus and the primary visual cortex of the brain, where the information is optimized to form our perceptions of the visual environment.

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Acknowledgments The authors are grateful to Dr. William H. Ridder for reading the text and providing helpful comments and Bryan Chen for assistance with drawings. See also: The Circadian Clock in the Retina Regulates Rod and Cone Pathways; Information Processing: Amacrine Cells; Information Processing: Bipolar Cells; Information Processing: Ganglion Cells; Information Processing in the Retina; Morphology of Interneurons: Amacrine Cells; Morphology of Interneurons: Bipolar Cells; Morphology of Interneurons: Horizontal Cells; Morphology of Interneurons: Interplexiform Cells; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods.

Further Reading Bloomfield, S. A. and Dacheux, R. F. (2001). Rod vision: Pathways and processing in the mammalian retina. Progress in Retinal and Eye Research 20: 351–384. Dowling, J. E. (1999). Retinal processing of visual information. Brain Research Bulletin 50: 317. Falk, G. and Shiells, G. (2006). Synaptic transmission: Sensitivity control mechanisms. In: Heckenlively, J. H., Arden, G. B., Nusinowitz, S., Holder, G., and Bach, M. (eds.) Principles and Practice of Clinical Electrophysiology of Vision, pp. 79–91. Cambridge, MA: MIT Press. Fu, Y. and Yau, K. W. (2007). Phototransduction in mouse rods and cones. Pflugers Archiv. European Journal of Physiology 454: 805–819. Kolb, H. (2006). Functional organization of the retina. In: Heckenlively, J. H., Arden, G. B., Nusinowitz, S., Holder, G., and Bach, M. (eds.) Principles and Practice of Clinical Electrophysiology of Vision, pp. 47–64. Cambridge, MA: MIT Press. Kolb, H. and Famiglietti, E. V. (1974). Rod and cone pathways in the inner plexiform layer of cat retina. Science 186: 47–49. Masland, R. H. (2001). The fundamental plan of the retina. Nature Neuroscience 4: 877–886. Nickle, B. and Robinson, P. R. (2007). The opsins of the vertebrate retina: Insights from structural, biochemical, and evolutionary studies. Cellular and Molecular Life Sciences 64: 2917–2932. Pugh, E. N., Jr. and Lamb, T. D. (1993). Amplification and kinetics of the activation steps in phototransduction. Biochimica et Biophysica Acta 1141: 111–149. Schmidt, T. M., Taniguchi, K., and Kofuji, P. (2008). Intrinsic and extrinsic light responses in melanopsin-expressing ganglion cells during mouse development. Journal of Neurophysiology 100: 371–384. Volgyi, B., Deans, M. R., Paul, D. L., and Bloomfield, S. A. (2004). Convergence and segregation of the multiple rod pathways in mammalian retina. Journal of Neuroscience 24: 11182–11192. Wassle, H. (2004). Parallel processing in the mammalian retina. Nature Reviews Neuroscience 5: 747–757.

Anatomy and Regulation of the Optic Nerve Blood Flow R Ehrlich, A Harris, and A M Moss, Indiana University, Indianapolis, IN, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Anastamosis – A network of streams that both branch out and reconnect forming a communication between two blood vessels or other tubular structures. Autoregulation – The intrinsic ability of a system to maintain constant blood flow despite changes in perfusion pressure and local vascular parameters to maintain homeostasis. Choriocapillaris – A layer of capillaries in the choroid immediately adjacent to Bruch’s membrane. Central retinal artery – A branch of the ophthalmic artery which pierces the optic nerve close to the globe, sending branches to the internal surface of the retina. Extraocular muscles – A group of six muscles that control the movements of the eye, including the superior, inferior, lateral, and medial recti, and the superior and inferior obliques. Fenestration – From the Latin word for window (fenestra), a fenestration is an opening in a wall or membrane. Hemodynamics – The study of the forces generated by the heart and the flow of blood through the cardiovascular system. Ophthalmic artery – A branch of the internal carotid artery which enters the orbit through the optic canal, along with the optic nerve, to supply structures in the orbit. Poiseuille’s law – A physical law that describes slow, viscous, incompressible flow through a circular cross section. It states that for a laminar, nonpulsatile fluid flow through a uniform straight tube, the vascular resistance is inversely proportional to the fourth power of the radius of a vessel, and is directly proportional to the blood viscosity and length of the vessel. Retrobulbar vessels – Blood vessels behind the eye.

Introduction A thorough understanding of vascular anatomy is critical to appreciate the physiology of the optic nerve head (ONH). The study of blood flow and the metabolism of

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the eye are also important in understanding the role of the circulatory system in various eye diseases. The arterial supply to the optic nerve has been widely investigated; however, the precise anatomy of the anterior optic nerve microvasculature remains difficult to ascertain. Detailed assessment of this anatomy is limited by its small vessel caliber, its complex three-dimensional structure, and the relative inaccessibility of the microvascular bed. Although the evaluation of ocular hemodynamics continues to improve with the development of new imaging technologies, current techniques for measuring optic nerve blood flow do not directly evaluate the optic nerve. We summarize the vascular anatomy of the ONH, retina, and choroid, the regulation of blood flow in the ONH, and several imaging techniques used to measure blood flow in the eye.

Anatomy of the Vascular Supply The ophthalmic artery (OA), which is the first branch of the internal carotid artery, provides the vast majority of the ocular blood supply (Figure 1). The OA enters the orbit through the optic canal and, in most individuals, runs inferolaterally to the optic nerve. After coursing nasally and anteriorly, the OA runs superior to the optic nerve, where it gives off most of its major branches. These branches include vessels to each of the extraocular muscles, the central retinal artery (CRA), and the posterior ciliary arteries (PCAs) (Figure 2). There are usually two to three PCA trunks, each dividing into approximately 10–20 short PCAs before, or, occasionally after, penetrating the sclera. The short PCAs supply the posterior choriocapillaris, peripapillary choroid, and the majority of the anterior optic nerve. The medial and temporal long PCAs pierce the sclera about 3–4 mm nasally and temporally from the optic nerve (Figure 2). They then travel anteriorly within the suprachoroidal space, along the horizontal meridians of the globe. Typically, the long PCAs divide in the vicinity of the ora serrata to supply the iris, ciliary body, and the anterior region of the choroid. The CRA branches directly from the OA to pierce the medial aspect of the optic nerve sheath approximately 10–15 mm behind the globe. The CRA courses adjacent to the central retinal vein (CRV) through the center of the optic nerve. It emerges from the optic nerve within the globe, where it branches into four major vessels: the arteriola nasalis retinae superior, arteriola nasalis retinae inferior, arteriola temporalis retinae superior, and arteriola temporalis retinae inferior.

Anatomy and Regulation of the Optic Nerve Blood Flow

Arteria ciliaris anterior

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Arteria supratrochlearis

Arteria dorsalis nasi

Arteria lacrimalis

Arteria ethmoidalis anterior Arteria ciliaris posterior longa Arteria ethmoidalis posterior Arteria ciliaris posterior brevis Arteria centralis retinae Arteria ophthalmica

Arteria carolis interna

Figure 1 Schematic depicting the general vascular organization and the arterial feeds of the major and microciliary processes. The anterior and posterior arterioles that branch off the major arterial circle of the iris (MAC) supply the capillaries of the major and minor processes, respectively. Several smaller branches off the anterior arteriole feed into the marginal capillaries of the major process. Branches of the posterior arteriole feed the internal capillaries (ICs) of the major process. Blood supply to the capillaries of the minor processes is derived from more than one posterior arteriole. Anastomoses (green arrowheads) occur between the lateral branches, some marginal or central capillaries of the major processes and the basally located capillaries that extend posteriorly (white star). From Morrison, J. C. and van Buskirk, E. M. (1984). American Journal of Ophthalmology 97: 372–383 in Figure 11.25f in Bron, A. J., Tripathi, R. C., and Tripathi, B. J. (1997). The choroid and uveal vessels. In: Wolff’s Anatomy of the Eye and Orbit, 8th edn., ch. 11. London: Chapman and Hall Medical.

The vasculature of the eye can be divided into two distinct systems: the retinal system and the uveal system. The retinal system provides blood flow to the inner twothirds of the retina. The choroid and ciliary body are nourished by the uveal system. The retinal pigment epithelial layer, which is located between the retina and choroid, actively exchanges nutrients and metabolic waste products between the retina and the choroid. Thus, the outer layers of the retina receive their blood flow via the uveal system. Retina The retina receives its arterial blood supply from two distinct sources. The CRA provides blood flow to the inner two-thirds of the retina. The CRA branches on the surface of the optic disk, typically producing four main trunks which lie within the nerve fiber layer. Each trunk supplies its respective quadrant of the retina. The outer

one-third of the retina, including the photoreceptors and bipolar cells, receives nourishment from the underlying choroid, specifically the choriocapillaris. Nutrients are actively transported between the choroid and retina via the retinal pigment epithelium. In approximately 30% of the people, a cilioretinal artery is present. Typically a branch of a ciliary artery, this vessel supplies a variably sized region of the retina temporal to the optic nerve. When present, the cilioretinal artery is an end artery, and therefore its territory receives no additional blood supply from any other vessels. Retinal capillaries run parallel to the retinal nerve fiber layer, eventually coalescing into retinal veins, which empty into the CRV. The CRV exits the eye through the optic nerve, running parallel to the CRA. Once in the optic nerve, the CRV receives additional intraneural tributaries, and eventually empties into the superior ophthalmic vein. Although the CRV is normally the only outflow channel

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Arteria and vena centralis retinae

Long posterior ciliary artery (arteria ciliaris posterior longa)

Short posterior ciliary artery (arteria ciliares posteriores breves)

Posterior ciliary artery (arteria ciliaris posterior)

Figure 2 Drawing depicting the vascular territories of the ciliary processes. The first terroritory (outlined in the green box) includes the anterior arterioles, the lateral branches, and that feed the lateral branches, and the branches that drain into the basally located venules (white star). The second territory (indicated by the green arrows), includes the marginal capillaries and the capillary network (shown as short connections) that connect to these and the internal capillaries (IC) of the major process. The third territory (outlined in the purple box) includes the capillaries that branch off the posterior arterioles, and the vasculature of the minor processes. According to some authors the vessels in the posterior third of the major processes also fall within the third vascular territory. From Morrison, J. C. and van Buskirk, E. M. (1986). Transactions of Ophthalmology Society 105: 13 in Figure 10.28b in Bron, A. J., Tripathi, R. C., and Tripathi, B. J. (1997). The posterior chamber and ciliary body. In: Wolff’s Anatomy of the Eye and Orbit, 8th edn., ch. 10. London: Chapman and Hall Medical.

for retinal circulation, potential anastamoses exist between the retinal and choroidal circulation. These alternate pathways are significant in the case of a CRV occlusion.

Choroid The choroid supplies the outer retina with nutrients and maintains the temperature and volume of the eye. The choroidal circulation, which accounts for 85% of the total blood flow in the eye, is a high-flow system with relatively low oxygen content. The choroidal circulation is controlled mainly by sympathetic innervation and is considered not to be autoregulated. This lack of autoregulation makes the choroid more dependent on the ocular perfusion pressure. The short PCAs supply the posterior choroid and the peripapillary region, while the anterior parts of the choroid are supplied by the long PCAs and the anterior ciliary artery. The anterior ciliary artery is a branch from the OA which accompanies the rectus muscle anteriorly to supply the iris and the anterior choriocapillaries. The outer choroid, known as Haller’s layer, is composed of large caliber, nonfenestrated, vessels. The inner choroid is referred to as Satler’s layer, and is composed of significantly smaller vessels. The choriocapillaries of the innermost choroid are composed of richly anastomotic, fenestrated capillaries. The capillaries of the choriocapillaries are

separate and distinct from the capillary bed of the anterior optic nerve. Venous drainage from the choriocapillaries is primarily through the four vortex veins. Minor drainage also occurs through the ciliary body and the anterior ciliary vein. Venous anastomoses are frequent in the choroid. The vortex veins drain into the inferior and superior ophthalmic veins, which then exit the orbit through the superior and inferior orbital fissures, respectively. Optic Nerve The anterior optic nerve is divided into four anatomical regions: the superficial nerve fiber layer, the prelaminar layer, the laminar region, and the postlaminar region (Figure 3). The arterial supply to the ONH is derived from branches of the OA. The short PCAs penetrate the perineural sclera at the posterior aspect of the globe to supply the peripapillary choroid and anterior ONH. The circle of Zinn-Haller is a noncontinuous arterial circle surrounding the ONH within the perineural sclera. Formed by a network of small branches of the short PCAs, the circle of Zinn-Haller provides multiple perforating branches to various regions of the anterior optic nerve, peripapillary choroid, and pial arterial system. The capillaries of the anterior ONH are nonfenestrated, contain tight junctions, and form a rich anastomotic plexus. Some investigators surmise that the division of the short PCAs into branches


Superficial nerve fiber layer region

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Retina

Prelaminar region

Choroid

Lamina cribrosa region

Retrolaminar region

Posterior ciliary artery Central retinal artery and vein Figure 3 Left: anatomical regions of the optic nerve. Right: blood vessels of the anatomical regions of the optic nerve.

that supply the choroid and those supplying the ONH form the watershed zone near the ONH. The superficial nerve fiber layer, which is continuous with the nerve fiber layer of the retina, receives its blood supply from recurrent arterioles arising from branches of the retinal arteries (Figure 4). These vessels, referred to as epipapillary vessels, originate in the peripapillary nerve fiber layer and run toward the center of the ONH. The temporal nerve fiber layer may receive additional arterial contribution from the cilioretinal artery when present. Immediately posterior to the nerve fiber layer is the prelaminar region, which lies adjacent to the peripapillary choroid. In this region, ganglionic axons are grouped into bundles, surrounded by glial tissue septa, as they prepare for passage posteriorly through the lamina cribrosa. The prelaminar region is supplied primarily by branches of the short PCAs and, when present, by branches of the circle of Zinn-Haller (Figure 5). The amount of choroidal contribution may be difficult to determine, as there are branches from both the circle of Zinn-Haller and the short PCAs which course through the choroid and ultimately supply the optic nerve in this region. These vessels do not originate in the choroid, but merely pass through it. The choroid contributes little, if any, blood supply to this area of the ONH.

The laminar region is continuous with the sclera and is composed of fenestrated connective tissue lamellae which allow the passage of neural fibers through the sclera. This region, called the lamina cribrosa, receives its blood supply either from centripetal branches of the short PCAs or from branches of the circle of Zinn-Haller (Figure 6). These branches pierce the outer aspect of the lamina cribrosa before branching centrally to form an intraseptal capillary network throughout connective tissue. The larger peripapillary choroidal vessels occasionally contribute small arterioles to the lamina cribrosa region. The retrolaminar region lies posterior to the lamina cribrosa, and is discernible by the beginning of the axonal myelination. Surrounded by the meninges of the central nervous system (CNS), the retrolaminar region is supplied primarily by branches of the pial arteries and the short PCAs (Figure 7). The pial system is an abundant anastomotic network fed by the OA, the circle of Zinn-Haller, and recurrent branches of the short PCAs. The pial branches are located within the pia matter and extend centripetally to perfuse the axons of the optic nerve. In addition, the CRA occasionally contributes small branches within the retrolaminar optic nerve. Like that of the retina, the venous drainage from the ONH is through the CRV. In the superficial nerve fiber

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Superficial nerve fiber layer region

Epipapillary vessels

Central retinal artery and vein

Figure 4 Schematic of the blood supply to the superficial nerve fiber layers.

Prelamina region

Retina

Choroid

Central retinal artery and vein Figure 5 Schematic of the blood supply to the prelaminar region of the optic nerve.

layer, blood is drained by small, converging veins that empty into the CRV. In the other layers of the ONH, centripetal veins serve as tributaries, eventually emptying into the CRV. In the prelaminar region, there is also a noteworthy contribution from the peripapillary choroidal veins. Small portions of the peripheral region of the ONH may partially drain into the pial venous network, which ultimately joins together with the CRV as well.

Histology of Blood Vessels in the Optic Nerve The anterior optic nerve is composed of nerve axons, neuroglia, blood vessels, and connective tissue. Largecaliber arteries of the optic nerve contain a muscularis layer, composed of multiple layers of smooth muscle, surrounded by the adventitia. The latter consists of


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Lamina cribrosa region

Circle of Zinn-Haller

Lamina cribrosa

Sclera

Short posterior ciliary arteries

Central retinal artery and vein

Figure 6 Schematic of the blood supply to the lamina cribrosa region.

Retrolaminar region

Pial system Sclera

Posterior ciliary artery Central retinal artery and vein Figure 7 Schematic of the blood supply to the retrolaminar region of the optic nerve.

circumferential collagen fibers which blend with fibers from the perivascular space. On its luminal surface, the muscularis layer is separated from the endothelium by an inner elastic layer. The basement membrane lies

between the endothelial cells and blends with the internal elastic lamina. Arterioles of the optic nerve are much smaller in diameter, and have a single layer of smooth muscle.

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They also possess minimal to no elastic lamina, but have a relatively dense reticular lining. The adventitia is continuous with collagen fibers of the intravascular space, and the endothelium is covered by a basement membrane. Precapillaries and capillaries have a very thin wall and their basement membranes stain positively with the periodic acid-Schiff reaction. The endothelium, basement membrane, and mural cells of the venous structures are similar to that of the capillaries. The veins of the optic nerve are composed of an inner lining of endothelial cells, elastic fibers, thin adventitia, and intermittently, irregularly spaced, smooth muscle cells. When the venules enlarge, the basement membrane thickens accordingly. Within the anterior ONH, the histology of the vasculature resembles that of the CNS, as the vessels contain a nonfenestrated endothelium with tight junctions. Capillaries predominate within the anterior ONH, and larger vessels are seldom visualized. Posteriorly, larger arterioles may be seen entering the lamina cribrosa. These capillaries and arterioles lack the internal elastic lamina and elastic tissue in the media that is characteristic of the larger vessels of the laminar and retrolaminar regions.

Regulation of Ocular Blood Flow Autoregulation is the intrinsic ability of a system to maintain constant blood flow despite changes in perfusion pressure and local vascular parameters. The intrinsic control of blood flow involves chemical secretion by the cells in the immediate vicinity of the blood vessels. Within the eye, autoregulation is defined as local vascular constriction or dilation to alter vascular resistance, thereby maintaining a constant nutrient supply in response to perfusion pressure changes. Perfusion pressure is equal to the difference between the mean arterial pressure (MAP) and the venous pressure. The MAP is defined as the diastolic blood pressure plus one-third of the difference between systolic and diastolic blood pressure. Since the pressure in the central vein is normally slightly higher than the intraocular pressure (IOP), the IOP is used as an estimate of ocular venous pressure: 2 Mean ocular perfusion pressure ¼ MAP IOP 3 1 MAP ¼ Diastolic BP þ ðSystolic BP Diastolic BPÞ 3

The maintenance of appropriate ocular blood flow is challenged by physiological changes in IOP, blood pressure, ocular perfusion pressure, and local tissue metabolic demands. As per Poiseuille’s law, vascular resistance is inversely proportional to the fourth power of the radius of a vessel, and is directly proportional to the blood viscosity

and length of the vessel. In the eye, vascular resistance is therefore dependent on the regulation of vessel diameter. In normal subjects, autoregulation is usually maintained until the IOP reaches approximately 40–45 mmHg. Failure of stable blood flow regulation may lead to ischemic damage of the optic nerve or retinal ganglion cells, which likely contributes to further impairment in vascular regulation. Several mechanisms, including neurogenic-, metabolic-, myogenic-, humoral-, and endothelial-mediated factors, have been demonstrated to play a role in the vascular regulation of ocular blood flow. Fed primarily by the CRA, the retinal system is generally a low-flow, constant rate system that supplies a highly metabolically active tissue. Although it may only account for as little as 15% of the total ocular circulation, the retinal circulation is capable of providing relatively constant blood flow over a substantial range of IOPs. The retinal and anterior optic nerve head do not possess direct autonomic innervations. Although the retinal and optic nerve head have adrenergic and cholinergic receptors their role remains unclear. Consequently, retinal blood flow is locally autoregulated. Several vasoactive molecules mediate retinal vascular autoregulation. Endothelial tone is determined by the balance between the vasoconstricting and vasodilating effects of secreted factors. Nitric oxide (NO) is produced by the oxidation of L-arginine by endothelial-derived nitric oxide synthase, which is present in both a constitutively active, membrane-bound form, and an inducible, cytosolic form. NO diffuses to nearby pericytes and smooth muscle, where it activates guanylyl cyclase, leading to the increase of cGMP and subsequent vasodilation. There are numerous stimuli for the production of NO, including increased shear force, bradykinins, insulin-like growth factor 1, acetycholine, and thrombin. Additionally, NO also inhibits platelet aggregation, platelet granule secretion, and leukocyte adhesion. Vasoconstriction of the retinal microvasculature is stimulated by several vasoactive molecules. Endothelins, the most potent vasoconstricting agents known, are molecules that bind to receptors on pericytes and smooth muscle cells. A second vasoconstrictive substance is angiotensin II. Angiotensinogen is an inactive molecule that is constitutively produced by the liver. In response to physiologic stimuli, the kidneys release renin, which converts angiotensinogen into angiotensin I. Angiotensin converting enzyme (ACE), which is present on the surface of luminal endothelial cells, converts angiotensin I to angiotensin II, and also inactivates bradykinin. Once in its active form, angiotensin II moderates retinal vasoconstriction through the activation of smooth muscle cells and pericytes. The choroidal vasculature is controlled extrinsically through hormonal influence and stimulation from the autonomic nervous system. It is characterized as a highflow, variable-rate system which is tightly regulated by


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the autonomic nervous system. The outer, larger vessels are composed of nonfenestrated endothelium, while the inner, smaller vessels form a richly anastomotic network of fenestrated capillaries. The vascular tone of the choroid is dominated by the sympathetic nervous system. Neurons course from the cranial cervical ganglion to the vascular bed, where vasoconstriction is mediated by the release of neuropeptide Y. The parasympathetic nervous system plays only a moderate and poorly defined role in the regulation of vascular tone. The presence of choroidal autoregulation is controversial and classically considered absent, though some autoregulatory response was reported during perfusion pressure changes.

calculate the mean flow velocity (MFV). An index of resistance (RI) can be calculated as RI = (PSV – EDV)/ PSV (Figure 8). Further research is necessary to determine the usefulness and application of the RI as it applies to the retrobulbar vasculature. Until recently, one critical limitation of this imaging technique had been that no quantitative information on vessel diameter was obtained, and therefore the calculation of total blood flow or flux was not possible. A recently developed analysis technique has made it possible to determine the diameter of the OA, allowing volumetric blood flow to be assessed. Further research is necessary to apply this technique to the assessment of blood flow through the other major vessels.

Technology for Measuring Ocular Blood Flow

Angiography

Color Doppler Imaging Although originally developed for monitoring blood flow in the heart, carotid arteries, and peripheral vasculature, color doppler imaging (CDI) has proven to be useful in the study of retrobulbar vessels as well. By combining B-scan ultrasound images with velocity of measurements calculated from the Doppler shift of moving erythrocytes, CDI can be used to assess the velocity of blood flow through the retrobulbar vessels. The peak systolic velocity (PSV) and end diastolic velocity (EDV) can be measured and used to

Using sodium fluoroscein, angiography allows direct visualization of retinal blood flow. Most techniques measure the amount of time that it takes for the fluoroscein to pass through the retinal circulation. Using this technique, data can then be used to assess blood velocity through the retinal and optic disc circulation. Choroidal vessels are visualized in a similar fashion using indocyanine green, which is selected due to its increased binding to plasma proteins, preventing leakage from vessels to the surrounding tissue. Using videoangiography and scanning laser ophthalmoscopy, the arterio-venous passage time and retinal circulation time can be determined. This technology

Figure 8 Color Doppler image of the central retinal artery and vein taken with a 7.5-MHz linear probe. The patient is placed comfortably in a half-supine position. An ultrasound probe is placed on closed eyelid and the optic nerve shadow of the optic nerve is identified. The vessels sampled include the ophthalmic artery, central retinal artery, and the nasal and temporal short posterior ciliary arteries. The Doppler-shifted spectrum (time–velocity curve) is displayed at the bottom of the image. Red and blue pixels represent blood movement toward and away from the transducer, respectively. The peak of the wave represents the peak systolic velocity (PSV) and the lower part of the wave the end diastolic velocity (EDV). The resistive index is calculated (PSV – EDV/PSV).

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also has limitations, as it is based on the assumption that all of the blood of an area supplied by a specific artery is drained by a single corresponding vein.

diameter and therefore cannot be used to measure volumetric flow. Laser Doppler Flowmetry

Blue Field Entotpic Technique The blue field entoptic phenomenon is produced by the different absorption of red and white blood cells when the retina is illuminated with blue light. Red blood cells absorb the short wavelength light, while passing white blood cells do not, thereby allowing the flux of the perimacular white blood cells to be estimated. This technique is limited by the assumption that leukocyte flux is proportional to retinal blood flow. Laser Doppler Velocimetry Laser Doppler velocimetry (LDV) is a technique that uses the optical Doppler shift of light to measure the blood flow velocities in retinal arterioles and venules. The Doppler shift of light is directly proportional to the blood velocity when the vessel is illuminated with a laser beam. The flow velocity in the vessel can be extrapolated from the range of frequency shifts of the power spectrum of the reflected laser light. The maximum frequency shift corresponds with the maximum velocity in the center of the vessel, assuming laminar flow.

The scattering theory for light in tissue, formulated by Bonner and Nossal, assumes a randomization of light directions impinging on the erythrocytes. By directing a laser light on vascularized tissue that contains no large vessels, relative mean velocity of erythrocytes and blood volume can be calculated. Through two-dimensional mapping of the optical Doppler shift, blood flow to the juxtapapillary retina and ONH can be accurately evaluated. There is, however, significant variation in scattering of light between test subjects, likely a result of varying vascular densities and orientations. Thus, this technique can be used to compare changes in a given subject, but has less use in comparison of values between subjects. Laser Doppler flowmetry can be combined with scanning laser tomography to provide a two-dimensional map of blood flow to the optic nerve and surrounding retina. The Heidelberg retina flowmeter (HRF) is one such commercially available system (Figure 9). This technique, however, is most sensitive to blood flow changes in the superficial layers of the ONH, and therefore provides only limited information about the deeper regions. This limits the ability to account for the retinal blood flow that is supplied by the choriocapillaris from the uveal system.

Retinal Vessel Diameters The aforementioned techniques can be utilized to provide information about ocular blood velocity, but lack the ability to calculate flux or flow rate. To determine blood flow, it is necessary to accurately measure the diameter of the vessel through which the blood is flowing. There are now commercially available systems that permit real-time assessment of retinal vessel diameter. The retinal vessel analyzer (RVA) is composed of a fundus camera and a sophisticated computer system which record vessel size in real time. One such system is the Canon Laser Doppler blood flowmeter, which combines the techniques of LDV and RVA. This approach is still limited to the study of larger vessels, and can only be performed on patients with clear ocular media.

Pulsatile Ocular Blood Flow Based on the changes in ocular volume and pressure during the cardiac cycle, it is possible to estimate pulsatile ocular blood flow. The pulse amplitude, which is the maximum IOP change during a cardiac cycle, is measured using a modified pneumotonometer. Alternatively, the

Laser Speckle Technique When the rough surface of the fundus is illuminated by coherent light, the backscatter of light produces a rapidly varying pattern. The rate of variation of this pattern produced by this laser speckle phenomenon can be measured to compute an estimate of the velocity of blood flowing through the retinal vessels. This laser speckle technique is limited by the fact that it provides only velocity information, as it cannot determine vessel

Figure 9 Confocal scanning laser Doppler flowmetry (Heidelberg retinal flowmeter) of optic nerve head and peripapillary retina. The patient is seated with the chin and forehead against the bar. The picture acquisition is performed without the need to dilate the patient’s eye. The conventional 40 40 pixel measurement window collects flow values in arbitrary units from the entire retina except for large vessels.


ocular fundus pulsation amplitude can be determined by calculating the maximum change in distance between the retina and the cornea during a cardiac cycle. These two values have been shown to be useful in the calculation of pulsatile ocular blood flow, but lack information of the nonpulsatile component of ocular blood flow.

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pathophysiology, and will therefore lead to advancements in both diagnosis and treatment. See also: Optic Nerve: Inherited Optic Neuropathies; IOP and Damage of ON Axons; Ischemic Optic Neuropathy; Optic Nerve: Optic Neuritis; Retinal Ganglion Cell Apoptosis and Neuroprotection.

Optical Doppler Tomography This technique combines the high-resolution cross-sectional imaging of optical coherence tomography with laser Doppler to measure velocity of blood flow in retinal arteries in real time.

Future Studies Each of the previously discussed technologies quantifies some aspect of ocular blood flow. It is impossible, however, to interpret the impact of any single blood flow parameter measured within a single vascular bed on total retinal metabolism. The measurement of ocular blood flow is only a surrogate assessment of the metabolic status of the retina. Direct measurement of retinal tissue oxygenation would reveal the true impact of ischemia on retinal ganglion cell health and function. New and emerging tools that assess metabolic parameters may help to reveal the relationship between reductions in ocular blood flow and tissue hypoxia. For example, Michelson and colleagues conducted a study in which they used imaging spectrometry to measure the oxygen saturation in retinal arterioles and venules in patients with glaucomatous optic neuropathy. In all examined eyes, the arteriolar oxygen saturation and the retinal arterio-venous differences in oxygenation were found to significantly correlate with the area of the patient’s optic rim. Eyes with normal tension glaucoma, but not those with primary open angle glaucoma, showed significantly decreased arteriolar oxygen saturation. Although further advancements are still needed, these metabolic assessment tools may be very valuable in the evaluation of retinal hypoxia and in elucidating the effects of ocular ischemia. Obtaining accurate measurements of ocular tissue metabolism will greatly improve our understanding of disease

Further Reading Bron, A. J., Tripathi, R. C., and Tripathi, B. J. (1997). The choroid and uveal vessels. Wolff’s Anatomy of the Eye and Orbit, 8th edn. London: Chapman and Hall Medical. Drexler, W. and Fujimoto, J. G. (2008). State-of-the-art retinal optical coherence tomography. Progress in Retinal and Eye Research 27: 45–88. Hardarson, S. H., Harris, A., Karlsson, R. A., et al. (2006). Automatic retinal oximetry. Investigative Ophthalmology and Visual Science 47: 5011–5016. Harris, A., Jonescu-Cuypers, C. P., Kagemann, L., Ciulla, T. A., and Krieglstein, G. K. (2003). Atlas of Ocular Blood Flow. Philadelphia, PA: Elsevier. Harris, A. and Rechtman, E. (2008). Optic nerve blood flow measurement. In: Yanoff, M. and Duker, J. (eds.) Ophthalmology, 3rd edn., ch. 10.8, section 2, pp. 52–55. Edinburgh: Elsevier. Hayreh, S. S. (2001). Blood flow in the optic nerve head and factors that may influence it. Progress in Retinal and Eye Research 20: 595–624. Hayreh, S. S. (2008). Patholophysiology of glaucomatous optic neuropathy: Role of optic nerve head vascular insufficiency. Journal of Current Glaucoma Practice 2: 6–17. Mackenzie, P. J. and Cioffi, G. A. (2008). Vascular anatomy of the optic nerve head. Canadian Journal Ophthalmology 43: 308–312. Michelson, G. and Scibor, M. (2006). Intravascular oxygen saturation in retinal vessels in normal subjects and open-angle glaucoma subjects. Acta Ophthalmologica Scandinavica 84: 289–295. Morrison, J. C. and van Buskirk, E. M. (1984). American Journal of Ophthalmology 97: 372–383. Orgu¨l, S. and Cioffi, G. A. (1996). Embryology, anatomy, and histology of the optic nerve vasculature. Journal of Glaucoma 5: 285–294. Orgu¨l, S., Gugleta, K., and Flamer, J. (1999). Physiology of perfusion as it relates to the optic nerve head. Survey of Ophthalmology 43: S17–S26. Schmetterer, L. and Garhofer, G. (2007). How can blood flow be measured? Survey of Ophthalmology 52: 134–138. Simon, B., Moroz, I., Goldenfeld, M., and Melamed, S. (2004). Scanning laser Doppler flowmetry of nonperfused regions of the optic nerve head in patients with glaucoma. Ophthalmic Lasers, Surgery, and Imaging 34: 245–250.

Animal Models of Glaucoma S I Tomarev, National Institutes of Health, Bethesda, MD, USA Published by Elsevier Ltd., 2010.

Glossary BAC – Bacterial artificial chromosome. It is a DNA construct based on a functional fertility plasmid, used for cloning in bacteria. The bacterial artificial chromosome’s usual insert size is 150–350 kbp. BAX – Proapoptotic BCL2-associated X protein. BCL2 is an integral outer mitochondrial membrane protein that blocks the apoptotic death of some cells. Retrobulbar space – The area located behind the globe of the eye. Synechia – An eye condition where the iris adheres to either the cornea (anterior synechia) or the lens (posterior synechia). Tonometry – The procedure to determine the intraocular pressure. TUNEL – Terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling for detection of DNA fragmentation resulting from apoptotic programmed cell death.

models, they allow the comparison of processes leading to RGC death induced by different initial insults. Such comparative analysis may lead to the identification of changes that are specific to glaucoma versus changes that are involved in more general RGC dysfunction. While none of the existing animal models is perfect, some of the existing models have been successfully used to uncover important features of glaucoma pathology in humans. Several factors should be considered in selecting a particular animal model of glaucoma for experimentation: (1) the similarity of the model visual system to the human eye; (2) the similarity in the time course of pathological changes in the model and human eyes; (3) ability to apply genetic manipulations; (4) training necessary to produce affected animals; (5) the size of the eye; (6) availability and difficulties of methods of analysis; (7) availability of animals; and (8) cost. This article briefly describes available animal models of glaucoma with emphasis on the strengths and weaknesses of each model.

Mammalian Models Glaucoma is a complex disease, the initiation and progression of which involves interactions between different parts of the eye and brain. It is difficult to perform experiments directed toward elucidating pathogenic molecular mechanisms and potential treatments for glaucoma in human subjects and, as a rule, only postmortem material can be used for biochemical analysis. Experiments in cell culture or organ culture systems may only partially reproduce the complexity of the natural ocular environment. It is now well recognized that animal models may provide a very useful tool for understanding the underlying molecular mechanisms involved in glaucoma and for identifying new genetic components of the disease, including both causative and modifier genes. In addition, appropriate animal models are used to develop and test new regiments of glaucoma treatment as a prerequisite for clinical trials in humans. A number of animal models of glaucoma have been developed over the years. Since elevated intraocular pressure (IOP) is the most important risk factor in glaucoma, most of the animal models of glaucoma are based on elevation of IOP by surgical procedures or by genetic manipulations. Several models used to study death of the retinal ganglion cells (RGCs) include optic nerve crush or transaction, intravitreal injection of excitory amino acids (glutamate and N-methyl-D-aspartic acid (NMDA)), or retinal ischemia. Although these are not true glaucoma

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Primate Models of Glaucoma Monkey and human eyes are very similar both anatomically and functionally, making monkey models very attractive to study different eye pathologies including glaucoma. IOP in monkeys is measured using the same equipment that is used to measure IOP in humans. Moreover, tonometry and visual-field analysis can be performed in conscious, trained monkeys. This is an important factor since it is well documented that general anesthesia that is necessary to measure IOP in most other animal models results in rapid ocular hypotension. The main disadvantage of monkey models is that experiments with monkeys are expensive and require a highly skilled team of investigators. Moreover, large numbers of animals are required to assess effects of elevated IOP on the optic nerve head (ONH) and retina because of genetic variations between animals. Several approaches have been used to develop pressureinduced glaucoma models in nonhuman primates. The most common method of IOP elevation in the monkey was originally developed more than 30 years ago and involves circumferential laser photocoagulation treatment of the trabecular meshwork. Several laser sessions are normally required to produce a sustained elevation of IOP. In the treated eyes, IOP rises several days after the laser treatment, normally to between 25 and 60 mmHg, and may last for more than a year. Other methods that have

Animal Models of Glaucoma

been used to produce elevated IOP elevation in monkeys are less consistent than laser coagulation. They include injection of ghost red cells, latex microspheres, cross-linked polyacrylamide gels, or enzymes into the anterior chamber or application of topical steroids. A non-IOP-related monkey model of glaucoma involves the delivery of endothelin1 to the retrobulbar space through osmotic pump for 6–12 months; this induces ischemia and leads to the preferential loss of large RGC axons. Ischemia-induced focal axonal loss is similar to human glaucoma and this model may reproduce some aspects of normal tension glaucoma. A number of important observations have been made using the monkey photocoagulation model. Apoptosis as the primary mechanism of glaucomatous RGC death was first demonstrated in this model before later being confirmed in other models and in human glaucoma. Multifocal electroretinogram (ERG) techniques were used in monkeys to demonstrate that not only RGCs but also cells in the inner and outer nuclear layers are damaged in advanced glaucoma. The monkey glaucoma model has been successfully used to study changes in retinal gene expression patterns after the induction of ocular hypertension. It is also being used to efficiently test new drugs and techniques to reduce IOP. For instance, recombinant adenoviral delivery of the human p21WAF-1/cip-1 gene to cause cell cycle arrest before filtration surgery in ocular hypertensive monkey eyes has shown a beneficial effect in long-term control of IOP. Rodent Models of Glaucoma Several rodent models of glaucoma have been developed over the last 20 years and new models are at different stages of development in several laboratories. These models have proven useful because the drainage structures of the rodent eye are similar to those in humans. Their utility was enhanced further by the development of new methods to measure IOP and analyze glaucomatous changes in these small eyes. Rodent models, and especially mouse models, are relatively cheap and allow extensive genetic manipulations. Rodent models are preferred when a significant number of animals are required to conduct genetic screens or to test different drugs and agents for neuroprotective or IOPlowering effects. One of the main disadvantages of rodent models is that there are anatomical differences between rodent and human eyes, including the arterial blood supply to the ONH and the absence of a well-developed, collagenous lamina cribrosa. These variations, as well as differences in general physiology, may explain why expression of certain genes in mouse and human eyes (e.g., mutated myocilin) have differential effects. Rat Models Rats are easy to handle. The relatively large size of their eyes allows multiple noninvasive IOP measurements

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in awake trained animals with commercially available equipment. The TonoPen was the instrument of choice for IOP measurements for many years but has recently been superseded by an induction/impact tonometer, marketed as the TonoLab rebound tonometer. This instrument is easy to operate and can be used in both rats and mice. Several rat models of pressure-induced glaucoma have been developed over the last 15 years. IOP elevation in the rat eye may be achieved by injection of hypertonic saline solution into the episcleral vein that leads to sclerosis of the aqueous humor outflow pathway. Sustained IOP elevation occurs 7–10 days after injection in most but not all rats. The saline injection generally produces a range of IOP elevation in different animals from a very minimal rise to twofold increase over IOP in control eyes, which can remain elevated for up to several months. Cauterization of two or more of the four large episcleral veins is another method of IOP elevation. In this model, IOP elevation occurs very quickly and there are some indications that this procedure impedes blood outflow from the globe and leads to ischemia. Reports indicate that IOP elevation may last from several weeks to several months without requiring retreatment. IOP increase can be also achieved by laser photocoagulation of the trabecular meshwork with or without injection of Indian ink into anterior chamber. Intracameral injection of hyaluronic acid or latex microspheres is another method of IOP elevation in rats. However, the repeated weekly injections required by this method may produce undesirable effects and are labor consuming. Topical application of dexamethasone for 4 weeks may also be used to induce ocular hypertension. These methods of chronic IOP elevation in rats are accompanied by death of the RGCs by apoptosis, optic nerve degeneration, and ONH remodeling similar to those observed in glaucoma in humans. Acute ocular hypertension, on the other hand, may be produced in rats by cannulation of the anterior chamber with a needle attached to a saline reservoir. Although such treatment leads to retinal ischemic injury, it has been suggested that this model mimics acute angleclosure glaucoma in humans. A mutant rat strain with a unilateral or bilateral globe enlargement and IOPs that range from 25 to 45 mmHg have been described. In this strain, cupping of the ONH as well as reduction in the number of RGCs progress with age. Unfortunately, this strain was obtained from the Royal College of Surgeons colony that has a mutation in the receptor tyrosine kinase gene, leading to degeneration of the photoreceptors. This drastically limits the utility of this strain to study phenomena that are specific to glaucoma and not confounded by other neurodegenerative processes. Rat models of glaucoma have been used to study effects of elevated IOP on the ERG, changes in the gene expression patterns in the retina, RGCs and optic nerve, and

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changes in the protein spectrum of the retina. Rat models also are often used to study neuroprotection. For instance, the hypertonic saline model was used to demonstrate for the first time that agents targeting multiple phases of the amyloid-b pathway provide a therapeutic avenue in glaucoma management. Mouse Models Mouse models of glaucoma recently have become very popular. Although most mouse models of glaucoma are based on the elevation of IOP, information about IOP is essential even for the models that do not include experimental IOP manipulation. The mouse eye is much smaller than the human eye, and devices designed for tonometry in humans do not produce reliable data in the mouse. Thus, new methods to measure IOP in mice have been developed and, as a result, the development and acceptance of mouse models of glaucoma have been accelerated. Currently, several invasive and noninvasive methods of IOP measurements in mice exist. The oldest method remains as one of the most reliable and accurate methods and does not depend upon the mechanical properties of the cornea. It involves the insertion of a glass microneedle connected to a pressure transducer into anterior chamber of the eye. However, this procedure cannot be performed too frequently in the same eye, as adequate time is required for corneal wound healing. In addition, cannulation tonometry is technically difficult and training is required to develop sufficient expertise to obtain reliable IOP readings. Cannulation tonometry was used to demonstrate that common mouse strains exhibit different average IOPs in the range between 10 and 20 mmHg. Other methods of IOP measurements in mice were later developed including noninvasive techniques (TonoLab tonometer). Noninvasive techniques allow multiple IOP measurements within short periods of time without extensive training. Pressure-induced mouse models

Surgical approaches similar to those that were used to produce elevated IOP in rats have also been developed in mice. Significant elevation of IOP in the C57BL/6J mouse eye is accomplished by combined injection of indocyanine green dye into the anterior chamber and diode laser treatment of the trabecular meshwork and episcleral vein region. IOP in operated eyes is significantly elevated 10 days after the surgery but returns back to normal 60 days after the procedure. Histological analysis of the treated eyes 65 days after the surgery revealed development of anterior synechia, loss of RGCs, thinning of all retinal layers, and damage to the optic nerve structures without evidence of prominent cupping. A reduction in the function of all retinal layers, as assessed by ERG studies, indicates that this model produces more dramatic

changes in the retina compared to glaucoma in humans. Elevation of IOP may also be induced by argon laser photocoagulation of the episcleral and limbal veins in C57BL/6J mouse eyes or by cauterization of three episcleral veins in CD1 mouse eyes. In one study, mean IOP in the eyes that underwent laser treatment was about 1.5 times higher than in control eyes for 4 weeks. RGC loss was 22.4 7.5% at 4 weeks after treatment with the majority of terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)positive apoptotic cells detected in the peripheral areas of the retina. Episcleral vein cauterization produced a maximum IOP elevation within 2–9 days after the procedure, which decreased progressively after that to baseline values in the following 24–33 days. This was associated with a 20% decline in the number of RGCs 2 weeks after the surgery. The DBA/2J strain has become a popular mouse model of secondary-angle-closure glaucoma and is one of the best-characterized mouse models of glaucoma in general. DBA/2J mice have mutations in two genes, Tyrp1 and Gpnmb, which lead to pigment dispersion, iris transillumination, iris atrophy, and anterior synechia. IOP is elevated in most mice by the age of 9 months. IOP elevation was accompanied by the death of the RGCs, optic nerve atrophy, and optic nerve cupping. Although no group of the RGCs appears especially vulnerable or resistant to degeneration, fan-shaped sectors of cell death and survival radiating from the ONH have been detected. It has been suggested that axon damage at the ONH might be a primary lesion in this model. Several important observations have been made using DBA/2J model. It was shown that proapoptotic protein BAX is required for RGC death but not for RGC axon degeneration in this model of glaucoma, suggesting that BAX may be a candidate human glaucoma susceptibility gene. Unexpectedly, high dose of g-irradiation accompanied with syngenic bone marrow transfer protected RGCs in DBA/2J. Similar to the results obtained with rat and monkey models, genes involved in the glial activation and immune response are activated in DBA/2J retina as shown by array hybridization. Complement component C1q is upregulated in the retina in several animal models of glaucoma and human glaucoma with timing, suggesting that complement activation plays a significant role in glaucoma pathogenesis. Recent data suggest that complement proteins opsonize central nervous system synapses during a distinct window of postnatal development and that the complement proteins C1q and C3 are required for synapse elimination in the developing retinogeniculate pathway. In DBA/2J mice, C1q relocalizes to adult retinal synapses at an early stage of glaucoma prior to obvious neurodegeneration. These data indicate that C1q in adult glaucomatous retina marks synapses for elimination at early stages of disease, suggesting that the complement cascade mediates synapse loss in glaucoma.


Another DBA/2 substrain, DBA/2NNia, also develops elevated IOP and demonstrates RGC loss and optic nerve degeneration when aged. However, depletion of cells in the inner and outer nuclear layers and significant damage of the photoreceptor cells in 15-month-old mice have also been observed. Transgenic and knock-out approaches have been used to prospectively develop several mouse models of glaucoma. The main advantage of these approaches is that animals within a particular line produce more uniform responses in terms of IOP elevation and damage to the retina and optic nerve as compared to surgically induced models. A large number of animals may be obtained and no training is needed to produce affected mice. Several lines of transgenic mice have been developed that contain BAC DNAs with a Tyr423His point mutation in the mouse or Tyr437His point mutation in the human MYOCILIN (MYOC) genes. Tyr437His mutation in the MYOC gene leads to severe glaucoma cases in humans, and mouse Tyr423His mutation corresponds to this human mutation. However, expression of mutated mouse or human myocilin in the eye-drainage structures of mice leads to moderate (about 2 mmHg at daytime and 4 mmHg at nighttime) elevation of IOP which is much less dramatic than IOP elevation in humans carrying the same mutation in the MYOC gene. Since these mice demonstrate progressive degenerative changes in the peripheral RGC layer and optic nerve with normal organization of the drainage structures, it has been suggested that these mice represent a mouse model of primary open-angle glaucoma. Another model of primary open-angle glaucoma was developed by the expression of a mutated gene for the a1 subunit of collagen type I. This mutation blocks the cleavage of collagen by matrix metalloproteinase-1. Transgenic mice expressing mutated collagen demonstrate elevated IOP which increases to a maximum of 4.8 mmHg greater than controls at 36 weeks. A transgenic model of acute angle-closure glaucoma was developed by expression of calcitonin-receptor-like receptor under the control of a smooth muscle a-actin promoter. Overexpression of this receptor in the papillary sphincter muscle results in enhanced adrenomedullininduced sphincter muscle relaxation that leads to abrupt transient rises in IOP in some mice up to a mean level of about 50 mmHg between 30 and 70 days of age. Although the aberrant ocular functions of adrenomedullin and calcitonin-gene-related peptide have not been associated with the pathogenesis of human acute glaucoma, it has been suggested that adrenomedullin and its receptor in the iris sphincter may present novel targets for the treatment of angle-closure glaucoma. Normal-tension mouse models

Mice deficient in the glutamate transporters GLAST or EAAC1 show RGC death and typical glaucomatous

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damage of the optic nerve without elevation of IOP. It has been shown that the glutathione levels are decreased in Mu¨ller cells of GLAST-deficient mice, while administration of glutamate receptor blocker prevents loss of RGCs. RGCs are more sensitive to oxidative stress in EAAC1deficient mice. These mice represent a model of normal tension glaucoma and are currently being used to develop therapies directed at IOP-independent mechanisms of RGC loss. Developmental mouse models Defects in genes involved in the development of the anterior eye segment may lead to relatively rare developmental glaucomas, which account for less than 1% of all human glaucoma cases. Several genes have been implicated in congenital glaucoma and anterior segment dysgenesis. They include CYP1B1, FOXC1, FOXC2, PITX2, LMX1b, and PAX6. Although Cyp1b1 knock-out mice do not develop elevated IOP, they have ocular abnormalities similar to defects in humans with primary congenital glaucoma: small or absent Schlemm’s canal, defects in the trabecular meshwork, and attachment of the iris to the trabecular meshwork and peripheral cornea. Foxc1–/– mice die at birth, while Foxc1+/– animals are viable but have defects in the eye-drainage structures in the absence of IOP changes. Similar eye defects are observed in Foxc2+/– mice. It has been suggested that Foxc1+/– and Foxc2+/– mice are useful models for studying anterior segment development and its anomalies, and they may allow identification of genes that interact with Foxc1 and Foxc2 to produce a phenotype with elevated IOP and glaucoma. Transgenic mice overexpressing the ocular development-associated gene (ODAG) in photoreceptors under the control of mouse Crx promoter exhibit gradual protrusion of the eyeballs with dramatically increased IOP that is not attributable to mechanical block of the aqueous humor outflow. These transgenic mice demonstrate optic nerve atrophy and impaired retinal development. All retinal layers of these transgenic mice are affected, thereby differentiating this model from a typical glaucomatous retina where morphological changes are detected only in the RGC layer.

Other Mammalian Models Several other mammalian models of glaucoma have been developed. Pig eyes are relatively large and, although the drainage outflow system of the pig eye is slightly different from that of the human eye, the porcine retina is more similar to the human retina than that of other large mammals (i.e., dog, goat, and cow). Cauterization of three porcine episcleral veins leads to a 1.3-fold elevation of IOP that is apparent 3 weeks after the surgery and persists for at least 21 weeks. It has been shown that endothelium leukocyte adhesion molecule 1 (ELAM-1), a molecular marker

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for human glaucoma, is also elevated in the trabecular meshwork of pigs with elevated IOP. Rabbits are a standard ophthalmic animal model for glaucoma filtration surgery and are often used for the development of new devices (e.g., drainage implants and degradable biopolymers) and medical therapies including gene therapy. At the same time, due to the unique anatomy of the rabbit eye, laser-induced elevation of IOP, like that in the monkey eye, is difficult to achieve. Alternatively, application of glucocorticoids has been successfully used to induce ocular hypertension in rabbit model. In addition, a line of rabbits with congenital glaucoma has been developed. Thick subcanalicular tissues and the deposition of extracellular matrix in the trabecular meshwork appear to contribute to the ocular hypertension exhibited by this model. Several purebred dogs develop glaucoma with high frequency. Among North American breeds, the highest prevalence of primary glaucoma is observed in the American cocker spaniel (5.52%), basset hound (5.44%), and chow chow (4.70%), exceeding that in humans. Lens displacement resulting in secondary glaucoma is common in terrier breeds. The high prevalence of the glaucomas in these canine breeds suggests a genetic basis of pathophysiology. It has been reported that topical application of corticosteroid induces reproducible elevation of IOP in the cow. The large amount of tissues available from the cow eye makes this model useful for biochemical studies.

implicated in glaucoma. It has been shown that wdr36 functions in ribosomal RNA processing and interacts with the p53 stress-response pathway, while olfactomedin 1 is essential for optic nerve growth and targeting of the optic tectum. Thus, zebrafish system may be very useful to complement studies with other model organisms, but by itself should be used with caution to study glaucoma.

Other Nonmammalian Models Open-angle glaucoma characterized by elevated IOP can be induced in domestic chickens or in Japanese quails when they are reared under continuous light. Besides, an unknown autosomal dominant mutation in a Slate line of domestic turkeys has been identified that leads to secondary angle-closure glaucoma. Although these models might be useful to study certain aspects of glaucoma in humans, one should remember that structural and physiological differences between human and bird eyes complicate direct comparison. Drosophila eyes have been suggested as a useful system for the discovery of genes that are associated with glaucoma. However, the general organization of human and Drosophila eyes are very different and data obtained with Drosophila may not always be relevant to glaucoma in humans.

Conclusion Nonmammalian Models Zebrafish The zebrafish is an excellent model system to study complex diseases as it allows one to combine forward and reverse genetic approaches. The general organization of the zebrafish eye is similar to the human eye, although the fine details of individual ocular structures are rather different. In particular, there are significant differences in the organization of the iridocorneal angle between zebrafish and mammals. They include the trabecular meshwork and lack of iris muscles as well as ciliary folds in zebrafish as compared to mammals. Even with these limitations in mind, zebrafish have been used as a model organism for glaucoma studies. An accurate method exists to measure IOP in zebrafish which is based on servo-null electrophysiology. Using this method, baseline IOP differences have been demonstrated in genetically distinct zebrafish strains. Among tested strains, the long fin strain (LF) had the highest IOP (20.5 1.2 mm Hg) while the Oregon AB strain (AB) has the lowest IOP (10.8 0.3 mm Hg). At the same time, these differences in IOP do not lead to detectable defects of the retina or in visual function. Zebrafish have also been used to determine the function of several genes (foxc1, lmx1b, wdr36, olfactomedin 1, and olfactomedin 2)

Animal models have already provided interesting new information about potential mechanisms of glaucoma in humans. However, even in monkey models which most closely mimic the human form of the disease, the time course of changes in the glaucomatous eyes may be significantly accelerated as compared with human glaucomatous eyes. Indeed, all of the previously discussed systems are, after all, just models of human glaucoma. Reactions to the same insult (IOP, expression of the same mutated protein, etc.) may be somewhat different between various animal models and humans. Results obtained with these models should not automatically be applied to human condition and should be confirmed by testing in human subjects when possible. Nevertheless, information on molecular mechanisms of glaucoma obtained using animal models might be extremely valuable to develop new therapeutic approaches for glaucoma treatment and prevention in humans.

Further Reading Anderson, M. G., Libby, R. T., Gould, D. B., et al. (2005). High-dose radiation with bone marrow transfer prevents neurodegeneration in an inherited glaucoma. Proceedings of the National Academy of Sciences of the United States of America 102: 4566–4571.

Animal Models of Glaucoma Baulmann, D. C., Ohlmann, A., Flu¨gel-Koch, C., et al. (2002). Pax6 heterozygous eyes show defects in chamber angle differentiation that are associated with a wide spectrum of other anterior eye segment abnormalities. Mechanisms of Development 118: 3–17. Harada, T., Harada, C., Nakamura, K., et al. (2007). The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma. European Journal of Clinical Investigation 117: 1763–1770. Iwata, T. and Tomarev, S. (2008). Animal models for eye diseases and therapeutics. In: Conn, P. M. (ed.) Sourcebook of Models for Biomedical Research, pp. 279–287. Totowa, NJ: Humana Press. Levkovitch-Verbin, H., Quigley, H. A., Martin, K. R., et al. (2002). Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats. Investigative Ophthalmology and Visual Science 43: 402–410. Libby, R. T., Anderson, M. G., Pang, I., et al. (2005). Inherited glaucoma in DBA/2J mice: Pertinent disease features for studying the neurodegeneration. Visual Neuroscience 22: 637–648.

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McMahon, C., Semina, E. V., and Link, B. A. (2004). Using zebrafish to study the complex genetics of glaucoma. Comparative Biochemistry and Physiology – Part C: Toxicology and Pharmacology 138: 343–350. Morrison, J. C., Johnson, E. C., Cepurna, W., and Jia, L. (2005). Understanding mechanisms of pressure-induced optic nerve damage. Retinal Eye Research 24: 217–240. Pang, I.-H. and Clark, A. F. (2007). Rodent models for glaucoma retinopathy and optic neuropathy. Glaucoma 16: 483–505. Rasmussen, C. A. and Kaufman, P. L. (2005). Primate glaucoma models. Journal of Glaucoma 14: 311–314. Senatorov, V., Malyukova, I., Fariss, R., et al. (2006). Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice. Journal of Neuroscience 26: 11903–11914. Smith, R. S., John, S. W. M., Nishina, P. M., and Sundberg, J. P. (eds.) (2002). Systematic Evaluation of the Mouse Eye. Boca Raton, FL: CRC Press. Weinreb, R. N. and Lindsey, J. D. (2005). The importance of models in glaucoma research Volume. Journal of Glaucoma 14: 302–304.

Blood–Retinal Barrier J Cunha-Vaz, AIBILI, Coimbra, Portugal ã 2010 Elsevier Ltd. All rights reserved.

Glossary iBRB – The inner blood–retinal barrier is a situation of restricted permeability established at the level of the retinal vessels, between the blood and retinal tissue, by the tight junctions (zonula occludents) between neighboring retinal endothelial vessels and the retinal endothelial cells themselves. oBRB – The outer blood–retinal barrier is a situation of restricted permeability established at the level of the retinal pigment epithelium, the blood, and the retinal tissue, by the tight junctions (zonula occludents) between neighboring retinal pigment epithelial cells and the retinal pigment epithelial cells themselves. Retinal leakage analyzer – A method developed to perform localized measurements of blood–retinal barrier fluorescein leakage using a confocal scanning laser ophthalmoscope modified to obtain fluorescence measurements in the vitreous. Tight junctions – Tight junctions are specialized junctions uniting neighboring cells by fusion of the outer leaflets of their cell membranes (zonulae occludentes) thus obliterating the intercellular space fusion and restricting paracellular diffusion. Uveitis – Uveitis is inflammation of the uvea, which is the vascular layer of the eye sandwiched between the retina and the sclera. The uvea extends toward the front of the eye and consists of the iris, choroid layer, and ciliary body. Vitreous fluorometry – A method developed to measure the fluorescence resulting from the presence of fluorescein in the vitreous after intravenous administration. It is a direct indicator of the permeability of the blood–retinal barrier to fluorescein.

The entire eye must function as the organ for vision and is organized with two major goals: normal function of the visual cell and the need to maintain ideal optical conditions for the light to access the visual cells, located in the back of the eye. The blood–ocular barriers play a fundamental role in the preservation and maintenance of the appropriate environment for optimal visual cell function (Figure 1). The blood–ocular barriers include two main barrier systems: the blood–aqueous barrier and the blood–retinal

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barrier (BRB) (Figure 2), which are fundamental to keep the eye as a privileged site in the body by regulating the contents of its inner fluids and preserving the internal ocular tissues from variations which occur constantly in the whole circulation. The blood–ocular barriers must not only provide a suitable, highly regulated, chemical environment for the avascular transparent tissues of the eye, but also serve as a drainage route for the waste products of the metabolic activity of the ocular tissues. One of these barriers, the BRB, similar to the blood–brain barrier (BBB), is particularly tight and restrictive and is a physiologic barrier that regulates ion, protein, and water flux into and out of the retina. It is also important to realize that once inside these barriers there are no major diffusional barriers between the extracellular fluid of the retina and adjacent vitreous; nor does the vitreous body itself significantly hinder the diffusional exchanges between the posterior chamber and the retinal extracellular fluid. This means that the functions of both barriers, blood–aqueous barrier and BRB, influence each other and must work in equilibrium.

Blood–Retinal Barrier The presence of an intact BRB is essential for the structural and functional integrity of the retina and in clinical conditions where BRB breakdown occurs vision may be seriously affected. The BRB consists of inner and outer components (inner BRB (iBRB) and outer BRB (oBRB)) and plays by itself a fundamental role in of the microenvironment of the retina and retinal neurons. The BRB regulates fluids and molecular movement between the ocular vascular beds and retinal tissues and prevents leakage into the retina of macromolecules and other potentially harmful agents (Figure 3). The iBRB is established by the tight junctions (TJs) (zonulae occludentes) between neighboring retinal endothelial cells. These specialized TJs restrict the diffusional permeability of the retinal endothelial layer to values in the order of 0.14 10 5 cm s 1 for sodium fluorescein. The retinal endothelial layer functions as an epithelium and in this way is directly associated with its differentiation and with the polarization of the BRB function. This continuous endothelial cell layer, which forms the main structure of the iBRB rests on a basal lamina that is covered by the processes of astrocytes and Mu¨ller cells. Pericytes are also present, encased in the basal lamina, in

Blood–Retinal Barrier

close contact with the endothelial cells but do not form a continuous layer and, therefore, do not contribute to the diffusional barrier. Astrocytes, Mu¨ller cells, and pericytes are considered to influence the activity of the retinal endothelial cells and of the iBRB by transmitting to endothelial cells regulatory signals indicating the changes in the microenvironment of the retinal neuronal circuitry. The oBRB is established by the TJs (zonulae occludentes) between neighboring retinal pigment epithelial (RPE) cells. The RPE is composed of a single layer of RPE cells that are joined laterally toward their apices by

Blood

Blood RET

PC

45

TJs between adjacent lateral cell walls. The RPE resting upon the underlying Bruch’s membrane separates the neural retina from the fenestrated choriocapillaries and plays a fundamental role in regulating access of nutrients from the blood to the photoreceptors as well as eliminating waste products and maintaining retinal adhesion. The metabolic relationship of the RPE apical villi and the photoreceptors is considered to be critical for the maintenance of visual function. In both, iBRB and oBRB, the cell TJs restrict paracellular movement of fluids and molecules between blood and retina, and the endothelial cells and RPE cells actively regulate inward and outward movements. As a result, the levels in the blood plasma of aminoacids or fatty acids fluctuate over a wide range while their concentrations in the retina remain relatively stable. Inner Blood–Retinal Barrier

Vitreous AC

Figure 1 Schematic drawing of the blood–ocular barriers and main fluid movements. RET, retina; PC, posterior chamber; AC, anterior chamber.

Retinal endothelial cells The endothelial cells of retinal capillaries are not fenestrated and have a paucity of vesicles. The function of these endothelial vesicles has been described as endocytosis or transcytosis that are receptor mediated. Pinocytotic residues are selectively decreased in the BRB endothelial cells. Receptor-facilitated transport mechanisms are used to move materials across the BRB (Figure 4). Channelfacilitated transport using transmembrane proteins is another mechanism for diffusion of specific substrates across the BRB. The glucose transporter Glut 1 is a good example, supplying the neuronal tissue with necessary glucose. Disruption of the iBRB in pathological conditions

Retina-capillary blood Endothelium pericapillary glia

Inner BRB

Neurons

ECF

Junctions Pigment epithelium

Outer BRB

Connective tissue Choroid-capillary blood

Choroid endothelium

Figure 2 Schematic presentation of the inner and outer blood–retinal barriers (BRBs) and their relative location. ECF, extracellular fluid.

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is associated with increased vesicle formation and disrupted endothelial membranes. These alterations may develop before opening of the TJs is detected on ultrastructural examination. Retinal endothelial TJs

TJs or zonula accludentes of the retinal vascular endothelium are formed by fusion of the outer leaflets of adjacent endothelial cell membranes and were described for the first time in the retinal vessels in 1966. The TJ obliterates the interendothelial space and confers highly selective barrier properties to the capillaries. Diffusion of molecules from the lumen to the tissue is significantly restricted by TJ. These endothelial junctions have, like in the brain capillaries, extremely high electrical resistance, 1000–3000 ohm cm2. The TJ complex contains at least 40 proteins composing transmembrane and internal adapter proteins that regulated paracellular flux. Transmembrane proteins that make up the TJ are occludins, claudins, and junctional adhesion molecules (JAMs). Occludin is a 65-kDa protein and its changes correlate with permeability changes (a)

(b) Endothelium

Tight junctions

Vesicular transport

Junctional Channels opening

Astrocytes Figure 3 Pathways for solute movements across the inner blood–retinal barrier (retinal endothelial cells): (a) normal; (b) mechanisms of breakdown of iBRB.

making it a likely candidate in regulating the opening and closing of the TJ. Claudins regulate small charged molecules and ion permeability. JAM is part of a family of proteins and is associated with adhesion molecules. Numerous adapter proteins localize just below the membrane and act as TJ organizers and cytoskeleton anchors. It is important to realize that TJs are dynamic structures that can be regulated by signal transduction through cyclic AMP levels, tyrosine kinases, etc.

Mu¨ller cells, astrocytes, and pericytes A close spatial relationship exists between Mu¨ller cells and blood vessels in the retina suggesting a critical role for these cells in the formation and maintenance of the BRB, regulating the functions of barrier cells in the uptake of nutrients and in the disposal of metabolites under normal conditions. Barrier function is also impaired by matrix metaloproteinases (MMPs) from Mu¨ller cells as these MMPs lead to proteolytic degradation of the TJ protein occludin. Astrocytes originate from the optic nerve and migrate to the retinal nerve fiber layer during retinal vascular development. They are associated closely with the retinal vessels and help to maintain their integrity. Astrocytes are known to increase the barrier properties of the retinal endothelium by enhancing the expression of TJ protein Z0.1 and may moderate TJ integrity. Astrocytes are considered to play an important regulatory role in the function of the BRB. Finally, the pericytes have been shown to play a role in regulating vascular tone, secrete extracellular material, and being phagocytic. Pericytes are considered to play an accessory role in maintaining the integrity of the iBRB by inducing mRNA and protein expression of occludin and other protein junctions. There is evidence that pericytes interact with the endothelial cells contributing to their modulation.

Y

Vesicle

Tight junction

Y

Y

Y

Receptor mediated transcytosis

YY

Pinocytosis YY

Lysosome degradation

Adherence junction

Paracellular transport Figure 4 Transport mechanisms across retinal vascular endothelial cells. Modified from Philips, B. E. and Antonetti, D. A. (2007). Blood–retinal barrier. In: Joussen, A. M., Gardner, T. W., Kirchoff, B., and Ryan, S. J. (eds.) Retinal Vascular Disease, pp. 139–153. Berlin: Springer.


Outer Blood–Retinal Barrier RPE cells

The RPE cells transport water from the subretinal space or apical side to the blood or basolateral side. Therefore, the RPE has the structural properties of an ion-transporting epithelium. RPE cells regulate water content and lactic acid removal generated by the characteristic high metabolic rates in the retina. RPE cells transport water out of the retina and into the choroidal capillary plexus. The force generated by this water flux produces an adhesion force and helps to maintain retinal attachment. Water transport is linked with ion transport, organic anion transport, and other drainage mechanisms. This outward molecular movement is largely dependent on active ionic transport associated with a relevant high oncotic pressure in the choroid. RPE cells also have a fundamental role by transporting glucose and retinol, in the appropriate direction, from blood to the photoreceptors. RPE tight junctions

Paracellular movement of larger molecules is restricted by the TJ between neighboring RPE cells. The paracellular resistance is 10 times higher than the transcellular resistance, classifying the RPE as a tight epithelium. Occludin, claudins, and adapter proteins have been detected at the RPE TJ as in TJ elsewhere. The TJs of the RPE are anchored to the actin cytoskeleton of RPE cells, interact with signaling molecules, and are important for the establishment of cell polarity. In addition to TJ between RPE cells, the polarized distribution of RPE membrane proteins contributes to the function of the oBRB. The outer retinal layers are nourished from the blood circulation through the fenestrated capillaries of the choriocapillaris and to subserve this function there is a necessity of a large baso-apical molecular movement from choroid to retina. Waste products of retinal metabolism are transported to the choroid through the oBRB. Polarity of the outer and inner barriers: TJ modulation

Establishment of cell polarity is a characteristic of a tissue barrier. The endothelial cells of the retinal vessels and RPE develop distinct apical versus basal membrane surfaces. This cell polarity is associated with organization of the cytoskeleton, apical/basal cell membrane proteins, and organization of the junctional complexes between neighboring cells. It is these TJ protein complexes that allow the establishment of the polarities of the BRB, restricting paracellular diffusion of blood–barrier compounds into the neuronal tissues.

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Understanding the normal function of these TJs and the pathological changes that are induced by their alteration resulting in increased permeability is necessary to understand disease progression in retinal diseases such as diabetic retinopathy (iBRB primarily affected) and wet age-related macular degeneration (oBRB primarily affected). Understanding the role of relevant proteins such as occludins, claudins, and JAMs in BRB physiology and in retinal pathology will certainly contribute to improved management of retinal disease.

Other factors regulating the molecular movement in the eye

Molecular movement from the retinal and choroidal vascular systems into, out, and across them is complex and limited by a variety of other ocular structures. There is a continuous molecular movement of small molecules (mainly water) from the vitreous cavity into the inner retina and through RPE to the choroid. The major proportion of aqueous humor secreted by the ciliary body from its rich vascular supply provides a bulk flow of fluid through the anterior chamber of the eye but a smaller proportion enters the vitreous cavity where it is largely cleared across the retina and RPE to the choroidal circulation. Molecular movement from vitreous to choroid is slowed by the cortical vitreous with its high concentration of hyaluronic acid stabilized in a relatively dense type II collagen matrix and the internal limiting membrane (ILM) of the retina. The ILM offers resistance to the diffusion of macromolecules of 148 kDa but allows the passage of smaller molecules. Movement through the retina of molecules that have crossed the iBRB into the retina is largely through the extracellular tissue spaces. Similary, Bruch’s membrane that separates the basal RPE from the fenestrated capillaries appears, in health, to offer little resistance to molecular movement. The presence of fenestrations in the choriocapillaris allows the passage of even large molecules such as albumin into the extravascular spaces of the choroid. The choriocapillaris, therefore, contributes little to the oBRB. Molecular movement across the oBRB is, however, probably influenced by the very high rate of blood flow in the choroid. A possible explanation for the high blood flow in the choroid is that there is a need not only to supply oxygen and metabolites to the energy demanding retina and RPE, but also for the rapid removal of waste products of the retinal metabolism into the blood circulation. Finally, the ciliary body may have a relevant regulatory role in the overall maintenance of the retinal microenvironment. The large surface covered by the ciliary processes, their location where the aqueous and vitreous meet, and the multiple transport functions of the ciliary

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epithelium are all factors suggesting an important role for the ciliary body in the regulation of the inner ocular fluids. The microenvironment of the retina, which closely resembles brain extracellular fluid and is in equilibrium with the vitreous is, therefore, maintained by a variety of facilitated and active transport processes which are located mainly in the iBRB and oBRB with the retinal endothelial cells and RPE playing fundamental roles. The Blood–Retinal Barrier and Ocular Immune Privilege The immune response has developed and evolved to protect the organism from invasion and damage by a wide range of pathogens. With time, the immune system has developed destructive responses that are specific for pathogens as well as tissues. Such tissue injury might, however, have a devastating effect on the function of an organ such as the eye that needs to maintain optical stability. The existence of ocular immune privilege is dependent upon multiple factors such as immunomodulatory factors and ligands, regulation of the complement system within the eye, tolerance promoting antigen-presenting cells (APCs), unconventional drainage pathways, and, with particular relevance, the existence of the blood–ocular barriers. The blood–ocular barriers provide a relative sequestration of the anterior chamber, vitreous, and neurosensory retina from the immune system and create the necessary environment for the existence of ocular immune privilege. The evolution of immune privilege as a protective mechanism for preserving the function of vital and delicate

organs such as the eye has resulted in a complex system with multiple regulatory safeguards for the control of both innate and adaptative immurity. The consequences of inadvertent bystander tissue destruction by antigenmonspecific inflammation can be so catastrophic to the organ or host that a finely tuned regulatory system is needed to ensure the integrity of the ocular tissues and maintain optical relationships. There are also several lines of evidence that points to immunosuppressive functions in the BRB cells, RPE, and retinal endothelial cells. These immunosuppressive effects are apparently due to the secretion of a variety of soluble factors, such as cytokines and growth factors. Clinical evaluation of the blood–retinal barrier Fluorescein angiography, an examination procedure performed routinely in the ophthalmologist’s office, permits a dynamic evaluation of local circulatory disturbances and identifies the sites of BRB breakdown (Figure 5). It is, however, only semi-quantitative and its reproducibility depends on the variable quality of the angiograms. Vitreous fluorometry was developed as a method capable of quantification of both inward and outward movements of fluorescein across the BRB system in the clinical setting. Protocols were devised, tested, and dedicated instrumentation developed. With the development of vitreous fluorometry methodologies, a large number of clinical and experimental studies demonstrated convincingly the major role played by alterations of BRB in posterior segment disease.

50 45 40 35 30 25 20 15 10 5

Figure 5 Sites of fluorescein leakage into the vitreous identified by the retinal leakage analyzer in an eye with nonproliferative retinopathy of a patient with diabetes type 2. Blue indicates minimal leakage; red indicates maximum leakage.


In clinical situations, alterations of the BRB have been measured in pathologies of the RPE, aged-related macular degeneration, and macular edema, as well as in hypertension and diabetes. The clinical use of vitreous fluorometry, however, has declined because it offers only an overall measurement over the posterior role and because at the time of its development there were no drugs available for stabilizing the BRB. Nowadays, vitreous fluorometry is mostly used in experimental research and in drug development. More recently, retinal leakage mapping has been introduced to identify the sites of BRB breakdown. Further developments of this methodology based on confocal scanning laser ophthalmology (SLO-Retinal Leakage Analyzer) associated with improved optical coherence tomography imaging are expected to contribute to earlier diagnosis of BRB alterations in retinal disease as well as improved testing of the effect of new drugs that are now becoming available for treatment of retinal disease.

Blood–retinal barrier and macular edema

Macular edema is the result of an accumulation of fluid in the retinal layers around the fovea, contributing to vision loss by altering the functional cell relationship in the retina and promoting an inflammatory reparative response. Macular edema is only a nonspecific sign of ocular disease and not a specific entity. It should be viewed as a special and clinically relevant type of macular response to an altered retinal environment, in most cases associated with an alteration of the BRB. It occurs in a wide variety of ocular situations such as uveitis, trauma, intraocular surgery, vascular retinopathies, hereditary dystrophies, diabetes, age-related macular degeneration, etc. The increase in water content of the retinal tissue that characterizes macular edema may be initially intracellular or extracelullar. In the first case, also called cytotoxic edema, there is an alteration of the cellular ionic distribution. In the second case, more frequent and clinically more relevant, the extracellular accumulation of fluid is directly associated with an alteration of the BRB. In this later situation, the protective effect of the BRB is lost and the Starling law applies. When there is breakdown of the BRB, any changes in the equilibrium between hydrostatic and oncotic pressure gradients across the BRB contribute to further water movements and progression of the macular edema. It is also of great relevance to keep in mind that the BRB cells, retinal endothelial cells, and retinal pigment epithelial cells, are both the target and producer of ecosanoids, growth factors, and cytokines. Breakdown of the BRB leading to situations of macular edema may be mediated by locally released cytokines and induction of an inflammatory reparative response creating the conditions for further release of cytokines, growth factors, etc.

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Macular edema is also one of the most serious consequences of inflammation in the retinal tissue. Inflammatory cells can alter the permeability of the TJs that maintain the iBRB and oBRB. Cell migration may occur primarily through splitting the junctional complex or through the formation of channels or pores across the junctional complex. Macular edema has particular relevance for its frequency in diabetic retinopathy. Leukocyte adhesion to retinal vessels and breakdown of the BRB appear to be mediated by nitric oxide (NO). NO upregulates intercellular adhesion molecule-1 (ICAM-1) and promotes the downregulation of TJ protein expression.

Relevance of BRB to Treatment of Retinal Diseases When administered systemically, drugs must pass the BRB to reach therapeutic levels in the retina. Drug entrance into the retina depends on a number of factors, including the plasma concentration profile of the drug, the volume of its distribution, plasma protein binding, and the relative permeability of the BRB. To obtain therapeutic concentrations within the retina, new strategies must be considered such as delivery of nanoparticles, chemical modification of drugs to enhance BRB transport, coupling of drugs to vectors, etc. The BRB must be considered as a dynamic interface that has the physiological function of specific and selective membrane transport from blood to retina and active efflux from retina to blood for many compounds, as well as degradative enzymatic activities. From the viewpoint of drug delivery, designing drugs (including peptides) with greater lipophilicity to enhance BRB permeability seems to be an easy approach. However, such a strategy would not only increase the permeation into tissues other than the retina, but also decrease the bioavailability due to the hepatic first pass metabolism in the case of oral administration. Accordingly, for the development of retina-specific drug delivery systems for neuroactive drugs the most effective approach is to utilize the specific transport mechanisms active at the BRB. That would mean designing drugs that mimic the substrates to be taken by particular transporters or receptors existing in the BRB. Eye drops are generally considered to be of limited benefit in the treatment of posterior segment diseases. Newer pro-drug formulations that achieve high concentrations of the drug in the posterior segment may have a role in the future. Meanwhile, periocular injection is one modality that has offered mixed results. Finally, the last years have seen a generalized and surprising safe utilization of intravitreal injections, a form of administration that circumvents the BRB. Steroids and a variety of anti-VEGF drugs have been administered

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through intravitreal injections to a large number of patients without significant side effects and demonstrating good acceptance by the patients. Intravitreal injections can achieve high drug concentrations in the vitreous and retina preserving the BRB function and its crucial protective function. At present the major challenge appears to be the need to decrease the number of intravitreal injections which in the case of anti-VEGF treatments are given every 6 weeks to maintain efficacy. The search for safe slow-delivery devices or implantable biomaterials is ongoing but the invasive approach to treat retinal diseases appears to be the only effective way of reaching rapidly therapeutic levels in the retina in the presence of a functioning BRB. See also: Anatomy and Regulation of the Optic Nerve Blood Flow; Breakdown of the Blood–Retinal Barrier; Breakdown of the RPE Blood–Retinal Barrier; Developmental Anatomy of the Retinal and Choroidal Vasculature; Innate Immune System and the Eye; Macular Edema; Physiological Anatomy of the Retinal Vasculature; Retinal Pigment Epithelial–Choroid Interactions; RPE Barrier.

Further Reading Cocaprados, M. and Escribano, J. (2007). New perspectives in aqueous humor and secretion and in glaucoma: The ciliary body as multifunctional neuroendocrine gland. Progress in Retinal Eye Research 26: 239–262. Cunha-Vaz, J. G. (1979). The blood–ocular barriers. Survey of Ophthalmology 23: 279–296.

Cunha-Vaz, J. G., Faria de Abreu, J. R., Campos, A. J., and Figo, G. (1975). Early breakdown of the blood–retinal barrier in diabetes. British Journal of Ophthalmology 59: 649–656. Cunha-Vaz, J. G. and Maurice, D. M. (1967). The active transport of fluorescein by retinal vessels and the retina. Journal of Physiology 191: 467–486. Cunha-Vaz, J. G. and Maurice, D. M. (1969). Fluorescein dynamics in the eye. Documenta Ophthalmologica 26: 61–72. Cunha-Vaz, J. G. and Travassos, A. (1984). Breakdown of the blood–retinal barriers and cystoid macular edema. Survey of Ophthalmology 28: 485–492. Cunha-Vaz, J. G., Shakib, M., and Ashton, N. (1966). Studies on the permeability of the blood–ocular barrier. I. On the existence, development and site of a blood–retinal barrier. British Journal of Ophthalmology 50: 411–453. Kaplan, H. J. and Niederkorn, J. Y. (2007). Regional immunity and immune privilege. In: Niederkorn, J. Y. and Kaplan, H. G. (eds.) Immune Response and the Eye. Chemical Immubology Allergy vol. 92, pp. 11–26. Basel: Karger. Lobo, C., Bernardes, R., and Cunha-Vaz, J. G. (1999). Mapping retinal fluorescein leakage with confocal scanning laser fluorometry of the human vitreous. Archives of Ophthalmology 117: 631–637. Partridge, W. M. (1998). Introduction to the blood–brain barrier: Methodology and pathology. In: Partridge, W. M. (ed.) Introduction to the Blood–Brain Barrier: Methodology, Biology and Pathology, pp. 1–10. New York: Cambridge University Press. Peyman, G. A. and Bok, D. (1972). Peroxidase diffusion in the normal and laser-coagulated primate retina. Investigative Ophthalmology 11: 35–45. Philips, B. E. and Antonetti, D. A. (2007). Blood–retinal barrier. In: Joussen, A. M., Gardner, T. W., Kirchhof, B., and Ryan, S. J. (eds.) Retinal Vascular Disease, pp. 139–153. Berlin: Springer. Rapoport, S. I. (1976). Blood–Brain Barrier in Physiology and Medicine. New York: Raven Press. Reese, T. S. and Karnovski, M. J. (1967). Fine structural localization of a blood–brain barrier to exogenous peroxidase. Journal of Cellular Biology 34: 207–217. Shakib, M. and Cunha-Vaz, J. G. (1966). Studies on the permeability of the blood–retinal barrier. IV. Junctional complexes of the retinal vessels and their role on their permeability. Experimental Eye Research 5: 229–234. Strauss, O. (2005). The retinal pigment epithelium in visual function. Physiological Review 85: 845–881.

Breakdown of the Blood–Retinal Barrier S A Vinores, Johns Hopkins University School of Medicine, Baltimore, MD, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Fenestrations – Spaces between vascular endothelial cells that allow free fluid exchange between vessel and tissue. Fenestrations are characteristic of vessels found in tissues that do not have a blood–tissue barrier. Leukostasis – The adhesion of leukocytes to the vascular endothelium as part of an inflammatory reaction. Macular edema – Fluid accumulation, due to blood–retinal barrier (BRB) breakdown, in the area of the human or primate retina of highest visual acuity. Tight junctions – Also referred to as zonula occludens, tight junctions are complex arrangements of microfilaments and other proteins that connect retinal vascular endothelial (RVE) or retinal pigment epithelial (RPE) cells and restrict the flow between them. Tight junctions are an integral component of the blood–retinal, blood–brain, or blood–nerve barrier. Vesicular transport – The nonspecific transcellular transport of fluid and high-molecular-weight molecules from the luminal to the abluminal surface of the vascular endothelium by means of pinocytotic vesicles. Uveitis – An inflammation of the uvea, or the middle layer of the eye. The uvea consists of three structures: the iris, the ciliary body, and the choroid.

retinal pigment epithelial (RPE) cells, or at both sites. The BRB is established by the formation of tight junctions between the retinal vascular endothelial (RVE) cells and the RPE cells and a paucity of endocytic vesicles within these cells. The establishment and maintenance of the BRB is regulated by the perivascular astrocytes and pericytes, but the mechanism for this regulation is not entirely clear. Some studies have shown that cell to cell contact is necessary to establish and maintain the BRB, while others provide evidence that a soluble mediator is sufficient. BRB breakdown can result from a disruption of the tight junctions, which are composed of a complex network of junctional proteins, an upregulation of vesicular transport across the RVE or RPE, or by degenerative changes to the barrier-forming cells or to the regulatory cells, the pericytes and glia. In some cases, BRB breakdown is related to identifiable structural defects, such as loss of pericytes, astrocytes, or RPE cells or changes to the vascular endothelial cells, as would be caused by microaneurysm formation. In other cases, where retinal vascular leakage is diffuse, such as in uveitis, or when the leakage is remote from a lesion, such as a surgical wound or tumor, it is clear that diffusible factors are involved. Blood–tissue barriers exist only in the retina, brain, and nerve. Vascular endothelial cells in the choroid and in other tissues are fenestrated (Figure 1(a)), allowing large molecular weight molecules to freely pass from the blood to the tissue, and thus do not have a barrier function.

Tight Junctions

Introduction The blood–retinal barrier (BRB), which is analogous to the blood–brain barrier, maintains homeostasis in the retina by restricting the entry of blood-borne proteins from the retina and by maintaining strict ionic and metabolic gradients. When this barrier breaks down, excess fluid accumulates in the retina and this can result in macular edema, which is associated with ischemic retinopathies, including diabetic retinopathy (DR) and retinopathy of prematurity (ROP), ocular inflammatory diseases, retinal degenerative diseases, and a variety of other ocular disorders, or following ocular surgery. BRB breakdown can occur at the inner BRB, which is established at the retinal vasculature, at the outer BRB, which consists of the

Tight junctions or zonula occludens consist of complex arrangements of over 40 proteins in the peripheral cytoplasm and apical plasma membrane that connect RVE or RPE cells and restrict flow between them (Figure 1(b)). Occludin and the claudins (over 24 isoforms), which form the junctional strands and are believed to constitute the backbone of the tight junction, span the plasma membrane and bind junctional proteins in adjacent cells. Zonula occludens proteins 1, 2, and 3 (ZO-1, -2, and -3) are intracellular proteins that associate with the cytoplasmic surface of the tight junctions and organize the complex. The binding of ZO-1 to occludin establishes the tight junction. Other integral components of the junctional complex are the junctional adhesion molecules, tricellulin, cingulin, 7H6, and symplekin. A breach of the tight junctions (Figure 1(c)) can result from an alteration in the content of the junctional proteins, their redistribution, or their phosphorylation.

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(a)

Adenosine, prostaglandin E1 (PGE1), interleukin-1b (IL-1b), tumor necrosis factor-a (TNFa), and vascular endothelial growth factor (VEGF) appear to be capable of causing a morphological and functional opening of the RVE tight junctions. A significant number of interendothelial cell tight junctions appeared open along their entire length within 6 h of intravitreal injection of each agent into rabbits with TNFa showing the greatest effect (35.6% of the interendothelial cell junctions appeared open, morphologically). The effect of PGE1 on tight junctions appeared to be transient, that of VEGF and IL-1b were partially reversible by 24 h, and the effect of the adenosine agonist, N-ethylcarboxamidoadenosine was not reversible by 48 h. The demonstration of immunoreactive albumin, which would normally be confined to the lumens of vessels with a blood–tissue barrier, along the entire length of these junctions, from the luminal to the abluminal surface, suggests that they are also functionally open (Figure 2(c)).

Vesicular Transport

(b)

(c) Figure 1 (a) Choroidal vessels are fenestrated (arrow) and, therefore, do not form a blood–tissue barrier. (b) A morphologically closed tight junction (arrow) in a normal retinal vessel. Note close apposition of vascular endothelial cells and an intact junctional complex. (c) A morphologically open tight junction (arrow) in a retinal vessel. The space between the vascular endothelial cells allows vascular leakage through the junction.

Occludin content at the tight junction is higher in cells that have a tighter barrier and decreased occludin correlates with increased BRB permeability, but occludin knockout mice appear to form functional tight junctions, so the association is complex and not simply regulated by occludin. Increased occludin phosphorylation is also associated with increased BRB permeability. Altered expression of claudins can lead to changes in selectivity of the junctions and claudin-5 appears to be particularly important for maintenance of a functional tight junction.

Since the tight junctions restrict the flow of molecules across the BRB, a series of pumps, channels, and transporter molecules are necessary to transport specific essential molecules from the blood to the retina. The nonspecific transport of high molecular weight molecules and fluids across the RVE by way of pinocytotic vesicles (Figure 2(a)) or caveolae is referred to as vesicular transport (Figure 2(b)) and serves as a transcellular means of BRB breakdown. This mechanism appears to be the predominant means for BRB compromise associated with VEGF-A-induced hyperpermeability in monkeys and in DR in humans, rats, and rabbits. In addition to causing the opening of interendothelial cell tight junctions in the retina, adenosine, PGE1, IL-1b, TNFa, and VEGF also promote the formation of pinocytotic vesicles in RVE cells and the distribution of albumincontaining intraendothelial vesicles across the entire RVE cell and at both the luminal and abluminal surfaces suggests that active vesicular transport is occurring. Although infrequently seen, the vesiculo-vacuolar organelle, which is associated with VEGF in the vascular endothelium of tumors, was also evident in the RVE of VEGF-treated rabbits, but not monkeys, and is likely to play a role in VEGF-mediated vascular permeability. The effect of these mediators on the outer BRB is less clear.

Role of Inflammation Inflammation has been associated with BRB breakdown in DR, choroidal neovascularization (CNV) associated with age-related macular degeneration, aging, ocular inflammatory disease, and the administration of pro-inflammatory molecules. The increased adhesion of leukocytes to


(a)

(b)

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endothelial cells in the retina is associated with increased expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD18, and other adhesion molecules, which are upregulated by VEGF and other pro-inflammatory molecules in DR and other ocular disorders, and appear to be regulated, at least in part, by protein kinase C (PKC). Diabetic CD18 and ICAM-1 knockout mice have significantly fewer adherent leukocytes than diabetic mice with normal CD18 and ICAM-1 and the decreased leukostasis is associated with fewer damaged endothelial cells and reduced BRB breakdown, supporting the role of adhesion molecules in increased inflammation and the correlation of an inflammatory response with endothelial cell damage and permeability. It is not clear whether the same molecules that facilitate leukostasis also mediate BRB breakdown or if this is attributable to molecules secreted by the recruited leukocytes, or both, but there appears to be a direct correlation between increased leukostasis and vascular permeability in the retina and pro-inflammatory molecules, such as TNFa and IL-1b, are among the most potent inducers of BRB breakdown. Leukocyte adhesion to the diabetic vascular endothelium can promote endothelial apoptosis and inhibition of leukocyte adhesion to the retinal vessels can not only prevent endothelial degeneration, but also reduce the diabetes-associated loss of pericytes, which support the vascular endothelium and help to confer BRB integrity. Inflammation can also alter the distribution of astrocytes and their ensheathment of retinal vessels, leading to alterations in BRB integrity. Leukocytes have also been shown to cause a downregulation and redistribution of tight junctional proteins, which leads to a disruption of tight junctions and a transient breakdown of the BRB during retinal inflammation.

Molecular Mechanisms

(c) Figure 2 (a) Immunocytochemical staining for endogenous albumin shows the formation of pinocytotic vesicles (arrows) on the luminal surface of vascular endothelial cells in a 7 month galactosemic rat. Immunoreactive albumin is contained within the formed vesicles (top right). (b) Immunocytochemical staining for albumin demonstrates albumin filled vesicles throughout the cytoplasm of the vascular endothelial cells of a rabbit. The presence of these vesicles and the positive staining for albumin in the extracellular matrix (left) suggests that vesicles are actively transporting serum proteins across the endothelium and extruding their contents at the abluminal surface as a means of transcellular BRB breakdown. (c) Immunocytochemical staining for albumin along the entire length of the interendothelial cell junction (arrow) and in the basal lamina indicates that there is vascular leakage through the junction.

The induction of BRB breakdown is a complex process that is mediated, not by a single factor, but by the interaction of multiple factors operating through different receptors and signaling pathways. The list of molecules that have been identified as playing a role in BRB breakdown, which is by no means all-inclusive, includes VEGF, hypoxia-indicible factors-1 and -2 (HIF-1 and -2), placental growth factor (PlGF), TNFa, IL-1b, platelet-activating factor, adenosine, histamine, prostaglandins (PGE1, PGE2, and PGF2a), platelet-derived growth factors A and B (PDGF-A and -B), insulin-like growth factor-1 (IGF-1), ICAM-1, VCAM-1, P-selectin, and E-selectin. The key will be to determine what the initiating event is and which events are parts of the resulting cascade. By targeting the appropriate molecules, subsequent events leading to BRB failure may be blocked. The various isoforms of VEGF and PlGF are members of the VEGF family. VEGF-A, a potent inducer of vascular

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permeability, binds to both VEGF type-1 (fms-like tyrosine kinase-1 or Flt-1) and type-2 (kinase insert domaincontaining receptor, referred to as KDR in humans or Flk-1 in other species) receptors (VEGFR1 and VEGFR2), whereas PlGF binds only to VEGFR1, so comparing their activities may be a means of dissecting the respective roles of the VEGF receptor isoforms, since both receptors have been implicated in BRB breakdown. VEGFR1-mediated signaling appears to operate primarily through p38 MAPK (mitogen-activated protein kinase), while VEGFR2 signaling may be mediated through RAS, phosphoinositide 3-kinases (PI3K)/Akt, or phospholipase C (PLC)g, but the interaction of VEGFR1 and VEGFR2 is complex and much remains to be learned about this interaction. Both receptors are associated with vascular permeability and angiogenesis, but in some circumstances, VEGFR1 can act as a negative regulator for VEGFR2. VEGF is a key molecule in promoting increased retinal vascular permeability. This activity may be mediated, at least in part, by an upregulation of ICAM-1, E-selectin, and P-selectin as a means of facilitating its pro-inflammatory activity. VEGF-induced permeability showed a biphasic pattern with a rapid and transient phase followed by a delayed and sustained phase, the latter of which was blocked by antibodies to urokinase plasminogen activator or its receptor. VEGF receptor kinase inhibitors can suppress VEGF-mediated BRB breakdown, but this strategy shows that TNFa, IL-1b, and IGF-1 do not induce BRB leakage through an induction of VEGF, indicating that these mediators operate through distinct pathways that may also be targeted. Endothelial nitric oxide synthase activation and NO formation also appear to be implicated in VEGF-mediated vascular permeability, probably through activation of the serine/threonine protein kinase AKT/ PKB, which can lead to an increase in nitric oxide production and ICAM-1 upregulation. Deletion of the hypoxia response element of the Veg f promoter also suppresses BRB breakdown in oxygen-induced ischemic retinopathy, demonstrating that HIF-induced VEGF is critical in this process. TNFa and IL-1b are upregulated in DR and other ischemic retinopathies, as well as ocular inflammatory disease, and both molecules are associated with increased leukostasis and BRB breakdown. IL-1b has been shown to accelerate apoptosis of retinal capillary endothelial cells through activation of nuclear factor kappa light-chain enhancer of activated B cells and this is exacerbated in high glucose. IL-1b can also stimulate the production of reactive oxygen species, which in turn can induce the release of additional cytokines. Aspirin and etanercept are inhibitors of TNFa and each can reduce ICAM-1 levels, diabetes-related leukostasis, and BRB breakdown in diabetic rats without altering VEGF levels, showing that TNFa is involved in this process and that it operates through a distinct pathway from VEGF.

These data show that there are a number of potential target molecules for inhibitors to suppress BRB breakdown. The challenge will be to identify the best target or targets and develop the most effective therapeutic strategy.

Assessing BRB Breakdown A variety of methods exist for the quantitative and qualitative assessment of the BRB, but each method has its particular limitations and sensitivity, so the choice of methods will largely depend on whether quantitative or qualitative data are desired and on the nature of the tissue being evaluated, whether it be fixed tissue, patients in a clinical setting, or experimental animal models. Since no single method can provide a quantitative assessment with precise localization of the site of BRB breakdown, multiple approaches may be necessary to provide an overall perspective. In addition, some methods can produce precise data in experimental models or on tissue specimens, but are not appropriate for use in the clinic. To identify and compare factors that cause BRB breakdown and to evaluate the efficacy of new treatments designed to prevent or reduce macular edema, a reliable quantitative assay for assessing BRB function is essential. The most widely used protocols for the quantitative assessment of BRB breakdown utilize Evans blue or 3H-mannitol as tracers. With the Evans blue assay, the extracted dye is quantified in the retina following intravenous injection of the dye and subsequent perfusion with saline. A spectrophotometer, set at 620 nm, is used to quantify the leakage of dye into the retina. With the 3H-mannitol assay, a scintillation counter is used to determine the CPM/mg tissue, 1 h after an intraperitoneal injection of the tracer, and the data are expressed as a ratio of retina/lung or retina/kidney. Since the lung and kidney do not have a blood–tissue barrier, the ratio corrects for any variation in the amount of isotope injected or absorbed. These methods have been used to assess the BRB in several models of ocular disease and to determine the effect of various factors, agents, and genetic manipulations on the integrity of the BRB. Thus, these methods have been useful in identifying factors that initiate BRB compromise and for determining the relative efficacy of various agents at preventing or reducing BRB failure. Both methods produce highly reproducible results in an experimental setting, with the 3H-mannitol assay possibly being somewhat more sensitive due to the lower molecular weight of mannitol than Evans blue dye, but neither is applicable to the clinic. Vitreous fluorophotometry (VFP) is a more appropriate means of assessing BRB failure in a clinical setting. Although these methods can provide a quantitative assessment of the BRB, they cannot localize the site of leakage or provide any insight into the


mechanism, so alternative techniques are required to provide this information. Fluorescein angiography has been used extensively in the clinic to visualize BRB breakdown, but it does not allow resolution at the cellular level. Magnetic resonance imaging (MRI) enhanced by the paramagnetic contrast agent gandoliniumdiethylene-triaminetetraacetic acid has been used to localize and quantify BRB breakdown in living animals. MRI is not subject to the optical limitations of VFP and allows the investigator to distinguish between inner and outer BRB failure in the rabbit. Its resolution is not as great as that resulting from microscopic evaluation of fixed tissue, but MRI allows in vivo analysis, thus enabling the investigator to monitor progressive changes in BRB integrity within the same animal. The use of exogenous tracer substances can provide a higher resolution, but several limitations are associated with their use. The use of tracers is impractical for clinical studies, the introduction of exogenous material may alter BRB integrity, and retrospective studies cannot be done on archival tissues. The immunolocalization of endogenous albumin (Figure 3) or IgG can circumvent most of these limitations and offers the following advantages for BRB assessment. The technique can be used with fixed surgical, autopsy, or archival specimens, no exogenous substance is introduced, and it can be used at the light and electron microscopic (EM) levels. Although, by nature, this is not a quantitative method, it can show the location and extent of BRB breakdown and, if used at the EM level, it can demonstrate the means by which serum proteins are extravasated from the retinal vessels or may transverse the RPE layer. This technique has been used to assess the BRB in a variety of human and experimental ocular disorders, including DR, retinitis pigmentosa, vascular occlusive disease, neoplastic disease, ocular inflammation or infection, and other diseases that develop macular edema, but for which pathological defects do not reveal a cause for BRB breakdown. EM immunocytochemical staining for albumin reveals that BRB breakdown can occur by the opening of tight junctions between RVE or RPE cells (Figure 2(c)), by an upregulation of trans-endothelial vesicular transport (Figures 2(a) and 2(b)), or by increased surface membrane permeability of RVE or RPE cells resulting from degenerative changes associated with the disease process. It has also provided insights into how various factors, such as VEGF, TNFa, IL-1b, prostaglandins, adenosine, and others promote BRB breakdown. VEGF transiently opens some tight junctions and some leakage through the interendothelial cell tight junctions is induced by VEGF, but electron microscopy has revealed that the predominant mechanism for VEGF-A-induced hyperpermeability of the RVE in monkeys and diabetes-related BRB breakdown in humans, rabbits, and rats is an upregulation of pinocytotic vesicular transport.

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(a)

(b)

(c) Figure 3 (a) In a normal mouse, immunohistochemical staining for albumin shows that, within the retina, albumin is confined to the vessels, indicating an intact BRB, but diffuse staining is demonstrated in the choroid (bottom) due to the fenestrated vessels and the absence of a blood–tissue barrier. (b) In a mouse infected with coronavirus, vascular leakage is demonstrated from a retinal vessel by immunohistochemical staining for albumin. (c) Immunohistochemical staining (red) shows that albumin has leaked from retinal vessels in a VEGF transgenic mouse. Vinores, S. A., et al. (2001). Blood–retinal barrier breakdown in experimental coronavirus retinopathy: Association with viral antigen, inflammation, and VEGF in sensitive and resistant strains. Journal of Neuroimmunology 119: 175–182, with permission from Elsevier.

Inhibiting BRB Breakdown A variety of therapeutic approaches have shown success at inhibiting BRB breakdown, but generally not preventing

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it. Most of the agents currently in clinical trials target inflammatory processes or VEGF. Antibodies to key molecules, such as VEGF, PlGF, or TNFa, have been effective at suppressing BRB breakdown, as have inhibitors of these molecules. Drugs that block histamine receptors also reduce retinal vascular leakage in diabetic rats and humans. Bevacizumab (avastin), an anti-VEGF IgG1 antibody, Ranibizumab (lucentis), the Fab fragment of a humanized anti-VEGF antibody, Pegaptanib sodium (macugen), a VEGF aptamer, and VEGF trap, in which the binding domains of VEGFR1 and VEGFR2 are combined with the Fc portion of IgG to neutralize all VEGF family members, have all shown varying degrees of success in clinical trials for reducing macular edema by targeting VEGF. Corticosteroids inhibit BRB breakdown, but it is not clear whether this activity is mediated by their anti-inflammatory effect, which occurs, at least in part, through a downregulation of ICAM-1, their inhibition of VEGF expression, their induction of occludin and ZO-1 expression, their reversal of occludin phosphorylation, or a combination of these activities. Even though steroids may improve visual acuity, they carry a high risk of cataracts and glaucoma. The involvement of PKC in vascular permeability has been established and a PKC activator can promote BRB breakdown. PKC inhibitors can reduce retinal vascular permeability, particularly that mediated by VEGF or prostaglandins, but generalized inhibition of PKC is likely to have serious systemic consequences. A PKCb inhibitor (LY333531) was also effective at suppressing retinal vascular permeability and may have fewer complications.

Prospects for the Future As more studies are conducted, the complexity of BRB breakdown leading to macular edema becomes increasingly apparent. This process is not attributable to a single factor or event, but the interaction of an undetermined number of initiating events that generates a cascade of subsequent events, ultimately leading to BRB failure. Since this is a multifactorial process, a multifaceted or pleiotropic approach that restores the homeostatic balance is more likely to suppress BRB breakdown than targeting a single pathway with an inhibitor. That would explain why the currently used monotherapies may lead to a reduction of macular edema and improved visual acuity, but generally not a total resolution of the disorder. To develop more effective therapeutic strategies, a better understanding of the basic mechanisms in the pathogenesis of BRB breakdown is imperative. In addition, the frequent injections, high cost, and the occasional side effects associated with current therapeutic approaches emphasize the need for more effective treatment that is less invasive, less costly, and has little or no side effects.

Conclusions BRB breakdown, leading to macular edema, occurs in a number of ocular disorders and can be due to structural changes or soluble mediators. Alteration in the content, distribution, or phosphorylation of junctional proteins can result in vascular leakage through the tight junctions. BRB breakdown can also result from an upregulation of transendothelial vesicular transport, which has been shown to be a major contributor to BRB failure caused by several mediators and in ocular disease models. Many of the same mediators can simultaneously promote opening of the tight junctions and upregulation of vesicular transport. Degenerative or structural changes to the RPE or RVE cells or to the pericytes and perivascular astrocytes that regulate the inner BRB can also lead to BRB breakdown. Inflammation promotes BRB breakdown, so the use of anti-inflammatory agents may be beneficial. BRB breakdown is not due to a single factor, but is a complex process involving multiple factors, receptors, and signaling pathways. Information on the molecular mechanisms is being revealed, but much remains to be learned. The complexity of the pathogenesis of BRB breakdown makes it likely that the greatest chance for success in preventing macular edema would be in targeting multiple molecules or pathways and a sensitive method for assessing the integrity of the BRB is necessary to monitor the efficacy of different therapeutic strategies.

Acknowledgment Dr. Vinores is supported by grant R01EY017164 from the National Eye Institute, National Institutes of Health. See also: Blood–Retinal Barrier; Breakdown of the RPE Blood–Retinal Barrier; Macular Edema; RPE Barrier; Retinal Vasculopathies: Diabetic Retinopathy; Retinopathy of Prematurity; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration.

Further Reading Antonetti, D. A., Lieth, E., Barber, A. J., and Gardner, T. W. (1999). Molecular mechanisms of vascular permeability in diabetic retinopathy. Seminars in Ophthalmology 14: 240–248. Erickson, K. K., Sundstrom, J. M., and Antonetti, D. A. (2007). Vascular permeability in ocular disease and the role of tight junctions. Angiogenesis 10: 103–117. Gardner, T. W. and Antonetti, D. A. (2008). Novel potential mechanisms for diabetic macular edema: leveraging new investigational approaches. Current Diabetes Reports 8: 263–269. Gardner, T. W., Antonetti, D. A., Barber, A. J., et al. (2002). Diabetic retinopathy: More than meets the eye. Survey of Ophthalmology 47(supplement 2): S253–S262. Hofman, P., Blaauwgeers, H. G. T., Tolentino, M. J., et al. (2000). VEGF-A induced hyperpermeability of blood–retinal barrier

Breakdown of the Blood–Retinal Barrier endothelium in vivo is predominantly associated with pinocytic vesicular transport and not with formation of fenestrations. Current Eye Research 21: 637–645. Joussen, A. M., Poulaki, V., Le, M. L., Koizumi, K., et al. (2004). A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB Journal 18: 1450–1452. Leal, E. C., Santiago, A. R., and Ambrosio, A. F. (2005). Old and new drug targets in diabetic retinopathy: From biochemical changes to inflammation and neurodegeneration. Current Drug Targets – CNS and Neurological Disorders 4: 421–434. Luna, J. D., Chan, C.-C., Derevjanik, N. L., et al. (1997). Blood–retinal barrier (BRB) breakdown in experimental autoimmune uveoretinitis: Comparison with vascular endothelial growth factor, tumor necrosis factor a, and interleukin-1b-mediated breakdown. Journal of Neuroscience Research 49: 268–280. Rizzolo, L. J. (2003). Development and role of tight junctions in the retinal pigment epithelium. International Review of Cytology 258: 195–234. Saishin, Y., Saishin, Y., Takahashi, K., et al. (2003). Inhibition of protein kinase C decreases prostaglandin-induced breakdown of

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the blood–retinal barrier. Journal of Cellular Physiology 195: 210–219. Vinores, S. A. (1995). Assessment of blood–retinal barrier integrity. Histology and Histopathology 10: 141–154. Vinores, S. A. (2007). Anti-VEGF therapy for ocular vascular diseases. In: Maragoudakis, M. E. and Papadimitriou, E. (eds.) Angiogenesis: Basic Science and Clinical Applications, pp. 467–482. Kerala, India: Transworld Research Network. Vinores, S. A., Derevjanik, N. L., Mahlow, J., Berkowitz, B. A., and Wilson, C. A. (1998). Electron microscopic evidence for the mechanism of blood–retinal barrier breakdown in diabetic rabbits: Comparison with magnetic resonance imaging. Pathology Research and Practice 194: 497–505. Vinores, S. A., Derevjanik, N. L., Ozaki, H., Okamoto, N., and Campochiaro, P. A. (1999). Cellular mechanisms of blood–retinal barrier dysfunction in macular edema. Documenta Ophthalmologica 97: 217–228. Xu, Q., Quam, T., and Adamis, A. P. (2001). Sensitive blood–retinal barrier breakdown quantitation using Evans blue. Investigative Ophthalmology and Visual Science 42: 789–794.

Breakdown of the RPE Blood–Retinal Barrier M E Hartnett, Moran Eye Center, University of Utah, Salt Lake City, UT, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Adherens junction – Cell–cell junction having transmembrane proteins called cadherins that connect to cadherins of epithelial cells through extracellular domains and to anchor proteins, known as catenins, through intracellular tails. Anchor proteins also bind to a continuous belt of actin filaments along cytoplasmic side of the cell plasma membrane, ultimately holding neighboring cells together. Focal adhesions – Cell–matrix junctions having transmembrane proteins called integrins that connect cells to extracellular matrix and also to the actin cytoskeleton. Transmembrane proteins can trigger signaling within and between adjacent cells. Inner blood-retinal barrier – It regulates the transport of fluid, ions, and metabolites between the neurosensory retina and the retinal vasculature. Na,K,ATPase – Enzyme providing active transport mechanism in the retinal pigment epithelial and other cells. By catalyzing an ATP-dependent transport of three Na+ ions out and two K+ ions into the cell, a transmembrane sodium gradient is created (necessary for other Na+ coupled transport systems) and an osmotic gradient (drives water toward the choroidal or basal side of the RPE and away from the subretinal space). Outer blood-retinal barrier – It regulates the transport of fluid, ions, and metabolites between the neurosensory retina and the choroidal vasculature. Tight junction – A cell–cell adhesion usually at the apical lateral aspects of polarized endothelia but can be present in other cells consisting of a number of proteins, including ZO-1, -2, -3, transmembrane protein, occludin, and claudins. The tight junction regulates paracellular permeability between adjacent cells and protein and lipid movement between apical and basal compartments of the cell.

Introduction The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier (BRB), which regulates the transport

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of fluid, ions, and metabolites between the neurosensory retina and the choroidal vasculature. The inner BRB is formed by retinal endothelial cells (RECs), which regulate the transport of fluid, ions, and metabolites between the neurosensory retina and the retinal vasculature. Together, the RPE and RECs collectively form the BRB that serves to exclude blood-borne substances from the neurosensory retina and regulates the ionic and metabolic gradients required for normal retinal function. The RPE barrier structures and functions will be broadly described below and for further detail, the reader is directed to other articles in this encyclopedia.

RPE Barrier Structure The RPE is a polarized monolayer located deep to the neurosensory retina with its apical processes adjacent to the photoreceptors and its basal aspect on Bruch’s membrane, a semipermeable collagen and elastin sandwich that separates the choroid from the RPE and neurosensory retina (Figure 1). The RPE helps to maintain the outer BRB through tight junctions that are located at the apical–lateral junctions of adjacent cells. Much of what is known about tight junctions was learned from studies of epithelial cells other than the RPE. Tight junction-associated proteins include ZO-1, -2, -3, and several transmembrane proteins, including junction adhesion molecules and occludin, which bind ZO-1 and -2 that then bind the cytoskeleton. Occludin also has extracellular domains, which bind to those of adjacent cells to form the tight junctions. Claudin proteins are also important in this structure and have tissue and regional specificity. There are a number of other proteins that are important in the development and function of the tight junction. Besides tight junctions, there are other points of contact between cells or cells and extracellular matrix. Adherens junctions have transmembrane proteins called cadherins that connect to cadherins of adjacent RPE cells through their extracellular domains and to anchor proteins, known as catenins, through intracellular tails. The anchor proteins also bind to a continuous belt of actin filaments along the cytoplasmic side of the cell plasma membrane. The resulting structure holds neighboring cells together. Focal adhesions have transmembrane proteins called integrins that connect cells to extracellular matrix and also to the actin cytoskeleton. Besides providing structure and connections, transmembrane proteins can trigger signaling pathways within and between adjacent cells.


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Light

Direction of fluid flow Vitreous Neurosensory retina

Photoreceptors Subretinal space

Sub - RPE space Bruch’s Bruch’smembrane membrane

Choroid

- Tight junction

Figure 1 Artist diagram of RPE monolayer with tight junctions at the apical lateral junctions (see inset of RPE/Bruch’s membrane/ choroid in lower left corner) and in relation to the layers within the macula (shown as a diagram of cross-section of macula on right) and eye. Light is shown coming from above. Overall direction of fluid through the eye is represented by arrow on left.

In addition, a number of active processes regulate the flow of ions, glucose, and amino acids across the RPE. For example, ion fluxes and signaling processes are synchronized between cells through gap junctions, such as connexin 43.

RPE Barrier Functions The RPE tight junctions regulate the passage of molecules across the paracellular pathway and this regulation depends on the selectivity and permeability of the tight junctions. The transcellular pathway requires pumps, such as Na,K-ATPase, channels, transporters, and metabolic modification to regulate transport across the cells of the monolayer. Breakdown of the RPE barrier may include disruption of the tight junction structure with reduced barrier properties of the RPE. To measure RPE barrier function in a living human, RPE barrier dysfunction is determined clinically by leakage of sodium fluorescein across the RPE seen on stereoscopic images of fluorescein angiograms following an intravenous injection of sodium fluorescein into the bloodstream. However, the distinction between dysfunction of the inner from the outer BRB is difficult in human diseases. This may be because broad BRB breakdown occurs from similar pathophysiologic mechanisms or because of limited resolution in optical imaging. Therefore, techniques are used in vitro to study the RPE barrier properties or in vivo to selectively poison the RPE and measure fluorescein leakage into the vitreous or eye in animal models (see below). When considering diseases associated with RPE barrier

breakdown, it is useful first to understand the fluid flow and forces within the eye.

Transport and Fluid Flow within the RPE and Eye The vitreous gel is within the eye adjacent to the inner retina, the ciliary body, zonule, and posterior lens capsule. The neurosensory retina includes retinal layers extending from the inner limiting membrane adjacent to the vitreous to the photoreceptors above the RPE. The subretinal space is the potential space between the photoreceptor outer segments and the apical processes of the RPE. The sub-RPE space is a potential space beneath the RPE basal infoldings and Bruch’s membrane. The choroid is deep to Bruch’s membrane (Figure 1). Historically, fluid tends to accumulate more easily in the subretinal than the sub-RPE spaces. Passive and active transport mechanisms within the RPE and ocular forces within the eye are important when considering the pathophysiology of disease (Figure 1). These mechanisms serve to maintain a directional flow from inside the eye, that is, vitreous, to the choroid. Passive transport mechanisms include intraocular pressure and the osmotic pressure from the choroid. However, studies have shown that the RPE reduces directional fluid movement from the subretinal space to the choroid. When the RPE barrier is damaged in animal models (such as through chemicals like sodium iodate), fluid egress from the subretinal space to the choroid is faster than in conditions in

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which the RPE has not been damaged. Therefore, in conditions in which there is increased fluid in the subretinal space and presumed RPE BRB breakdown, there is believed to be another pathophysiologic abnormality, such as increased perfusion pressure from the choroid from a vascular tumor, choroidal neovascular membrane, or hyperpermeable choroidal vessels, or reduced ability to accommodate extra fluid, such as with choroidal ischemia or inflammation.

RPE Na,K-ATPase pump Since the RPE restricts fluid movement from within the eye toward the choroid, it is believed that one or more active transport mechanism is necessary to prevent fluid from accumulating in the subretinal space. The Na, K-ATPase pump is an important active transport mechanism in the RPE. This enzyme is located at the apical surface of the RPE and it is believed that the asymmetric positioning of this enzyme with other transporters, such as chloride channels, is important in creating directional fluid flow. The enzyme catalyzes an adenosine triphosphate (ATP)-dependent transport of three Naþ ions out and two Kþ ions into the cell. This transmembrane sodium gradient is necessary for functions of other Na+ coupled transport systems and creates an osmotic gradient, which helps drive water toward the choroidal or basal side of the RPE and away from the subretinal space. It has been shown in vitro using human RPE grown in polarized monolayers that Na,K-ATPase function is necessary for the structural integrity of tight junctions and their function. Inhibition of Na,K-ATPase using a K+ free media or with ouabain led to increased ionic and nonionic permeability in association with a reduction in the number of contact points in the tight junctions as seen by transmission electron microscopy. Thus, Na,K-ATPase function is necessary to reduce paracellular permeability.

Methods to Assess RPE Barrier Structure and Function

Actin (red) Occludin (green)

y x Figure 2 Immunolocalization of occludin (green), actin (red), and Hoechst nuclear stain (blue) in human fetal RPE cultured for >1 month.

important in the RPE tight junction function, and the reader is directed to other articles of this encyclopedia. When viewed with electron microscopy, it is noted that functional tight junctions have multiple contact points between cells, and that when functional assays show reduced tightness of the barrier, the number of contact points was reduced. The protein components of the tight junction and phosphorylation or activation of certain component, such as occludin, can be determined by immunoprecipitation and/or Western blot analysis. Barrier Function Electrophysiologic methods are used to measure barrier function. Transepithial electrical potential (TEP) measures the ion gradient across the monolayer generated by energy-driven ion pumps that regulate passage across the cells. Transepithelial electrical resistance (TER) measures resistance of substances through the paracellular space mainly through the fine structure of the tight junction structure. Permeability to nonionic compounds such as inulin or mannitol can be measured.

Tight Junction Structure

Assays That Measure Barrier Properties of RPE

ZO-1 staining is useful to define the hexagonal architecture of the RPE monolayer in vitro, but does not indicate that paracellular permeability is reduced and the barrier is tight. RPE is cultured in specific media to attain tight properties, and the tightness of the monolayer often correlates with the duration in culture and the immunolocalization of occludin to the cell junctions (Figure 2). However, lack of occludin can be associated with a functional tight junction and from this it has been shown that other proteins, including the claudins, are important. Therefore, ZO-1, occludin, and claudins, as well as other proteins, are

In vitro. Measures of barrier function can vary depending on the RPE cell type, the components making up the media, and the duration of time in culture. Although a TER of 100 O cm2 is sufficient to exclude movement from the apical and basal compartments of a monolayer of RPE, many investigators strive to obtain culture conditions in which the RPE monolayer develops a TER of 500 O cm2. However, in RPE-choroid explants, a TER of 200 O cm2 has been reported. Therefore, it remains unclear whether a higher TER than that in vivo indicates a physiologic condition or one in which the structure of the tight junction is


altered by an abnormal microenvironment. It is important in studies that compare the effects of agents on a barrier properties that the cell culture conditions be standardized and replicable. Ex vivo. Explants of the RPE and choroid have been used in modified Ussing chambers to study the movement of compounds and to determine the barrier properties. In vivo. Assessing the BRB function is performed with several assays that measure the amount of a substance leaked into the retina. In animal studies, quantitative assays include comparing blood concentration of Evans’ blue dye to that measured within the retina after administration of a known concentration, or measuring the extravasation of albumin or inulin into the retina. In addition, qualitative and semi-quantitative assessment (i.e., measuring the area of fluorescein leakage) of fluorescein angiograms can be performed. However, it is difficult to distinguish leakage from the inner versus the outer BRB using these methods. Clinical assays. Since direct measure of specific functions of the outer BRB is not possible in humans in vivo, in vitro testing and fluorescein angiography or vitreous fluorophotometry are performed. Vitreous fluorophotometry measures fluorescein leakage into the vitreous by measuring fluorescence. Fluorescein angiography. When sodium fluorescein (376 Da) dye is activated with blue light (490 nm), it emits fluorescence in the green wavelength (520–530 nm). In a fluorescein angiogram, the dye is injected as a bolus (usually 500 mg/5 mL over 7 s) into the antecubital vein, and images of the fundus are obtained digitally or on film over time. At 10 s following injection, a choroidal flush from filling of the choroid that is partly blocked by the RPE melanin occurs. Nearly simultaneously, filling of the retinal arterioles occurs. Subsequently, the veins fill (Figure 3(a)). Areas of abnormal hyperfluorescence can occur from loss of the RPE melanin or cell (known as window defects because of permitted fluorescence from the underlying choroidal vasculature) or from filling of abnormal vessels or pooling or leakage into spaces where fluid is not naturally present. Hypofluorescence can occur from blockage by pigment or blood, or from nonperfusion within any of the vascular structures in the eye (choroid, retinal vasculature, vessels of the optic nerve). Stereoangiography is helpful to determine the relative position within the retina where abnormal fluorescence was seen. Currently, clinicians gain additional data from optical coherence tomography (OCT). OCT. Light is directed through a dilated pupil and reflects off different optical interfaces corresponding to layers within the retina (Figure 3(b)). Disorganization of the retinal architecture, invasion of tissue, and fluid-like cysts can occur within layers and be used in conjunction with the fluorescein angiogram to diagnose and manage several diseases (see below). Spectral domain OCT has provided better image quality than time domain OCT.

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00:30 FA

(a)

(b) Figure 3 (a) Normal mid-phase fluorescein angiogram showing optic nerve at left (slightly out of focus) and filling of retinal arterioles and veins. (b) Spectral domain optical coherence tomogram (OCT) of adult macula showing reflective layers of retina (red – highly reflective) and natural foveal depression.

Clinical Conditions Associated with Breakdown of the RPE Barrier Dysfunction of the BRB is believed to disrupt the normal health and function of the neurosensory retina in several diseases and lead to reduced visual function. RPE barrier dysfunction is diagnosed usually by the presence of hyperfluorescence in the deeper retina, unlike in normal, as determined by fluorescein angiography (cf. Figures 3(a) and 4(a)). Occasionally, vitreous fluorophotometery is used, but this method does not distinguish leakage from the inner or outer BRB. Leakage of fluorescein from the RPE can appear as mild hyperfluorescence and staining, but may lead to intraretinal edema, seen as cystoid macular edema, shown by hyperfluorescence in early frames at the level of the RPE with petalloid hyperfluorescent within the retina in late frames (Figure 4(b)). However, most causes of CME are believed to involve several factors including leakage from the inner BRB and Muëller cell dysfunction.

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03:02

(a) 13

00:29 FA

(b)

Early frame FA

05:35 FA

Late frame FA

Figure 4 (a) Deep hyperfluorescence in macula of left eye seen on recirculation phase of fluorescein angiogram demonstrating subtle leakage at the level of the RPE barrier. (b) Early angiogram shows minimal hyperfluorescence surrounding the avascular zone of the macula, but in the recirculation frame of the angiogram, hyperfluorescence takes on a petalloid appearance in a patient with cystoid macular edema.

Inflammation/Infection Inflammation reduces the BRB function and affects both the inner BRB of the RECs and the outer BRB of the RPE. Clinically, any disease that has an infectious or inflammatory component can compromise the BRB. Viral infections (such as human immunodeficiency virus (HIV), cytomegalovirus (CMV), Herpes) and bacteria (such as Bacillus cereus) have been shown to disrupt the tight junctions of the RPE in vitro. Factors such as tumor necrosis factor (TNF)-a, interleukins (IL)-6, -8, -1 disrupt tight junction barrier functions in vitro or in vivo. The RPE has been shown to have chemokine receptors, such as CXCR4, which can be activated by its ligand, stromalderived factor (SDF-1), to release chemokines such as monocyte chemotactic protein-1 (MCP-1) and IL-8. Chemokines, like MCP-1, can attract leukocytes. Contact of RPE with monocytes results in additional secretion of IL-8 and MCP-1 through several pathways involving mitogen-activated protein kinases. Such pathways may

amplify the effects of inflammation on BRB breakdown. As a model of human uveitis, experimental autoimmune uveoretinitis (EAU) is induced in animals by immunization with different proteins. For example, EAU can be induced in Lewis rats by immunization with S-antigen or in mice using peptides to interphotoreceptor retinoidbinding protein. A number of events occur in the EAU model and one is the movement of leukocytes into the retina. It has been shown that transient loss of ZO-1 occurs in RPE from IL-1 and MCP-1. (Transient loss of occludin-1 in RECs within venules was believed to play a role in permitting leukocyte diapedesis in the EAU model.) Inflammation in conditions such as Vogt Kayanagi Harada’s disease, or choroidal inflammatory conditions can reduce RPE barrier function in humans. Age-Related Macular Degeneration Age-related macular degeneration (AMD) is a leading cause of nonreversible blindness worldwide. Clinical


evidence of RPE BRB dysruption is seen in fluorescein angiograms showing hyperfluorescence from the choroidal vascular normally blocked by healthy RPE, but where loss of melanin or RPE cells has occurred, as in atrophic AMD (Figure 5). Hyperfluorescence can also occur from leaky blood vessels that appear to disrupt the RPE barrier and lead to fluid accumulation in neovascular AMD (Figure 6). OCT provides additional evidence in neovascular AMD as to the location of the fluid: beneath the RPE (sub-RPE), above it (subretinal), or within the retina itself (intraretinal) (Figure 7). Neovascular AMD occurs in about 10% of AMD cases but accounts for 90% of severe vision loss. 01:04

10 Figure 5 Presence of hyperfluorescence in late frame of fluorescein angiogram because of loss of RPE melanin, which naturally blocks fluorescence of underlying choroid.

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Most of the neovascular AMD begins as ill-defined hyperfluorescence and late leakage noted on fluorescein angiography and had been initially called occult choroidal neovascularization (CNV). Later, occult CNV was characterized histopathologically as type 1 CNV, indicating that CNV remained beneath the RPE (Figure 6). This is in contrast to classic or well-defined CNV, characterized histopathologically as type II CNV in which the CNV has entered the neurosensory retinal space (compare Figures 6 and 8). (This distinction is useful as a model but in reality is not so clear-cut, since clinically there are often mixed types.) However, studies have provided support that when vision declines from neovascular AMD, more than half of the time it is from the transition from occult to classic CNV. This prompts the hypothesis that contact of the ECs with the RPE or its matrix can trigger signaling pathways leading to the release of angiogenic or chemotactic growth factors and the migration of choroidal ECs and movement of fluid into the neurosensory retina. These events would appear to involve a breakdown in the RPE BRB properties. Many stimuli that are believed to be involved in the pathophysiology of AMD also reduce BRB properties as determined in in vitro assays described earlier. These include, for example, oxidative stress (can disorganize tight junction proteins including occludin), inflammation (can reduce TER and disrupt tight junctions), activation of complement factors (RPE possess complement 5a receptors and can be stimulated by complement 5a to release several cytokines which can then regulate leukocyte function during inflammation), and contact of RPE or its extracellular matrix with endothelial cells (reduce TER and increase permeability of RPE through release of soluble forms of vascular

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(c) Figure 7 (a) Time domain optical coherence tomogram from patient with normal macula showing good definition of macular layers and normal foveal contour. (b) Optical coherence tomogram from patient with neurosensory retinal choroidal neovascularization, showing disruption of ordered architecture of the retinal layers, increased hyperreflectance (red) in layer near RPE/Bruch’s membrane and intraretinal cysts, indicating intraretinal fluid (arrowhead). (c) Optical coherence tomogram showing subretinal fluid (dotted arrow – beneath the photoreceptors and above the RPE) and sub-RPE fluid (solid arrow – beneath the RPE and above Bruch’s membrane).

neovascularization that grows above the inner limiting membrane into the vitreous (Figure 9). However, fluorescein staining at the level of the RPE is seen on fluorescein angiograms of patients with diabetes. Also, hyperglycemia has been shown to impair the function of tight junctions of the RPE in vitro. Diabetic retinopathy also impairs vision through the development of macular edema, which occurs when there is fluid and solutes that leak from the vasculature into the neurosensory retina (Figure 10). It can occur through a breakdown of the inner and potentially outer BRBs. Besides the finding that hyperglycemia can reduce TER in cultured RPE, animal models of diabetic retinopathy have found that there is reduced Na,K-ATPase activity in the RPE. So, once retinal blood vessels leak fluid, lipids, and protein into the neurosensory retina, mechanisms to transport fluid and compounds out of the retina also appear to be impaired in the diabetic state. Inflammation has been shown to play a role in the pathophysiology of diabetic retinopathy. In animal models, leukostasis or adherence of white blood cells to retinal capillaries has been found and postulated to be a mechanism of later capillary nonperfusion and endothelial damage, which precede the development of proliferative diabetic retinopathy and macular edema. Furthermore, diabetes can also cause a choroidal vasculopathy associated with leukostasis, which may cause choroidal ischemia and later angiogenesis both of which can interfere with intrinsic ocular flow from the vitreous toward the choroid. Proliferative Vitreoretinopathy

endothelial growth factor (VEGF)). All these stimuli can lead to increased secretion of VEGF, which also increases permeability of blood vessels and RPE and choroidal endothelial migration, proliferation, and chemotaxis, all processes believed important in the development of neurosensory retinal choroidal neovascularization. Furthermore, RPE can release other cytokines that recruit leukocytes, including macrophages that can then release VEGF, or that interact with other processes important in the pathogenesis of AMD.

Proliferative vitreoretinopathy (PVR) occurs when cellular contractile membranes develop on the surface of the retina and contract it and pull open breaks in the retina, which can lead to complex retinal detachments. It is the most common cause of failed retinal detachment repair. Vitreous fluorophotometry readings in animal models of PVR show a breakdown in the BRB associated with released cells into the vitreous cavity. It is believed that RPE cells, serum, and other factors have access to the vitreous cavity and are responsible for further breakdown of the BRB and growth of preretinal membranes. The treatment for PVR, currently, is surgical, requiring vitrectomy and stripping of the membranes from the retina, and then methods to reattach the retina and create a permanent chorioretinal adhesion. However, ongoing research may provide medical means to prevent the formation of preretinal membranes.

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In diabetic retinopathy, the inner BRB is impaired and is most easily appreciated clinically on fluorescein angiography, as leakage from microaneurysms, dilated capillaries, intraretinal microvascular abnormalities, and

Several drugs, including thioridazine (Mellaril), thorazine, hydroxychloroquine, and chloroquine, have been shown to cause vision loss and toxicity with pigmentary changes. The exact effects on the RPE and BRB are unclear.


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Figure 9 (a) Color images of right eye of patient with diabetic retinopathy previously treated with laser (examples of pigmented laser spots shown by arrows). (b) Fluorescein angiogram of same eye showing intraretinal microvascular abnormalities (arrow) and areas of avascular retina with dilated capillaries, an irregular foveal avascular zone, and microaneurysms (arrowhead). Dotted arrow shows area of hyperfluorescence from leaking neovascularization likely growing above the inner limiting membrane.

Central Serous Retinopathy Central serous retinopathy (CSR) is a clinical disease often occurring in young to middle-aged individuals, although it can also manifest or recur and become chronic in later life. Symptoms include reduced vision or inability to focus, and clinical examination shows the presence of a neurosensory retinal detachment within the macula. Fluorescein angiography shows focal areas of RPE leaks (Figure 11) or diffuse RPE disturbances. In chronic CSR, RPE decompensation occurs (Figure 12) and can lead to chronic subretinal leakage and accumulation of subretinal

Figure 10 Example of intraretinal edema in a time domain optical coherence tomogram from a patient with diabetic macular edema. Note that the RPE reflective layer is intact in contrast to Figure 7(b) in which invasion of cells into the neurosensory retina has occurred in neovascular AMD.

and intraretinal fluid. Although cases of CSR usually resolve without permanent vision loss, recurrent or chronic CSR can lead to permanent loss of visual acuity. Even when a sole leak appears to be present, CSR is believed to be associated with broad RPE dysfunction, because if only one area of dysfunction were present, the Na,K-ATPases of surrounding healthy RPE would pump out fluid from the subretinal space.

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a type A personality and is believed to be related to elevated cortisol and epinephrine, which affect the autoregulation of the choroidal circulation. In early studies, adult Japanese monkeys that received multiple daily (>30) injections of intravenous adrenalin developed serous retinal detachments and leaking RPE spots by fluorescein angiography similar in appearance to that seen in CSR. An intramuscular injection of prednisolone led to the same findings on fluorescein angiography but required fewer doses of adrenalin.

Retinitis Pigmentosa

6 Figure 12 RPE decompensation showing broad area of hyperfluorescence in fluorescein angiogram from chronic longstanding central serous retinopathy in 60 year old male.

Angiograms using indocyanine green dye, which permits visualization of the choroidal vasculature, show that areas of choroidal hypoperfusion and later choroidal hyperpermeability are present in CSR. Although one might suspect inflammation to be a cause of the hyperpermeability, treatment with steroids can severely worsen CSR and should be avoided. Corticosteroids can affect the expression of adrenergic receptor genes and it is thought that this contributes to the overall effect of catecholamines on CSR. Some have postulated that the pathology may involve the adrenocorticotrophic hormone. Another unusual aspect of CSR is that treatment of a sole RPE leak on fluorescein angiography can hasten resolution of the serous detachment, even though it is believed that broad RPE dysfunction is present. Besides corticosteroids, hypertension also increases the risk. CSR has long been believed to be associated with breakdown in the BRB particularly of the RPE. The cause remains unknown but it is associated with increased stress and

Evidence of abnormalities in the localization of ZO-1, beta-catenin, and other associated adherens proteins in the rho/ mouse, a model of autosomal dominant retinitis pigmentosa, provides support for tight junction and adherens junction-associated protein modifications in retinitis pigmentosa. Furthermore, in retinitis pigmentosa, there is cystoid macular edema often associated with hyperfluorescence of the RPE cells on fluorescein angiography, suggesting a breakdown of the RPE barrier. Toxins such as sodium iodate selectively poison the RPE and have been used to test the role of the RPE BRB in animal models in which fluid had been injected into the subretinal space or for studies in PVR. Although the toxin is selective for RPE, it poisons the RPE and therefore affects all functions of the RPE, not just the tight junctions. Urethrane was used to test the BRB in earlier studies and was reported to lead to inhibition of intervesicular transport across endothelia and loss of RPE.

Growth Factors Besides the role of VEGF in reducing RPE barrier properties, other growth factors play a role. Insulin-like growth factor-1 (IGF-1) can induce VEGF-related RPE barrier breakdown. Also, hepatocyte growth factor (HGF) can lead to disassembly of tight and adherens junctions in


association with reduced barrier properties. HGF has also been shown to be increased in human PVR.

Studies to Increase the Function of the BRB Carbonic anhydrase inhibitors lead to acidification of the subretinal space, which, in turn, leads to an increase in chloride ion transport into the choroid, thus eliminating water from the subretinal space and retina and increasing the adhesiveness of the RPE. Carbonic anhydrase inhibitors have been associated with improved BRB function, based on reduced fluorescein leakage into the retina, in small clinical studies. However, larger clinical trials have shown a smaller benefit. See also: Breakdown of the Blood–Retinal Barrier; Photopic, Mesopic and Scotopic Vision and Changes in Visual Performance; Phototransduction in Limulus Photoreceptors.

Further Reading Alberts, B., Johnson, A., Lewis, J., et al. (2002). Cell junctions, cell adhesion, and the extracellular matrix. In: Alberts, B., Johnson, A., Lewis, J., et al. (eds.) Molecular Biology of the Cell, pp. 949–1009. New York: Garland Science. Bian, Z. M., Elner, S. G., Yoshida, A., and Elner, V. M. (2003). Human RPE-monocyte co-culture induces chemokine gene expression through activation of MAPK and NIK cascade. Experimental Eye Research 76: 573–583. Campbell, M., Humphries, M., Kennan, A., et al. (2006). Aberrant retinal tight junction and adherens junction protein expression in an animal model of autosomal dominant retinitis pigmentosa: The Rho(–/–) mouse. Experimental Eye Research 83: 484–492. Crane, I. J., Wallace, C. A., McKillop-Smith, S., and Forrester, J. V. (2000). CXCR4 receptor expression on human retinal pigment epithelial cells from the blood–retina barrier leads to chemokine

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secretion and migration in response to stromal cell-derived factor 1 alpha. Journal of Immunology 165: 4372–4378. Dibas, A. and Yorio, T. (2008). Regulation of transport in the RPE. In: Tombran-Tink, J. and Barnstable, C. (eds.) Ocular Transporters in Ophthalmic Diseases and Drug Delivery, 1st edn., pp. 157–184. Berlin: Springer/Humana Press. Fubuoka, Y., Strainic, M., and Medof, M. E. (2003). Differential cytokine expression of human retinal pigment epithelial cells in response to stimulation by C5a. Clinical and Experimental Immunology 131: 248–253. Hartnett, M. E., Lappas, A., Darland, D., et al. (2003). Retinal pigment epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism. Experimental Eye Research 77: 593–599. Hu, J. and Bok, D. (2001). A cell culture medium that supports the differentiation of human retinal pigment epipthelium into functionally polarized monolayers. Molecular Vision 7: 14–19. Jin, M., Barron, E., He, S., Ryan, S. J., and Hinton, D. R. (2002). Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor. Investigative Ophthalmology and Visual Science 43: 2782–2790. Lodish, H., Berk, A., Zipursky, S. L., et al. (2000). Transport across cell membranes. In: Molecular Cell Biology. Basingstokes: WH Freeman. Marmor, M. F. and Wolfensberger, T. J. (eds.) (1998). The Retinal Pigment Epithelium. New York: Oxford University Press. Penn, J. S., Madan, A., Caldwell, R. B., et al. (2008). Vascular endothelial growth factor in eye disease. Progress in Retina and Eye Research 27(4): 331–371. Rajasekaran, S. A., Hu, J., Gopal, J., et al. (2003). Na,K-ATPase inhibition alters tight junction structure and permeability in human retinal pigment epithelial cells. American Journal of Physiology – Cell Physiology 284: C1497–C1507. Rizzolo, L. J. (2007). Development and role of tight junctions in the retinal pigment epithelium. In: Jeon, K. W. (ed.) International Review of Cytology, a Survey of Cell Biology vol. 258, pp. 195–234. San Diego, CA: Elsevier. Sen, H. A., Robertson, T. J., Conway, B. P., and Campochiaro, P. A. (1988). The role of breakdown of the blood–retinal barrier in cellinjection models of proliferative vitreoretinopathy. Archives of Ophthalmology 106: 1291–1294. Xu, H., Dawson, R., Crane, I. J., and Liversidge, J. (2005). Leukocyte diapedesis in vivo induces transient loss of tight junction protein at the blood–retina barrier. Investigative Ophthalmology and Visual Science 46: 2487–2494. Yoshioka, H., Katsume, Y., and Akune, H. (1982). Experimental central serous chorioretinopathy in monkey eyes: Fluorescein angiographic findings. Ophthalmologica 185(3): 168–178.

Circadian Metabolism in the Chick Retina P M Iuvone, Emory University School of Medicine, Atlanta, GA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Basic helix–loop–helix-Per-ARNT-Sim (bHLHPAS) domain transcription factors – A family of transcription factors that heterodimerize and bind to E box enhancer elements in gene promoters. The family includes the clock gene products CLOCK, neuronal PAS domain protein 2 (NPAS2), and brain and muscle aryl hydrocarbon receptor nuclear translocator 1 (BMAL1), as well as the arylhydrocarbon nuclear receptor and the hypoxiainducible factors. Circadian rhythms – Changes in biological processes that occur on a daily basis; they are driven by autonomous circadian clocks; these rhythms provide selective advantage to organisms by allowing them to anticipate temporal changes in their environment. Clock-controlled genes – Genes that are regulated by circadian oscillators via clock gene transcription factors; the proteins encoded by clock-controlled genes are rhythmically expressed and generate rhythms of physiology. Clock genes – Genes that encode proteins that form the molecular basis of circadian oscillators; most clock gene proteins are transcription factors. Scotopic vision – Vision in dim light that is mediated by rod photoreceptors and rod bipolar cell pathways. Zeitgeber time – Time of day relative to the light–dark cycle; light onset corresponds to ZT0.

Introduction The chick retina has been used extensively to study ocular circadian rhythms. It displays particularly robust rhythms of clock gene expression, clock-controlled gene expression, and several biochemical and physiological clock outputs. In addition, embryonic chick retinal cells can be cultured, and they maintain light responsiveness and circadian rhythm generation in vitro. These cell cultures facilitate pharmacological and molecular studies of clock signaling pathways.

Clock Gene Expression The current model for the molecular basis of circadian clocks involves transcriptional–translational feedback loops

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that are comprised of highly conserved clock genes and the proteins that they encode (Figure 1). The clock gene proteins are characterized as positive and negative elements, based on their transcriptional activity. The positive elements include basic helix–loop–helix–PAS-domain (bHLH-PAS) transcription factors that heterodimerize and bind to circadian E box enhancer elements in promoters of other clock genes and clock-controlled genes. These positive elements include brain and muscle aryl hydrocarbon receptor nuclear translocator 1 (BMAL1; also called ARNTL, MOP3) and CLOCK. The BMAL1 heterodimerizes with CLOCK and the dimerized transcription factors stimulate the transcription of the genes encoding the negative elements, the cryptochromes (CRY1 and CRY2) and periods (PER1, PER2, and PER3). The CRY and PER proteins are imported into the nucleus and inhibit transactivation of their own promoters by BMAL1:CLOCK. In some clocks, neuronal PAS domain protein 2 (NPAS2, also called MOP4) can substitute for CLOCK and form active heterodimers with BMAL1. A second feedback loop involves E-box-mediated transcriptional activation of the orphan retinoic-acid-related receptor family genes, Rev-erba and Rora; the protein products of these genes contribute to the rhythmic regulation of Bmal1 gene transcription. These feedback loops, coupled with a variety of post translational modifications, generate daily rhythms of gene expression that ultimately generate physiological circadian rhythms. Most of the clock gene transcripts identified in mammalian circadian clocks have been identified in the chick retina and in cultured chick photoreceptors, and the regulation of their expression has been analyzed (Figures 2 and 3). Of the genes encoding the positive elements of the oscillator, Bmal1 and Npas2 transcripts show robust daily rhythms under light–dark cycles or in constant (24 h day1) darkness (Figure 2). These circadian rhythms peak near the time of subjective dusk (ZT12) in vivo and in vitro. In contrast, Clock mRNA appears to be constitutively expressed in dark–dark (DD). Transcripts encoding the negative elements, the cryptochrome and period proteins, also show rhythmic expression (Figure 3(a) and 3(b)). The Cry1 mRNA peaks in the middle of the day (ZT8) in vivo and in photoreceptor cultures. In vivo, the most robust rhythms of Cry1 mRNA expression are observed in the ganglion cell and photoreceptor cell layers. The Cry2 mRNA is also expressed in retina, but analyses of whole retina mRNA suggest that it is not rhythmically expressed. The Per2 mRNA expression is maximal in the early morning, while Per3 transcript level appears maximal late in the night.

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Figure 1 Circadian clockwork mechanism. Circadian clocks in a wide range of organisms are composed of two interdependent transcription–translation feedback loops that drive the periodic rhythms in the mRNA and protein levels of the clock components. In mammalian SCN, the first loop involves two bHLH-PAS-containing transcription factors, CLOCK (Cl) and BMAL1 (B). These transcription factors heterodimerize and activate the rhythmic transcription of three period genes (Per1-Per3, with Per1 and Per2 being critical to the circadian clock) and two cryptochrome genes (Cry1 and Cry2). The PER (P) and CRY (C) proteins complex with casein kinase 1 d and e (CKId/e), which phosphorylates PER. The resulting complex inhibits CLOCK/BMAL1-mediated transcription of period and cryptochrome genes, thus providing the negative feedback loop. The second loop involves CLOCK/BMAL1 driven rhythmic transcription of Rev-erba and Rora, members of the retinoic acid-related orphan nuclear receptor family. The phase of Rora expression closely resembles those of Per1 and Per2, and is opposite in phase with Rev-erba. The resultant REV-ERBa and RORa proteins (RE and R, respectively) compete for the same promoter element, RRE (Rev-erb/Ror element) and drive the rhythm in Bmal1 transcription. CLOCK/BMAL1 heterodimers also bind to circadian E-boxes in clock-controlled genes (CCGs), providing an output from the clock that drives rhythmic physiology. Adapted from Iuvone, P. M., Tosini, G., Pozdeyev, N., et al. (2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Progress in Retinal and Eye Research 24: 433–456, with permission from Elsevier.

Thus far, Per1 has not been identified in the chick retina, and the gene may be missing from the chicken genome. In addition to oscillating in a clock-dependent manner, Per2 and Cry1 are rapidly induced by light exposure (Figure 3(c) and 3(d)). It is generally thought that this induction is a mechanism for circadian clock entrainment by light. The rhythmic and light-regulated expression of circadian clock genes in the chick retina, particularly in cultured retinal cells, provides conclusive evidence that the retina contains autonomous circadian oscillators that can function independently of oscillators in the brain and pineal gland. Nevertheless, in the intact organism, retinal, pineal, and brain clocks are thought to interact to regulate physiology.

Circadian Regulation of Cyclic AMP in Retina Cyclic AMP is a ubiquitous second messenger molecule that regulates multiple aspects of cellular metabolism and

function. Effects of cyclic AMP are mediated by activation of cyclic AMP-dependent protein kinase (PKA), which phosphorylates proteins to regulate their function or activity. In so doing, cyclic AMP regulates intermediary metabolism, neurotransmission, and gene expression. Cyclic AMP can also affect cellular function by regulating cyclic nucleotidegated channels or activating Epac, a cyclic AMP-dependent Rap GTP exchange factor. The Rap is a guanine nucleotidebinding protein of the Ras family. In chick photoreceptor cell cultures, cyclic AMP levels are regulated by light and circadian clocks. Photoreceptor cyclic AMP levels are high in darkness and reduced by light exposure. Thus, when cells are exposed to a daily light–dark cycle, cyclic AMP fluctuates as a daily rhythm with high levels at night in darkness and low levels during the daytime in light (Figure 4). Exposure to light at night rapidly reduces cyclic AMP. The daily rhythm of cyclic AMP persists, albeit with reduced amplitude, when cells are transferred from a light–dark cycle to constant darkness. Thus, the combined effects of illumination and

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Figure 2 Temporal expression of positive modulators of the circadian clockwork system. Relative mRNA levels of Bmal1 (a) and Npas2 (b) in photoreceptor-enriched retinal cell cultures collected at the indicated Zeitgeber times (ZT) in light–dark (LD) and dark–dark (DD). Each data point represents clock gene transcripts normalized to hypoxanthine-guanine phosphoribosyl transferase (Hprt) mRNA and expressed relative to the lowest values in LD. The open horizontal bar at the X-axis represents times of light exposure; the black bars represent times of darkness. Analysis of variance (ANOVA) indicated significant rhythms of Bmal1 and Npas2 transcripts in LD and DD, with highest levels in the late day and early night. (c) Clock mRNA showed significantly higher values during the night (ZT 16) than during the day in LD; transcript levels increased on the first day of DD but there were no significant rhythms on DD1 or DD2. Reproduced from Chaurasia, S. S., Pozdeyev, N., Haque, R., et al. (2006). Circadian clockwork machinery in neural retina: Evidence for the presence of functional clock components in photoreceptor-enriched chick retinal cell cultures. Molecular Vision 12: 215–223, Copyright Molecular Vision 2006.

circadian influences interact to generate the daily rhythm of cyclic AMP. The regulation of cyclic AMP formation in chick photoreceptor cells is Ca2+-dependent, at least in part. Depolarization of the plasma membrane with high concentrations of extracellular K+ stimulates cyclic AMP formation. This effect requires Ca2+ influx through L-type voltage-gated Ca2+ channels. The plasma membrane of photoreceptors is partially depolarized in darkness and is hyperpolarized by light. Thus, the dark–light difference in cyclic AMP formation in photoreceptors is likely due to high Ca2+ conductance in darkness and decreased Ca2+conductance

following light exposure, due to closure of the voltage-gated channels. Accordingly, the circadian fluctuation in cyclic AMP levels is eliminated by nitrendipine, an L-type Ca2+ channel blocker. Cyclic AMP is synthesized from ATP by adenylyl cyclase. There are 10 isoforms of adenylyl cyclase that are regulated by multiple mechanisms. The findings described above indicate that photoreceptor cyclic AMP levels may be regulated by a Ca2+/calmodulin-stimulated cyclase. The type 1 and type 8 adenylyl cyclases are both stimulated by Ca2+/calmodulin and are both expressed in the chick retina. There is a circadian rhythm in the expression of


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Figure 3 Temporal expression of negative modulators of the circadian clockwork system. Circadian profiles of Cry1 (a) and Per2 (b) transcripts in the photoreceptor-enriched retinal cell cultures collected at the indicated Zeitgeber time (ZT) in light–dark (LD) and dark–dark (DD). Each data point represents clock gene transcripts normalized to Hprt mRNA, expressed relative to the lowest values in LD. Acute light exposure at night induces Cry1 (c) and Per2 (d) mRNA expression. On day 9 in vitro (DIV9) cells were kept in constant darkness until ZT 18, when one group of cells was collected. Another group of cells remained in darkness for an additional 2 h (solid symbol), while a third group of cells was exposed to light for 2 h prior to cell harvesting (open symbol). Exposure to light significantly increased Cry1and Per2 transcript levels. Reproduced from Chaurasia, S. S., Pozdeyev, N., Haque, R., et al. (2006). Circadian clockwork machinery in neural retina: Evidence for the presence of functional clock components in photoreceptor-enriched chick retinal cell cultures. Molecular Vision 12: 215–223, Copyright Molecular Vision 2006.

Adcy1, the transcript that encodes the type 1 adenylyl cyclase, in photoreceptor cell cultures and in the chick retina in vivo. In addition, there is a circadian rhythm in Ca2+/calmodulin-stimulated adenylyl cyclase activity in membranes prepared from photoreceptor cell cultures, with high activity at night. Thus, the circadian regulation of cyclic AMP is due to clock-controlled expression of Ca2+/calmodulin-stimulated adenylyl cyclase. The mammalian Adcy1 gene contains an E-box in its promoter that can be activated by BMAL1:CLOCK heterodimers, and a similar mechanism may contribute to the circadian regulation of Adcy1 expression in chick photoreceptor cells. However, this hypothesis has not yet been tested directly in the chick. The circadian rhythm of cyclic AMP formation may also be influenced by the clock-controlled expression and activity of the L-type Ca2+ channels in photoreceptors.

Circadian Regulation of Melatonin Biosynthesis Melatonin is a neurohormone that is synthesized in the retinal photoreceptor cells and in the pineal gland. Melatonin synthesis in retinas of most vertebrate species, including chicken, is regulated in a circadian fashion, with high levels at night in darkness. Melatonin functions in the retina to optimize nighttime visual function. Like cyclic AMP formation, melatonin levels are regulated by both illumination and circadian clocks. The key regulatory enzyme in melatonin biosynthesis is arylalkylamine N-acetyltransferase (AANAT). In chicken retina, AANAT activity undergoes a robust circadian rhythm with peak activity at night (Figure 5). Exposure to light at night causes a rapid decline in AANAT activity


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Figure 4 Circadian fluctuation of intracellular cAMP level. Cells were prepared from embryonic neural retinas and incubated for 8 days under LD. Illumination was switched from LD to DD before expected onset of light at the beginning of day 9 in vitro (DIV 9). White symbols represent cAMP level at Zeitgeber time (ZT) 10 in light; black symbols represent ZT 20 in darkness; gray symbols represent subjective day, ZT 10, in darkness. The horizontal white and black bars above the x-axis represent times of light and darkness, respectively. Level of cAMP was significantly higher at night than during the day in LD on DIV 8 and this fluctuation persisted in DD on DIV 9 and DIV 10. Reproduced from Ivanova, T. N. and Iuvone, P. M. (2003). Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: A mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells. Brain Research 991: 96–103, with permission from Elsevier.

(t½ 20 min), to insure that significant melatonin synthesis occurs in darkness only. Chicken AANAT activity is regulated by transcriptional and post-translational mechanisms (Figure 6). The retina displays daily rhythms of Aanat mRNA and AANAT protein in chickens exposed to a light–dark cycle or constant darkness. Levels of transcript, protein, enzyme activity, and melatonin all peak at night. The proximal promoter of the chicken Aanat gene contains a circadian E-box that can be activated by either BMAL1: CLOCK or BMAL1:NPAS2 heterodimers, and it is generally thought that this directly couples the circadian clock to the rhythmic expression of Aanat. In addition, the chicken Aanat 50 -flanking region contains cyclic AMP response elements. Thus, the circadian rhythm of cyclic AMP may also contribute to the rhythm of Aanat mRNA. The AANAT protein is regulated by PKA-dependent phosphorylation and proteasomal degradation (Figure 6). The AANATcontains two consensus PKA phosphorylation sites. When cyclic AMP levels are high at night, AANAT is phosphorylated. Phospho-AANAT binds to 14-3-3 proteins, which are ubiquitous signaling proteins involved in

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Figure 5 Daily rhythm of retinal AANAT activity: effects of light. (a) AANAT activity fluctuates during the 12 h light–12 h dark cycle (filled circles). Unexpected light exposure at night (open circles) rapidly inhibits activity. (b) AANAT activity in constant (24 h day1) darkness. The activity rhythm persists on the second day of constant darkness (filled circles). The rhythm is phase advanced by a 6 h light pulse from 18 to 24 h 2 days prior to sampling in constant darkness (open circles). Filled bars on the xaxis represent darkness; open bars represent light. Activity was measured in retinal homogenates of 2-week old chickens. Reproduced from Iuvone, P. M. and Alonso-Go´mez, A. L. (1998). Melatonin in the vertebrate retina. In: Christen Y., Doly, M., and Droy-Lefaix, M. -T. (eds.) Retine, Luminiere, et Radiations, vol. 9, pp. 49–62. Paris: Irvinn; and Iuvone, P. M., Tosini, G., Pozdeyev, N., et al. (2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Progress in Retinal and Eye Research 24: 433–456, with permission from Elsevier.

multiple cellular functions. The interaction of 14-3-3 with AANAT increases the affinity of the enzyme for its substrate, increasing catalytic activity. Exposure to light at night causes a very rapid decrease in AANAT activity and protein level (t½ 20 min) without any initial change of Aanat mRNA (Figure 6). The decrease of AANAT is accompanied by a similarly rapid decline in melatonin levels, insuring that melatonin only functions in darkness. The decrease of protein and activity results from the lightinduced decrease of cyclic AMP levels, resulting in dephosphorylation of AANAT, unbinding of 14-3-3, and rapid proteolytic degradation of the enzyme. The degradation can be blocked by proteasome inhibitors, indicating that this is a proteasome-dependent event. During the daytime, multiple mechanisms cooperate to keep melatonin biosynthesis at a minimum (Figure 6). Low levels of cyclic AMP appear to play key roles in these mechanisms. The Aanat transcript levels are low, presumably due to reduced cyclic AMP-directed transcriptional activation and suppression by Cry1 of E-boxmediated transactivation (Figure 6). In addition, the low levels of cyclic AMP during the daytime, especially in light, favor the dephosphorylated state of the AANAT protein, resulting in its rapid degradation by proteasomes. The majority of AANAT in the chick retina is expressed at night in photoreceptor cells. However, there is some

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Figure 6 Model for circadian clock- and light-regulated melatonin biosynthesis in photoreceptor cells (see text for detailed description of the model). The left side of the figure depicts processes occurring in light while the right side shows processes occurring at night in darkness. Abbreviations: AANAT, arylalkylamine N-acetyltransferase; AC1, type 1 Ca2+/calmodulin-stimulated adenylyl cyclase; CaM, calmodulin; cAMP, cyclic adenosine 30 ,50 -monophosphate; CRE, cAMP-responsive element; pAANAT, phosphorylated AANAT; pCREB, phosphorylated cAMP response element-binding protein; PKA, cAMP-dependent protein kinase, with permission from Elsevier.

evidence that small amounts of AANAT may be expressed in ganglion cells during the daytime. The role of ganglion cell-derived AANAT is not known.

Other Rhythms of Gene Expression and Metabolism in the Chick Retina Circadian clocks regulate phospholipid metabolism in the chick retina. Daily rhythms of phospholipid labeling with either 32P or [3H]glycerol have been observed in chicks kept on a light–dark cycle or in constant darkness. The 32 P labeling of total phospholipids in photoreceptors and ganglion cells peaks in the late night. The major phospholipid labeled under these conditions is phosphotidylinositol. In contrast, labeling with [3H]glycerol peaks midday, with phosphatidylcholine as the major labeled species. Melanopsin (Opn4) is a photopigment first discovered in Xenopus melanocytes, where it mediates photic regulation of melanin pigment aggregation. Subsequent cloning of orthologous melanopsin genes revealed that it is expressed in mammalian retina exclusively in a small subset of ganglion cells that are intrinsically photosensitive. These ganglion cells project to the suprachiasmatic nucleus (SCN) of the hypothalamus, the inferior olivary nucleus, and other brain regions involved in nonimage forming vision. The intrinsically photosensitive ganglion

cells mediate the pupillary light reflex and photic entrainment of the circadian oscillator in the SCN. In contrast to mammals, chickens express two melanopsin genes, designated Opn4x and Opn4m because of their sequence homology to the Xenopus and mammalian melanopsin genes, respectively. The Opn4x and Opn4m transcripts are more widely distributed than their mammalian counterpart. Chicken melanopsin transcripts are found in a subpopulation of cells in the ganglion cell layer, and also in photoreceptors, retinal pigment epithelium (RPE) cells, inner nuclear layer (INL) cells, the pineal gland, and areas of the brain known to contain deep brain photoreceptors. The Opn4x is rhythmically expressed in the chicken retina in light–dark cycles and in constant darkness. Patterns of rhythmicity differ among retinal layers; Opn4x peaks in the morning in RPE and INL cells, but at night in photoreceptors. Similar to the retinal photoreceptors, Opn4x also peaks at night in chicken pinealocytes, which are directly photosensitive and contain a circadian clock that is entrained by light. The regulation and localization of Opn4 is consistent with the hypothesis that this novel photopigment plays a role in circadian regulation in the retina and pineal gland. Iodopsin is the photopigment of red-sensitive cone photoreceptors of avian species. Iodopsin mRNA levels fluctuate as a circadian rhythm in retinal photoreceptors in vivo and in vitro, in cultured photoreceptors, and in

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retinal explants. The rate of transcription of the iodopsin gene peaks late in the subjective day in constant darkness, 3 h before the beginning of the subjective night, and mRNA levels peak early in the subjective night. The functional significance of the circadian rhythm of iodopsin mRNA is yet to be determined. Ion channels in chicken cone photoreceptors are also subject to circadian regulation. There is a circadian rhythm in the affinity of the cone cyclic nucleotide-gated channel for cGMP, with highest affinity during the subjective night. In addition, L-type Ca2+ channels are regulated in a circadian fashion. The Ca2+ currents and immunoreactivity for a1C and a1D calcium channel subunits are greater at night than during the day. There is also a rhythm of a1D mRNA level. More details on rhythms of ion channels can be found elsewhere in this encyclopedia.

Conclusion The chick retina is a remarkably rhythmic tissue, with robust circadian control of gene expression, metabolism, physiology, and melatonin synthesis. Most attention has been paid thus far to photoreceptor rhythms, but inner retinal neurons also express clock genes and are likely to be subject to circadian control. The ability to generate retinal cell cultures, which maintain their circadian properties and can be manipulated pharmacologically and genetically, suggests that the chick retina will continue to be a valuable model system for exploring the circadian organization of the retina.

Acknowledgments The author is grateful to the past and present members of his laboratory, especially Rashidul Haque, Nikita Pozdeyev, Shyam Chaurasia, and Tamara Ivanova, and to David Klein and the members of his laboratory, who contributed greatly to the body of knowledge contained within this article. The author also thanks Gianluca Tosini for his collaborative contributions and for critical comments and suggestions on the article. Research in the author’s laboratory is funded by the National Institutes of Health EY004864 and EY06360, and by Research to Prevent Blindness. See also: The Circadian Clock in the Retina Regulates Rod and Cone Pathways; Circadian Photoreception; Circadian Regulation of Ion Channels in Photoreceptors; Circadian Rhythms in the Fly’s Visual System; Fish Retinomotor Movements; Limulus Eyes and Their Circadian Regulation; Neurotransmitters and Receptors: Dopamine Receptors; Neurotransmitters and Receptors: Melatonin Receptors.

Further Reading Bailey, M. J., Beremand, P. D., Hammer, R., et al. (2004). Transcriptional profiling of circadian patterns of mRNA expression in the chick retina. Journal of Biological Chemistry 279: 52247–52254. Bellingham, J., Chaurasia, S. S., Melyan, Z., et al. (2006). Evolution of melanopsin photoreceptors: Discovery and characterization of a new melanopsin in nonmammalian vertebrates. PLoS Biology 4: e254. Bailey, M. J., Chong, N. W., Xiong, J., and Cassone, V. M. (2002). Chickens’ Cry2: Molecular analysis of an avian cryptochrome in retinal and pineal photoreceptors. FEBS Letters 513: 169–174. Bernard, M., Iuvone, P. M., Cassone, V. M., et al. (1997). Avian melatonin synthesis: Photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina. Journal of Neurochemistry 68: 213–224. Chaurasia, S. S., Haque, R., Pozdeyev, N., Jackson, C. R., and Iuvone, P. M. (2006). Temporal coupling of cyclic AMP and Ca/calmodulin-stimulated adenylyl cyclase to the circadian clock in chick retinal photoreceptor cells. Journal of Neurochemistry 99: 1142–1150. Chaurasia, S. S., Pozdeyev, N., Haque, R., et al. (2006). Circadian clockwork machinery in neural retina: Evidence for the presence of functional clock components in photoreceptor-enriched chick retinal cell cultures. Molecular Vision 12: 215–223. Chaurasia, S. S., Rollag, M. D., Jiang, G., et al. (2005). Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): Differential regulation of expression in pineal and retinal cell types. Journal of Neurochemistry 92: 158–170. Chong, N. W., Bernard, M., and Klein, D. C. (2000). Characterization of the chicken serotonin N-acetyltransferase gene. Activation via clock gene heterodimer/E box interaction. Journal of Biological Chemistry 275: 32991–32998. Chong, N. W., Chaurasia, S. S., Haque, R., Klein, D. C., and Iuvone, P. M. (2003). Temporal-spatial characterization of chicken clock genes: Circadian expression in retina, pineal gland, and peripheral tissues. Journal of Neurochemistry 85: 851–860. Garbarino-Pico, E., Carpentieri, A. R., Contin, M. A., et al. (2004). Retinal ganglion cells are autonomous circadian oscillators synthesizing N-acetylserotonin during the day. Journal of Biological Chemistry 279: 51172–51181. Guido, M. E., Pico, E. G., and Caputto, B. L. (2001). Circadian regulation of phospholipid metabolism in retinal photoreceptors and ganglion cells. Journal of Neurochemistry 76: 835–845. Hamm, H. E. and Menaker, M. (1980). Retinal rhythms in chicks – circadian variation in melatonin and serotonin N-acetyltransferase. Proceedings of the National Academy of Sciences of the United States of America 77: 4998–5002. Haque, R., Chaurasia, S. S., Wessel, J. H., III, and Iuvone, P. M. (2002). Dual regulation of cryptochrome 1 mRNA expression in chicken retina by light and circadian oscillators. NeuroReport 13: 2247–2251. Iuvone, P. M. and Alonso-Go´mez, A. L. (1998). Melatonin in the vertebrate retina. In: Christen, Y., Doly, M., and Droy-Lefaix, M.-T. (eds.) Retine, Luminiere, et Radiations, vol. 9, pp. 49–62. Paris: Irvinn. Iuvone, P. M., Brown, A. D., Haque, R., et al. (2002). Retinal melatonin production: Role of proteasomal proteolysis in circadian and photic control of arylalkylamine N-acetyltransferase. Investigative Ophthalmology and Visual Science 43: 564–572. Ivanova, T. N. and Iuvone, P. M. (2003). Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: A mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells. Brain Research 991: 96–103. Iuvone, P. M., Tosini, G., Pozdeyev, N., et al. (2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Progress in Retinal and Eye Research 24: 433–456. Pierce, M. E., Sheshberadaran, H., Zhang, Z., et al. (1993). Circadian regulation of iodopsin gene expression in embryonic photoreceptors in retinal cell culture. Neuron 10: 579–584. Pozdeyev, N., Taylor, C., Haque, R., et al. (2006). Photic regulation of arylalkylamine N-acetyltransferase binding to 14-3-3 proteins in retinal photoreceptor cells. Journal of Neuroscience 26: 9153–9161.

Central Retinal Vein Occlusion S S Hayreh, University of Iowa, Iowa City, IA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Demographic – The statistical study of a population, including geographical distribution, sex and age composition, and birth and death rates. Electroretinography – The recording of the changes in electric potential in the retina by stimulating it by light. Fluorescein fundus angiography – The visualization of blood vessels in the interior of the eye following intravenous injection of fluorescein. Glaucoma – An eye disease caused by an increase in eye pressure, which causes changes in the optic nerve and loss of vision. Hematological – Dealing with the blood and bloodforming tissues. Histopathological – Dealing with the minute structure of diseased tissues. Lamina cribrosa – The perforated portion of the back part of the white of the eye (sclera) through which nerve fibers from the retina exit. Multifactorial – Related to, or arising through the action of many factors. Neovascularization – The formation of abnormal new blood vessels. Ophthalmoscopy – The examination of the interior of the eye with the instrument called ophthalmoscope. Panretinal photocoagulation – The application of an intense beam of laser light to the entire retina. Pathogenesis – The mechanisms of development of a disease. Perimeter – An apparatus used to test the visual field.

Retinal vein occlusion is the most common retinal vascular occlusive disorder. In general, there is a tendency to regards this as one disease; that is not only incorrect but also causes much confusion. From the point of view of pathogenesis, clinical picture, prognosis, and management, retinal vein occlusion in fact consists of six distinct clinical entities that are categorized as follows: 1. Central retinal vein occlusion (CRVO), which comprises a. nonischemic CRVO, and b. ischemic CRVO.

2. Hemi-central retinal vein occlusion (HCRVO), which comprises a. nonischemic HCRVO, and b. ischemic HCRVO. 3. Branch retinal vein occlusion, which includes a. major BRVO, and b. macular BRVO. It is beyond the scope of this article to discuss all the six types of retinal vein occlusion; hence, we restrict our discussion only to CRVO. Over the last 150 years, a large volume of literature has accumulated on the subject of CRVO. The objective of this article is to provide a brief review of the current state of our knowledge on the subject.

Pathogenesis A good understanding of the pathogenesis of a disease is fundamental to a full grasp of the clinical features of the disease and its logical management. There is almost a universal tendency to blame one or two factors as causative factor(s) in the development of CRVO, but association does not necessarily mean there is a cause-and-effect relationship. Available evidence strongly suggests that the pathogenesis of CRVO, like many other ocular vascular occlusive disorders, is a multifactorial process. It seems that some risk factors predispose an individual or an eye to CRVO (predisposing risk factors), while others act as the final insult and produce clinically evident disease (precipitating risk factor(s)). Only when an eye and an individual have the critical number of risk factors required for the development of CRVO, does the CRVO develop. This must explain why bilateral CRVO is rare. Once this basic concept of multifactorial causation is understood, one can attach appropriate significance to the various risk factors. The various risk factors for CRVO may be divided into the following three categories: Local. Two local factors are particularly important: 1. The central retinal vein and central retinal artery lie in the center of the optic nerve, surrounded by a fibrous tissue envelope (Figure 1). In elderly persons, sclerotic changes in the central retinal artery and the fibrous tissue envelope compress the thinwalled central retinal vein, resulting in narrowing of its lumen. This produces circulatory stasis. According to Virchow’s triad, slowing down of the blood stream causes stagnation thrombosis.

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Figure 1 Histological sections (Masson’s trichrome staining) showing the central retinal vessels and surrounding fibrous tissue envelope, as seen in a transverse section of the central part of the retrolaminar region of the optic nerve, in a normal rhesus monkey (above) and in a rhesus monkey with experimental arterial hypertension, atheroselerosis, and glaucoma (below). CRA, Central retinal artery; CRV, central retinal vein; FTE, fibrous tissue envelope.

2. It is well established that CRVO is significantly more common in patients with raised intraocular pressure (IOP) and glaucoma. Systemic. A significant association of CRVO has been reported with arterial hypertension, diabetes mellitus, cardiovascular disease, atherosclerosis, and thyroid disease. Hematological. The literature is full of reports of hematological abnormalities in CRVO. The author recently critically reviewed the literature dealing with these and found no definite pattern – often the negative findings outweighed the positive ones. The idea of hematologic factors playing a role in CRVO is essentially based on the assumption that those hematological disorders, which play a role in development of systemic venous thrombosis (e.g., deep vein thrombosis), must also do so in CRVO. All the available evidence, however, indicates that the hematologic risk factor responsible for major systemic venous thrombosis occurs only sporadically in CRVO. Furthermore, CRVO is extremely rare in patients with systemic venous thrombosis. Moreover, the presence of a particular hematologic disorder in a patient does not necessarily mean it has a cause-andeffect relationship with CRVO. In view of this, there is no particular reason for conducting a detailed hematological investigation in all patients with CRVO.

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Figure 2 Schematic representation of blood supply of the optic nerve. A, arachnoid; C, choroid; CRA, central retinal artery; Col. Br., collateral branches from other orbital arteries to the optic nerve; CRV, central retinal vein; D, dura; LC, lamina cribrosa; ON, optic nerve; PCA, posterior ciliary artery; PR, prelaminar region; R, retina; S, sclera; SAS, subarachnoid space. Adapted from Hayreh, S. S. (1974). Transactions – American Academy of Ophthalmology and Otolaryngology 78: OP240–OP254, with permission from American Academy of Ophthalmology.

Site of Occlusion in CRVO Based on histopathological studies, there is a widespread misconception that the site of occlusion in CRVO is invariably at the lamina cribrosa. However, all the available anatomical, experimental, and clinical evidence (particularly fluorescein fundus angiography) shows that the actual site of occlusion in the central retinal vein is typically in the optic nerve, at a variable distance posterior to the lamina cribrosa, and not at the lamina cribrosa (Figure 2). The farther back the site of occlusion, the more collaterals are available, and the less severe is the retinal venous stasis. Thus, in nonischemic CRVO the site of occlusion most likely is farther back in the optic nerve, whereas in ischemic CRVO it is closer to the lamina cribrosa.

Demographic Characteristics CRVO is more common in middle-aged and elderly persons, and patients with ischemic CRVO tend to be older than those with nonischemic CRVO. Contrary to the prevalent impression, CRVO is not at all rare in young persons, and the incidence in persons under the age of 45 years has been reported as high as 18%. Thus, no age is immune. In our series of 620 consecutive CRVO cases, 81% were nonischemic and 19% ischemic CRVO. The Kaplan– Meier estimate of the cumulative proportion of eyes that developed nonischemic CRVO in the fellow eye is about 6% within 1 year and 7% within 5 years from onset in the first eye; for ischemic CRVO it is 5.6% at 2.8 years.


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(b) Figure 3 Fundus photograph (a) and OCT (b) of a nonischemic CRVO eye with resolution of retinopathy, except for the cystoid macular edema (arrow).

Clinical Features With regard to symptoms, patients with nonischemic CRVO may have no symptoms and it may be detected as an incidental finding on a routine ophthalmic examination. Retinal venous stasis with mild retinal hemorrhages per se is asymptomatic. Occasionally there may be a history of episodes of transient visual blurring before constant visual deterioration. Almost invariably, it becomes symptomatic only when there is involvement of the foveal region by development of macular edema (Figures 3 and 4) and rarely by hemorrhages. Therefore, the most common complaint is gradual development of central visual blurring, usually more marked on waking up in the morning, improving to a variable extent after a few hours or in the afternoon. In ischemic CRVO, on the other hand, there is always marked deterioration of vision. While the diagnosis of CRVO is not difficult because of its classical clinical features (Figures 5 and 6), the main problem is differentiation of nonischemic from ischemic CRVO, which is crucial for the correct management of CRVO. This is because nonischemic CRVO is a comparatively benign condition, with permanent central

Figure 4 Late phase of fluorescein fundus angiogram of an eye with nonischemic CRVO showing classical petaloid pattern of cystoid macular edema.

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(b) (b) Figure 5 Fundus photograph (a) and fluorescein fundus angiogram showing intact retinal capillary network (b) of an eye with nonischemic CRVO. Reproduced from Hayreh, S. S. (1994). Indian Journal Ophthalmology 42: 109–132.

scotoma as the major complication in some eyes, but no ocular neovascularization (NV). In sharp contrast to this, ischemic CRVO is a blinding disease, with high risk of development of anterior segment NV, particularly neovascular glaucoma, which often results in blindness or even loss of the eye. Thus, the two types of CRVO can be compared to benign and malignant tumors.

Differentiation of Ischemic from Nonischemic CRVO Ophthalmologists have almost universally used ophthalmoscopic and fluorescein angiographic appearances to evaluate and manage CRVO and to differentiate ischemic from nonischemic CRVO. However, these two morphological tests have much lower sensitivity and specificity to differentiate the two types of CRVO compared to the four functional tests – visual acuity, peripheral visual fields plotted with a Goldmann perimeter, relative afferent

Figure 6 Fundus photograph (a) and fluorescein fundus angiogram showing complete nonperfusion of retinal capillary network (b) of an eye with ischemic CRVO. Reproduced from Hayreh et al. (1983). Ocular neovascularization with retinal vascular occlusion III. Incidence of ocular neovascularization with retinal vein occlusion. Ophthalmology 90: 488–506.

pupillary defect, and electroretinography. Table 1 gives sensitivity and specificity of various functional tests to differentiate ischemic from nonischemic CRVO. On fluorescein fundus angiography, to differentiate ischemic from nonischemic CRVO, the presence of a 10 disk area or more retinal capillary obliteration has been regarded as the gold standard in practically all the reported studies, but there are several serious problems with this criterion, including the following: 1. During the early, acute stages of CRVO, to provide reliable information on retinal capillary obliteration, angiography has many serious limitations, including extensive retinal hemorrhages, poor-quality angiograms, inability to perform angiography for a variety of reasons, the time lag of several weeks after the onset of CRVO before retinal capillary obliteration is visible, and other limitations. A study showed that fluorescein angiography provided reliable information at best in only


50–60% of cases during the early, acute phase, which is clinically unsatisfactory for early management. 2. Most importantly, a criterion of a 10 disk area or more of retinal capillary obliteration has been widely advocated as the definitive yardstick for diagnosis of ischemic CRVO. However, this is an invalid criterion to differentiate nonischemic from ischemic CRVO. A multicenter study showed that eyes with 95%) in the photoreceptor cell population. Photoreceptor maturation is completed between the second and the third postnatal week (arrow). In the human (b), S-opsin is synthesized first, followed shortly after by overlapping rhodopsin and L-/M-opsins. Rhodopsin production increases dramatically after birth, coincidental with outer segment formation and maturation. Cone and rod photoreceptors are rearranged after birth, with packing of cones in the fovea (dashed arrow). Outer segments continue to expand until they reach their mature size by the third year of age (solid arrow). B, birth; OS, outer segment.

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greater proportion of rods than cones at the edge of the pure cone area. The mature fovea becomes evident by 1 year of age with the characteristic packaging of cones in the center. Rod outer segments continue to grow in length well into the first postnatal years until the human retina reaches full maturation by approximately 3–5 years of age.

Factors Affecting Photoreceptor Genesis In the stereotypical progression of cell differentiation in the retina, cone genesis is initiated from common proliferating RPCs, in a sequential manner after RGCs and before horizontal and amacrine neurons. Rod birth is initiated later compared to cone genesis, but it precedes bipolar and Mu¨ller glia cells. However, the time intervals during which full complements of cone and rod photoreceptors are generated largely overlap (see Figure 1). Lineage and birth-dating analyses of mouse retina indicate that photoreceptor cell-fate decisions are made at the time of terminal mitosis; however, additional investigations are necessary. Two key elements control the commitment and differentiation of photoreceptors: gene-regulatory networks that confer competence to the RPC and dictate differentiation events (Figures 2 and 3), and extrinsic factors that modulate transcriptional cascades in differentiating RPCs and later modulate cell–cell communication (Figure 2). In addition, epigenetic mechanisms (such as chromatin modifications) appear to play a significant role in directing photoreceptor specification and differentiation.

Early Stages in Photoreceptor Development From RPC to Photoreceptor Precursor At the time of their last mitosis and prior to exiting the cell cycle, RPCs fated to become photoreceptors upregulate the expression of paired-class homeodomain transcription factor orthodenticle protein homolog 2 (OTX2) (Figure 2). In the mouse, OTX2 is first detected at E11.5–12.5 in the retinal neuroblastic layer (Figure 3). OTX2 expression increases thereafter, concomitantly with early cone and rod development, and persists in the postnatal mouse retina in bipolar and photoreceptor cells. Otx2 plays an important role as an early factor that specifies photoreceptor cell fate, and probably as a late factor that may promote terminal differentiation by participating in upregulation of photoreceptor-specific genes. When Otx2 is conditionally knocked out in the developing mouse eye, photoreceptors do not develop and are replaced by amacrine-like cells. Furthermore, when Otx2 is ubiquitously expressed in RPCs, all cells follow the photoreceptor fate. In humans, OTX2

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S-cone precursor Figure 2 A model of photoreceptor development in the murine retina. Rods, S and M cones are generated from a common pool of retinal progenitor cells (RPCs), which exit the cell cycle at specific times during retinogenesis. A synergistic interaction between intrinsic and extrinsic factors progressively restricts cell fate, biasing individual cells toward differentiation into a unique mature cell type. Some of the known molecules involved are illustrated here. Proteins that appear to be major contributors to specification/determination of cell fate are shown in bold. Expression of OTX2 and CRX defines the postmitotic pool of cells fated to become photoreceptors (photoreceptor precursors). Expression of NRL and its target NR2E3 determines rod photoreceptor cell fate, whereas their absence leads to cone fate. NRL/NR2E3-expressing rod precursors progress through multiple transition stages and become functional rod photoreceptors when rhodopsin and phototransduction protein synthesis occurs and outer segments are mature. Photoreceptor precursors that do not express NRL/NR2E3 progress toward the cone lineage. Upon downregulation of TRb2 and RXRg, cone precursors become S cone precursors and synthesize S-opsin. For M cone precursors to develop, S-opsin expression must then be repressed by the heterodimer TRb2/RXRg, and M-opsin synthesis initiated by another TRb2-containing complex. S- and M-opsin expression followed by outer segment maturation complete cone differentiation. The current model supports the existence of a default S-cone pathway that requires active inhibition by NRL/NR2E3 and by TRb2/RXRg to allow differentiation of rods and M cones, respectively. The roles of other cell intrinsic and extrinsic factors are further described in the text. Arrows indicate active promotion and truncated lines indicate inhibition of a developmental stage. Dotted lines indicate tentative roles. See the text for explanation of abbreviations.

mutations are associated with retinal diseases, such as anophthalmia, microphthalmia, and coloboma. At the molecular level, OTX2 is shown to activate the promoter of cone–rod homeobox (CRX) transcription factor, a closely related homeodomain transcription factor. The two transcription factors (OTX2 and CRX) appear to promote completion of photoreceptor differentiation programs (Figure 2). CRX does not contribute to photoreceptor cell specification, but is essential for terminal differentiation and maintenance. In Crx-null mice, despite normal photoreceptor genesis, morphogenesis is incomplete as outer segments fail to elongate, and photoreceptors do not produce a full complement of phototransduction proteins. These abnormal photoreceptors undergo synaptogenesis, but their synaptic endings appear malformed and eventually degenerate. Mutations in the human CRX result in retinopathies, including Leber

congenital amaurosis (LCA), cone–rod dystrophy, and retinitis pigmentosa (RP). Another protein suggested to participate in photoreceptor cell development is the basic helix-loop-helix (bHLH) transcription factor NeuroD1 (Figure 2). NeuroD1 is expressed in developing and differentiated rod and cone photoreceptors. Its targeted deletion in the mouse, however, causes only a modest decrease in photoreceptor number. NeuroD1 acts in combination with another bHLH transcription factor, MASH1, which may contribute to regulating the timing of photoreceptor differentiation as its targeted deletion in mouse leads to delay in photoreceptor development. Very early, at the time of photoreceptor specification, postmitotic precursor cells become committed toward rod or cone fates. The Maf family basic motif-leucine zipper transcription factor NRL and its target, the

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Figure 3 Stages of photoreceptor development in murine retina. Developmental stages are characterized by unique markers. Postmitotic photoreceptor precursors (a) can be detected by immunohistochemistry (IHC) with anti-OTX2 antibodies (green). Intermediate stages leading to cone or rod precursors are less delineated. However, cone precursors are characterized by the expression of TRb2 and RXRg, while rod precursors can be distinguished for NRL and NR2E3 expression. When the lacZ reporter gene driven by the Trb2 promoter is expressed in transgenic mice (b), cone precursors, normally expressing TRb2, express LacZ (cyan). Similarly, rod precursors in a transgenic mouse expressing green fluorescent protein (GFP) under control of Nrl promoter (c) become fluorescent (green). Mature photoreceptors are identified by expression, at the RNA level first and protein level after, of their characteristic opsins and phototransduction proteins. Rod (d) and cone (e) photoreceptors are visualized by IHC with antirhodopsin (red) and anticone arrestin (green) antibodies, respectively. Areas labeled with an asterisk in (a)–(e) are enlarged in the inlets. Images are provided by Dustin Hambright ((a), (d), and (e)), Li Jia and Douglas Forrest (b), and Jerome Roger (c). RPE, retinal pigment epithelium; NBL, neuroblastic layer; GCL, ganglion cell layer; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; E, embryonic day; P, postnatal day; DAPI, 4’-6-diamidino-2-phenylindole (nuclear staining); CAR, cone arrestin.

photoreceptor-specific orphan nuclear receptor NR2E3, are the primary determinants of rod versus cone cell-fate determination. It appears that S cone cell fate is specified as a default pathway and that inhibition of this pathway by NRL/NR2E3 is permissive to rod specification (Figure 2).

From Cone Precursor to Cone Photoreceptor Cones are the first photoreceptor cell type to exit the cell cycle in all vertebrates (Figure 1). However, their differentiation takes a few days (mouse) to weeks (human) and is complete only after a majority of rods have become postmitotic. Cone precursors must go through an additional specification process that determines distinct cone subtypes (L, M, or S in humans, and M or S in mouse). Studies of cone-photoreceptor-fate determination rely 8w?>on the detection of specific opsins at the mRNA or protein level, thus overlapping with studies on opsin gene

regulation. In mouse and human cone precursors, S-opsin expression is detected first, followed by M-opsin expression (Figure 1). Variable numbers of photoreceptors go through a transitional state in which both opsins are expressed, before S-opsin is downregulated and M-opsin predominates. In the adult mouse retina, a prevalent population of photoreceptors coexpresses both pigments, whereas cone photoreceptors in the human retina express a single opsin. In mice, onset of cone opsin expression is regulated by numerous factors; these include CRX, members of the nuclear receptor family, thyroid hormone nuclear receptor beta 2 isoform (TRb2) and retinoid X nuclear receptor gamma (RXRg), and of the retinoic acid (RA) receptorrelated orphan receptor – RORa and RORb2. RORa and RORb2 are expressed in postmitotic cone-photoreceptor precursors and directly regulate S-opsin expression synergistically with CRX. Furthermore, RORa also appears to regulate the expression of M-opsin through direct binding to its promoter, highlighting a potentially more complex role in cone differentiation. Notably, Rorb2-null

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mice lack outer segments, suggesting RORb2 plays an additional role in promoting photoreceptor maturation. TRb2 and RXRg are also expressed in postmitotic cone precursors in the neonatal mouse retina (Figures 2 and 3) and act as transcriptional repressors of S-opsin expression. In fact, both receptors are downregulated concomitant with the initial onset of S-opsin expression and upregulated at a later stage in M cones in the dorsal retina to suppress S-opsin. Further evidence comes from studies of Rxrg-null and Trb2-null mice. Both nuclear receptors are necessary to repress S-opsin expression and probably act in concert on the S-opsin promoter. However, only TRb2 is required for M-opsin expression, making it a key regulator of M cone differentiation. Trb2 gene appears to be regulated by a transcriptional complex containing NeuroD1. Although NeuroD1 alone is not sufficient to initiate Trb2 expression, it is required for sustained gene activation, thus supporting its role in M cone differentiation. To date, there is no evidence involving RA in cone development in the mouse or human. On the other hand, TH has been suggested to regulate the ratio and patterning of cone-photoreceptor subtypes through changes in geographical distribution in the developing retina.

From Rod Precursor to Rod Photoreceptor NRL is the master regulator of rod cell fate and serves as the unique defining signature of newborn rod photoreceptors (see Figures 2 and 3). NRL induces the expression of NR2E3. Following this, NRL and NR2E3 – together with CRX – lead to the expression pattern typical of mature rod photoreceptors (Figure 2). In mice null for the Nrl gene, all rods are converted to cones. Targeted deletion of Nr2e3 in the mouse leads to enhanced S-cones with hybrid photoreceptors. Transgenic expression of Nr2e3 in Nrl-null mice suppresses cone differentiation and leads to the generation of rod-like photoreceptors, yet NR2E3 is not sufficient to produce functional rods. These data support a model in which active induction by NRL and NR2E3 in CRXexpressing photoreceptor precursors is required for rod cell-fate determination with simultaneous inhibition of the cone pathway (Figure 2). Mutations in NRL are associated with retinal degenerative diseases, including autosomal dominant and recessive RP, and retinopathies with varying phenotypes. Loss of NR2E3 in humans leads to enhanced S-cone syndrome, Goldmann–Favre syndrome, and similar retinopathies with increased S-cone function. The retinoblastoma (Rb) gene appears to be an important intrinsic regulator of rod development. In the postnatal mouse retina, Rb is expressed in mitotic RPCs, where it regulates timely exit from the cell cycle, and in

differentiating rod photoreceptors. When Rb is deleted, RPCs continue to proliferate and rod photoreceptors do not develop. Unlike Nrl- and Nr2e3-null mice, rod photoreceptors in Rb-null mice do not change their fate to cones (cone number remains unmodified). Rather, their development is arrested at the progenitor stage. It remains unclear from the current literature whether Rb is instructive or permissive for rod-photoreceptor cell fate. Several extrinsic factors contribute to signaling for rod development (Figure 2). Cell–cell interaction mediated by Notch1 is known to sustain the undifferentiated and proliferating state of RPCs, repressing neuronal fate in general. Recently, Notch1 signaling has been shown to specifically inhibit photoreceptor fate, that is, cone in the embryonic and rod in the postnatal mouse retina. This function of Notch1 may allow differentiation of other neuronal cell types as light detection evolved from uniquely photoreceptor-based, in lower species, to multiple cell-type-mediated, in higher species. Other extrinsic factors inhibiting rod fate are leukocyte inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), and epidermal growth factor (EGF). Taurine, secreted by RGCs, is suggested to stimulate rod photoreceptor production through glycine (Gly) and gamma aminobutyric acid (GABA(A)) receptors. The hypothesis that taurine and its receptor GlyRa2 have a role in promoting rod cell fate awaits further validation. Proper maturation and/or migration and integration of young rods into the outer nuclear layer (ONL) may require gradients of Indian Hedgehog (IHH) secreted by the retinal pigment epithelium (RPE), and Sonic Hedgehog (SHH) secreted by RGCs. IHH may also have a role in the specification of photoreceptor fate, possibly inducing Nrl in photoreceptor precursors. Similarly, fibroblast growth factor (FGF) family members – acidic FGF (FGF1) and basic FGF (FGF2) – are implicated in rod maturation and may induce NRL expression. The role of RA in promoting rod photoreceptor differentiation is still unclear, though it can induce Nrl expression and RA-responsive sites are present in the Nrl promoter. However, retinoic acid receptor (RAR)b2/ RARg2 double null mutant mice contain a normal photoreceptor complement. Activin A – a transforming growth factor beta (TGFb)like protein expressed by extraocular mesenchyme and RPE – promotes photoreceptor development in vitro and in vivo, and increases the number of photoreceptor cells in rat retinal cultures. In vitro, it causes RPCs to exit the cell cycle and biases them to become rods, but not cones. In vivo, mice with homozygous deletion of activinA show substantial decrease in the number of photoreceptors. Finally, growth differentiation factor 11 (GDF11) and related TGFb family members control the competence of mitotic progenitors to acquire rod cell fate. GDF11-null retinas have more RGCs at the expense of photoreceptors.


Maturation of Photoreceptors Cones are generated in mice during the prenatal period. Rod photoreceptor birth overlaps with the genesis of all retinal cell types, though most rods are born postnatally (Figure 1). Maturation of committed precursors to differentiated functional photoreceptors is a lengthy process and involves expression of cell-type-specific phototransduction genes, biogenesis of outer segments, and formation of synapses with specific interneurons. Expression of most photoreceptor-enriched genes depends on the synergistic or antagonistic actions of NRL, NR2E3, and CRX and their interaction with other regulatory proteins. In most instances, these proteins co-occupy the promoter/ enhancer regions of their target genes. Mutations in the target genes of NRL and CRX are associated with retinal dysfunction. Studies with transgenic and knockout mice, together with microarray and chromatin immunoprecipitation analysis, have yielded valuable information about gene-regulatory networks that guide photoreceptor differentiation and maturation. As NRL and its direct target nuclear receptor NR2E3 determine rod cell fate, these two transcription factors activate the expression of rod-specific genes and repress cone-gene expression. NRL, even in the absence of NR2E3, can activate all rod-specific genes (e.g., rhodopsin, PDE-a, PDE-b) but the repression of cone genes (e.g., S-opsin) is not efficient, leading to hybrid photoreceptors in mice expressing NRL but not NR2E3 (rd7 mice). NR2E3, on the contrary, can repress cone genes but it is unable to efficiently activate rod genes in the absence of NRL. Both NRL and NR2E3 interact with a multitude of regulatory proteins to accomplish transcriptional regulation. NRL is a highly phosphorylated protein and its function is modulated by several kinases. NRL also interacts with TATA-binding protein (TBP) and, presumably, brings the basal transcriptional machinery to target gene promoters. Recent studies have shown a key role for the protein inhibitor of activated STAT3 (PIAS-3) in the sumoylation of NR2E3, adding another level of control in gene expression and consequently rod differentiation. A combined action of NRL and NR2E3 is essential to generate functional rod photoreceptors. CRX, on the other hand, acts as an enhancer of both rod and cone genes. While photoreceptors are produced even in the absence of CRX, these cells do not elaborate outer segments because of the low expression of most, if not all, phototransduction and structural proteins. CRX is, therefore, necessary to produce functional photoreceptors. CRX also interacts with coactivator proteins that possess histone acetyltransferase (HAT) activity and recruits HATs to promoter/enhancer regions to acetylate histone H3, thereby inducing and maintaining chromatin configurations that facilitate binding of NRL, NR2E3, and RNA

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polymerase II. These recently described molecular mechanisms underscore the importance of as yet poorly understood epigenetic factors in determining retinal photoreceptor cell fate and maturation.

Current Research in Photoreceptor Development Cell interactions and intrinsic cellular mechanisms modulate gene expression and regulate photoreceptor differentiation. Among these, chromatin remodeling and small regulatory RNAs have attracted attention in recent years. Histone methylation/acetylation are key epigenetic modifications that govern chromatin dynamics. The role of chromatin-modifying activities in directing tissue-specific development is an active area of investigation. Histone acetylation is emerging as an important mechanism in regulating photoreceptor development. Acetylation of histone H3 by HATs – recruited by CRX – appears important in maintaining chromatin configurations permissive to NRL/NR2E3 transcriptional activity in developing rods. Furthermore, histone deacetylase 4 (HDAC4) activity is shown to promote the survival of newly differentiated photoreceptors. More recently, the chromatin-remodeling complex Baf60c has been identified in differentiating, but not mature, retinal cells. A family of three DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, partially cooperate to establish and maintain genomic DNA methylation patterns. The presence of high levels of Dnmt3a and of Dnmt3b in the mouse rostral neural tube, including the optic grooves and cranial neural folds at E8.5, and in the area of evaginating optic vesicles at E9.5 is suggestive of the role Dnmts play in eye development. Epigenetic chromatin-remodeling mechanisms are also active in retinal neuroblasts undergoing cell-fate commitment, and are likely to contribute to retina-restricted patterns of gene expression. For example, hypomethylation is suggested to modulate interphotoreceptor retinoid-binding protein (IRBP) gene activation during photoreceptor genesis. Little is known about the influence of methylation on retinal cell fate, yet it is plausible that DNA methylation is actively involved in establishing RPC competence. MicroRNAs (miRs) are short (18–24 nucleotides), noncoding, RNA sequences that modulate gene expression by binding the 30 (and 50 ) untranslated region (UTR) of their target RNAs, thus regulating their stability and translation. They originate as longer RNA transcripts that are processed and cleaved by the subsequent activity of two RNase III endonucleases – the Drosha-DGCR8 complex and Dicer. MiRs could play a gene-regulatory role in development (including retinal) that is comparable to that of transcription factors. For example, in the Drosophila eye,

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miR-7 is activated by EGF-receptor (EGFR) signaling in cells undergoing the initial steps in photoreceptor differentiation. Furthermore, conditional Dicer-null mutation in the developing retina results in apparently normal retinal structure – albeit interspersed with rosettes and fated to progressive degeneration – pointing to the role of miRs in normal photoreceptor genesis and function. Further studies are warranted to fully elucidate the role of miRs in photoreceptors in general and in their development in particular.

Conclusions When investigating cellular events and molecular mechanisms, the accuracy of models is only as good as the methods used to collect the data and the relevance to human retinal development of the animal model used. Future studies in other vertebrate species (e.g., zebrafish) and in human embryonic stem cells, with more sophisticated tools to dissect cell-fate specification/determination mechanisms, and the analysis of the vast amount of omic data available will permit integration of current photoreceptor development models to more closely represent the relevance to human disease. It is the authors’ auspice that this article be read as the starting point to stimulate the reader’s curiosity to further investigate the field of retinal developmental neurobiology for more in-depth understanding and up-to-date breakthroughs. See also: Coordinating Division and Differentiation in Retinal Development; Embryology and Early Patterning; Histogenesis: Cell Fate: Signaling Factors; Microvillar and Ciliary Photoreceptors in Molluskan Eyes; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Retinal Histogenesis; Zebra Fish–Retinal Development and Regeneration.

Further Reading Adler, R. and Raymond, P. A. (2008). Have we achieved a unified model of photoreceptor cell fate specification in vertebrates? Brain Research 4: 134–150. Chen, J., Rattner, A., and Nathans, J. (2005). The photoreceptorspecific nulear receptor Nr2e3 represses transcription of multiple cone-specific genes. Journal of Neuroscience 25: 118–129. Cheng, H., Aleman, T. S., Cideciyan, A. V., et al. (2006). In vivo function of the orphan nuclear receptor NR2E3 in establishing photoreceptor identity during mammalian retinal development. Human Molecular Genetics 15: 2588–2602. Fishman, R. S. (2008). Evolution and the eye: The Darwin bicentennial and the sesquicentennial of the origin of species. Archives of Ophthalmology 126: 1586–1592. Hatakeyama, J. and Kageyama, R. (2004). Retinal cell fate determination and bHLH factors. Seminars in Cell and Developmental Biology 15: 83–89. Hendrickson, A., Bumsted-O’Brien, K., Natoli, R., et al. (2008). Rod photoreceptor differentiation in fetal and infant human retina. Experimental Eye Research 87: 415–426. Lamba, D., Nelson, G., Kari, M. O., and Reh, T. A. (2008). Specification, histogenesis, and photoreceptor development in the mouse retina. In: Chalupa, L. M. and Williams, R. W. (eds.) Eye, Retina, and Visual System of the Mouse, pp. 299–310. Cambridge, MA: MIT Press. Livesey, F. J. and Cepko, C. L. (2001). Vertebrate neural cell-fate determination: Lessons from the retina. Nature Reviews Neuroscience 2: 109–118. Mears, A. J., Kondo, M., Swain, P. K., et al. (2001). Nrl is required for rod photoreceptor development. Nature Genetics 29: 447–452. Ng, L., Hurley, J. B., Dierks, B., et al. (2001). A thyroid hormone receptor that is required for the development of green cone photoreceptors. Nature Genetics 27: 94–98. Nishida, A., Furukawa, A., Koike, C., et al. (2003). Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development. Nature Neuroscience 6: 1255–1263. Oh, E. C., Khan, N., Novelli, E., et al. (2007). Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL. Proceedings of the National Academy of Sciences of the United States of America 104: 1679–1684. Onishi, A., Peng, G.-H., Hsu, C., et al. (2009). Pias3-dependent SUMOylation directs rod photoreceptor development. Neuron 61: 234–246. Roberts, M. R., Srinivas, M., Forrest, D., et al. (2006). Making the gradient: Thyroid hormone regulates cone opsin expression in the developing mouse retina. Proceedings of the National Academy of Sciences of the United States of America 103: 6218–6223. Zhang, J., Gray, J., Wu, L., et al. (2004). Rb regulates proliferation and rod photoreceptor development in the mouse retina. Nature Genetics 36: 351–360.

The Photoreceptor Outer Segment as a Sensory Cilium J C Besharse and C Insinna, Medical College of Wisconsin, Milwaukee, WI, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Axoneme – The cytoskeletal backbone of cilia and flagella composed of nine microtubule doublets aligned in a cylindrical array. Motile axonemes, generally referred to as 9 + 2 axonemes, contain a pair of central singlets and dynein arms. Basal body – A centriole that becomes associated with the cell membrane for the nucleation of a cilium. Doublet microtubules of cilia in most animals are extensions of triplets of the basal body. Centriole – A barrel-shaped or cylindrical organelle composed in most animals of nine triplet microtubules. A pair of centrioles, surrounded by an amorphous zone containing many proteins, constitutes the centrosome. Cilia – The extensions of the cell surface that have a plasma membrane and contain a cytoskeletal core of microtubules, called an axoneme, which extends out from the basal body. They may be motile, as in the airway epithelium in humans, or nonmotile and sensory in function. Cilia and flagella have the same organization, but flagella are generally motile and are longer. Intraflagellar transport – A microtubule-based trafficking pathway required for the formation and maintenance of cilia and flagella. Intraflagellar transport (IFT) involves the bidirectional transport of a protein adaptor complex composed of highly conserved IFT proteins. The pathway requires kinesin 2 family motors in the outward (anterograde) direction and a cytoplasmic dynein in the return (retrograde) direction. The pathway is equally important in cilia and flagella. Kinesins – The plus-end-directed motor proteins powered by adenosine triphosphate hydrolysis to walk along microtubules. These are involved in multiple cellular functions, including transport of cargo, mitosis, and meiosis. Many different genes encode kinesins with different properties and functions. Microtubules – The cytoskeleton tubules assembled from protofilaments composed of linear polymers of a- and b-tubulin heterodimers. These are asymmetric hollow cylinders with plus and minus ends that differ in polymerization rate and binding proteins. They form a dynamic network within the cell and organize themselves into complex structures, such centrioles, basal bodies, and cilia. Microvilli – The membrane protrusion from the cell surface composed of cytoplasm and dense bundles

of actin filaments serving to increase the surface area of the cell. Sensory cilia – The cilia that contain membrane receptors and signaling components. These often have a nonmotile 9 + 0 axoneme, but actively motile cilia may also serve a sensory function.

Introduction There are at least two fundamentally different designs for visual photoreceptors, generally referred to as the rhabdomeric type and the ciliary type. Rhabdomeric photoreceptors are widely represented among invertebrates and have been intensively studied in the horseshoe crab, fruit fly, and squid. Rhabdomeric photoreceptors concentrate the visual pigment for photon capture in a highly replicated array of microvilli, each containing an actin cytoskeletal core. The array of microvilli is called the rhabdomere. The visual pigment of ciliary photoreceptors is also concentrated in a photon capture organelle called the outer segment (OS). However, the OS is derived from the plasma membrane of a cilium and retains the microtubule-based cytoskeletal core, called an axoneme, which is common to all cilia. Often, rhabdomeric and ciliary photoreceptors are referred to as invertebrate and vertebrate types, respectively, but this distinction can be misleading. Both photoreceptor types appeared early in animal evolution and are present in multiple invertebrate phyla. Furthermore, some animals have both photoreceptor types, and the retina in the compound eye of the scallop contains a layered juxtaposition of both rhabdomeric and ciliary photoreceptors. Although vertebrate photoreceptors with true rhabdomeres have not been identified, the recent discovery of intrinsically light-sensitive ganglion cells (ipGCs) has led to the realization that their melanopsin photopigment and transduction pathway have features in common with rhabdomeric rather than ciliary photoreceptors. This suggests that ipGCs may have originated from an evolutionary ancient rhabdomeric precursor.

Turnover of the OS and Phototransduction Machinery The light-sensitive OS of vertebrate photoreceptors is an elegantly complex organelle that serves as the starting

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point for highly sensitive nighttime vision (rods) as well as high acuity, color vision (cones) in the daytime. The OS consists of a stack of membrane disks containing a photopigment (opsin) of the guanine-nucleotide-binding protein-coupled receptor (GPCR) family, and a large array of cytosplasmic, cytoskeletal, membrane, and membraneassociated proteins that are essential for phototransduction. The phototransduction cascade begins with photon absorption by the vitamin-A-derived chromophore of opsin and proceeds through activation of transducin, a guanine-nucleotide-binding protein (G-protein), which in turn activates a phosphodiesterase that reduces cytoplasmic cyclic guanosine monophosphate (cGMP) levels. The photoresponse is a hyperpolarization event that occurs when declining local cGMP levels result in closure of the cGMP-gated channel in the OS plasma membrane. The high sensitivity of the rod cells, which can respond to single-photon absorption events, and the rapid responses of cone cells in bright light, depend on the integrity of the disk stack and close juxtaposition of the protein components of the transduction cascade. Optimal function also depends on other OS proteins that support transduction such as membrane guanylyl cyclases (GC1 and GC2), which regenerate cGMP, a Na+/Ca2+ exchanger which regulates Ca2+ levels, anaerobic glycolysis, which supplies a portion of the adenosine triphosphate (ATP), and the pentose phosphate shunt which supplies nicotinamide adenine dinucleotide phosphate (NADPH), essential for conversion of the all-trans retinaldehyde to retinol in the visual pigment cycle. Two additional interdependent features of OS organization, essential for optimal visual function, are proper targeting of the phototransduction proteins to the OS and long-term maintenance through OS renewal. A striking feature of normal rods and cones is the high level to which phototransduction proteins are concentrated in the OS. Since those proteins are all synthesized at polyribosomes in the inner segment, a great deal of recent focus has been on those mechanisms that are essential for proper trafficking of OS proteins. This is a major problem throughout the life of the cell because OSs renew at a particularly high rate (see Figure 1). In the 1960s, Richard Young, then at the University of California at Los Angeles, thoroughly documented the fact that rod OSs are renewed through continuous new disk assembly adjacent to the inner segment. Furthermore, Young along with Dean Bok determined that OS length is maintained through a compensatory shedding of disks from the distal tip where they are phagocytized and degraded by the retinal pigment epithelium. In the 1970s, these same concepts were extended to include the cone OSs. The turnover of OSs is highly conserved, but occurs at very different rates in different species. In mice, rod OSs turnover once every 10 days.

The Photosensitive Organelle as a Sensory Cilium With the advent of transmission electron microscopy in the 1950s, photoreceptors were an early subject of analysis, particularly by Eduardo de Robertis in Argentina and Kiyoteru Tokuyasu and Eichi Yamada in Japan. Those early studies showed that developing photoreceptors have the basic organization of cilia. During early differentiation, a centriole pair moves to the plasma membrane where one member serves as a template for assembly of a microtubule cytoskeletal structure called an axoneme (Figure 2); the other centriole is generally seen next to the basal body as an accessory centriole. Centrioles consist of an array of nine triplet microtubules and each triplet has an A-, B-, and C-tubule. The A-tubule is a complete microtubule comparable to cytoplasmic microtubules, but the B subtubule is a partial microtubule built on the wall of the A-tubule. Likewise, the C-tubule is built on the wall of B. The centriole that associates with the plasma membrane is called a basal body because it serves as a template for outgrowth of doublet microtubules that form the axoneme. As a consequence of basal body templating, the axoneme grows out as an array of nine doublet microtubules that are direct extensions of the A- and B-tubules of the basal body. The photoreceptor and other sensory cilia are generally said to have a 9 + 0 axoneme, in contrast to motile cilia, which have a 9 + 2 axoneme; the 2 in the latter designation refers to a central pair of single microtubules within the core of the axoneme. Although the 9 + 2 axoneme is found in a wide array of motile cilia, 9 + 0 cilia are sometimes motile. For example, the rotatory cilia of the embryonic Henson’s node have a 9 + 0 axoneme. As the axoneme elongates in rodents, the plasma membrane expands at the distal end of the cilium, and membrane vesicles and tubules accumulate (Figure 2). At this early stage, the photopigment apoprotein, opsin, is localized within the plasma membrane of the distal end of the cilium, and membrane vesicles and tubules accumulate. This region expands in the early stages of OS formation, quickly taking the form of an orderly stack of membrane disks (Figure 2). Frog OSs, as described by S. E. Nilsson in 1964, exhibit ordered disks from the very beginning of differentiation. At later stages, and, presumably during early development, new membrane disks are thought to form as evaginations of the ciliary membrane. The most proximal part of the cilium emanating from the basal body, called the connecting cilium by Eduardo de Robertis in 1956, connects the photosensitive OS with the cells synthetic machinery in the inner segment; components of the phototransduction machinery in the OS are synthesized in the inner segment and must be transported to the OS through the connecting cilium.


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Figure 1 Conceptual diagram of a pulse chase autoradiography experiment revealing OS turnover in rod cells. Radioactive amino acids provided as a pulse at 1 (left) were incorporated into protein, mainly rhodopsin, in the inner segment (IS). Over the next few hours (2–3) radioactive protein was transported to the apical inner segment and disk-forming region at the connecting cilium (CC), and incorporated into newly formed disks creating a discrete radioactive band (4). Over the next 10 days (mammals), the band was gradually displaced toward the distal end of the OS (5–6) until discarded in a process called disk shedding (7). Discarded disks (7) are phagocytized by adjacent retinal pigment epithelium (not shown). From Young, R. W. (1967). The renewal of photoreceptor cell outer segments. Journal of Cell Biology 33: 61–72.

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Figure 2 Early development of the outer segment. After the last mitotic division the centriole pair (1, left) moves to the cell surface (2). One member of the centriole pair associates with the plasma membrane where it is called a basal body (BB); the second centriole is often seen adjacent to the basal body in EM images where it is called the accessory centriole (AC). The basal body nucleates the extension of doublet microtubules at the cell surface to elongate the cilium (3). In the early stages of cilium elongation membrane vesicles and tubules are seen in the ciliary cytoplasm (3–4). Finally, disks align perpendicular to the axoneme composed of doublet microtubules (5). Based on early EM analysis by Tokuyasu, K. and Yamada, E. (1959). The fine structure of the retina studied with the electron microscope. IV. Morphogenesis of outer segments of retinal rods. Journal of Biophysical and Biochemical Cytology 6: 225–230; De Robertis, E. (1960). Some observations on the ultrastructure and morphogenesis of photoreceptors. Journal of General Physiology 43(6) supplement, 1–13; and Greiner, J. V., Weidman, T. A., Bodley, H. D., and Greiner, C. A. M. (1981). Ciliogenesis in photoreceptor cells of the retina. Experimental Eye Research 33: 433–446.

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Figure 3 Structure of the ciliary axoneme of a mature photoreceptor. Longitudinal view is shown on the right and magnified cross sections at the position of the arrows on the left. The axoneme grows out of the basal body as an array of nine doublet microtubules. The microtubule plus ends extend distally and the minus ends are anchored in the basal body. The region adjacent to the basal body is a transition zone in which doublets are closely linked with the plasma membrane by Y-shaped crosslinkers (lower diagram on left); this region is often referred to as the connecting cilium. The region immediately distal to the transition zone is the site of new disc assembly. Within the OS the doublets loose their B-tubule to become singlets in the distal OS. This is shown as an abrupt transition on the right, but conversion to singlets appears to occur gradually leaving mixtures of singlets and doublets (middle diagram on left). The distal OS only has singlets.

The term connecting cilium refers to that portion of the cilium between the basal body and the disk-forming region of the OS. As originally demonstrated by Pal Ro¨hlich in 1976, the connecting cilium is actually comparable to the transition zone, a structure common to all eukaryotic cilia and flagella (Figure 3). Here, the plasma membrane is closely linked to the doublet microtubules of the axoneme by cross-linkers that extend from the doublet microtubule to the plasma membrane. These cross-linkers are stable structures that remain bound to the axoneme after detergent extraction and link the axoneme to cell surface glycoconjugates through transmembrane connections. Although these microtubule-membrane cross-linkers exhibit a conserved Y-shaped structure across many cilium types, their molecular composition has not been determined. Recently, a number of cilium proteins relevant to human photoreceptor

degenerative disease, such as retinitis pigmentosa guanosine triphosphate (GTP)ase regulator (RPGR) and RPGRinteracting protein 1 (RPGRIP), have been localized to the transition zone and some may be components of the cross-linking structures. Further high-resolution analysis of the composition of cross-linkers may lead to a better understanding of the function of connecting cilium proteins that are relevant to human disease. Distally, beyond the transition zone, the axoneme extends deep into the OS (Figure 3). This point requires emphasis because the term connecting cilium refers to the link between inner and OS and the term is often used with the implication that this is the entire cilium. However, both early electron microscopic studies and numerous immunocytochemistry studies have shown that photoreceptor axonemes extend through much of the length of the OS (Figure 4); in some cases, they extend all the way to the distal tip. The recent finding of extremely long axonemes in mouse, frog, and zebrafish OSs, along with earlier studies showing much shorter axonemes, suggests that they may vary significantly in their length. The reason for variability in axoneme length observed in various studies is not known. A possible explanation, however, is that the distal axoneme is dynamic and unstable, resulting in some cases in poor preservation for morphological studies. The principles governing these length variations in either rods or cones remain unknown, but are likely to be relevant to the finding that OSs maintain a relatively constant length through many cycles of OS turnover.

Evidence for Intraflagellar Transport in Photoreceptors Recently our work has demonstrated that the assembly of photoreceptor OSs depends on a highly conserved microtubule-based trafficking pathway called intraflagellar transport (IFT). IFTwas originally discovered in the motile flagella of the green alga Chlamydomonas rheinhartii and quickly extended to the sensory cilia Caenorhabditis elegans. The essential components of IFT are the kinesin and dynein molecular motors that drive movement along axonemal microtubules and multiprotein IFT particles that are thought to link cargo such as cytoskeletal and phototransduction proteins to the IFT motors (see Figure 5). For example, at least 16 different proteins assemble to form two large protein complexes referred to as IFT particles (Figure 5), and all of these proteins are highly conserved between C. rheinhartii and man. IFT transport is bidirectional. In anterograde IFT plus-end-directed motors of the kinesin 2 family move IFT particles with attached cargo toward the plus end of the axoneme, while in retrograde IFT a minus-end-directed dynein motor returns the IFT machinery for exchange with a pool in the cell body. Again, the pathway is highly conserved in that the same molecular


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Figure 4 Immunocytochemical labeling of the axoneme of cone OSs from zebrafish. An antibody to a-tubulin was used to label (red) the microtubules of the axoneme (large arrows). An immunofluorescence image is on the left and for orientation this image is merged with a phase image of the same cells on the right. The image includes a long single cone (upper left) and a double cone (lower right). Note that the axoneme staining is attenuated distally (small arrows), particularly in the long single cone. Magnification bars in lower right equal 8.91 mm. IS, inner segment; OS, outer segment.

motors identified in C. rheinhartii and C. elegans perform similar functions in virtually all eukaryotic cilia including those in man. For example, heterotrimeric kinesin II, assembled from the kinesin family member 3A and 3B (KIF3A, KIF3B) along with kinesin-associated protein 3 (KAP3) proteins, is the canonical anterograde motor, while a dynein containing the cytoplasmic dynein 2 heavy chain (DNCH2) is the canonical retrograde motor. A prominent role for IFT in photoreceptor OS formation is now well established. Four of the IFT proteins (IFT88, IFT57, IFT52, and IFT20) have been localized along photoreceptor axonemes and photoreceptor OS assembly defects have been fully characterized in rods of mice with a mutation in IFT88. This has been extended to cone cells with targeted, cone-specific deletion of IFT20, which results in disrupted OS assembly. A central feature of the IFTmodel is the multi-protein IFT particle (Figure 5). IFT particles containing IFT88, IFT57, IFT52, and IFT20 can be isolated from bovine photoreceptor outer segments. The photoreceptor IFT particle is large, fractionating in sucrose gradients at a peak size of 500–750 kDa and has properties remarkably similar to those originally described in C. rheinhartii. This implies that the photoreceptor particle contains additional IFT proteins for which antibodies have not yet been generated. Evidence for photoreceptor IFT is also based on analysis of its canonical motors. All three subunits of kinesin II as well as DNCH2 have been localized to photoreceptor axonemes. Furthermore, conditional deletion of the KIF3A subunit of kinesin II disrupts OS assembly, causes rhodopsin mislocalization, and results in photoreceptor cell death.

A Special Role for KIF17 in Photoreceptors The foregoing description of photoreceptor IFT describes conditions and expectation of a canonical IFTmodel that has

applicability in virtually all ciliated cells. However, a novel feature of photoreceptor IFT is the critical involvement of an additional kinesin motor, the homodimeric kinesin family member 17 (KIF17). As illustrated in Figure 5, both kinesin II and KIF17 are associated with the IFT particle along doublet microtubules in the proximal OS, but KIF17 alone is associated with movement of the IFT particle along singlet microtubules in the distal OS. Our recent work has demonstrated that KIF17 is required to form outer segments, but does not simply replace kinesin II function; both kinesins are required. An interesting feature of this work is that while reduced kinesin II function disrupts both photoreceptor and kidney cilium elongation, knockdown of KIF17 results in failed OS assembly with no apparent effect in the kidney. The importance of KIF17 is likely related to the presence of singlet microtubule extensions in photoreceptors (see Figure 3). While singlet extensions are prominent in photoreceptor cilia, as originally illustrated in the older EM literature, singlet extensions in kidney sensory cilia are either very short or not present at all. Work from the laboratory of Jonathan Scholey at the University of California at Davis has shown that the C. elegans KIF17 homolog, osmotic avoidance abnormal protein (OSM-3), is also required for sensory cilium elongation, specifically in cilia with singlet extensions. In C. elegans cilia OSM-3 serves as an accessory IFT motor along with kinesin II. Specifically, either kinesin motor can function and compensate for loss of the other motor in the proximal cilium, which contains doublet microtubules, but only OSM-3 can extend and move along the distal singlets. In fact, it was the existence of singlet extensions in photoreceptors that drew our attention to KIF17 and led to our finding of a prominent role of this accessory kinesin motor. However, the simple model for dual IFT kinesins in C. elegans does not fully explain findings in photoreceptors. The C. elegans model predicts that knockdown of KIF17 would result in short OSs that fail to elongate.

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consensus calcium, calmodulin kinase II (CaMKII) phosphorylation site in the KIF17 tail region, demonstrates that KIF17 plays a critical role in the distal OS in the control disk shedding (see Figure 1).

What Is the IFT Cargo?

Anterograde

Retrograde Dynein KIF17 Kinesin II IFT particle Cargo

Figure 5 Conceptual diagram of intraflagellar transport (IFT) in photoreceptors. (Left) Anterograde IFT uses the plus end directed kinesin motors, kinesin II and KIF17, to move IFT particles with attached ’cargo’ toward the distal, plus end of the axoneme (arrow). Note that the minus-end-directed motor, dynein, is illustrated as cargo for anterograde IFT. Based largely on work in C. elegans, it is hypothesized that kinesin II and KIF17, move cooperatively on doublets in the proximal outer segment and that KIF17 operates alone on singlets in the distal outer segment. (Right) Retrograde IFT uses a cytoplasmic dynein motor to return IFT particles and cargo to the inner segment. Note that kinesin II and KIF17 are illustrated as cargo for retrograde IFT. IFT complexes along with cargo and molecular motors are thought to assemble into large complexes in the inner segment in the region adjacent to the basal body.

Results from direct comparison of mutated forms of the KIF3B subunit of kinesin II and KIF17 suggest that the motors carry out nonredundant functions and suggest that, in addition to a role for KIF17 in distal cilium extension, the motor may also be involved in transport of proteins that are essential for disk assembly in the proximal outer segment. Although knockdown of KIF17 results in ablation of OS formation, a recent mutagenesis study of a

The requirement for IFT in ciliogenesis has led to the general idea that the IFT particle serves as an adaptor to link IFT cargo to the requisite IFT motors. Some of the early evidence for cargo relates to the motors themselves. Since the axoneme plus end is at the distal end of the cilium, the minus-end-directed dynein motor required for retrograde IFT cannot move from the cell body to the distal tip on its own and available data suggest that it is cargo during anterograde IFT. Likewise, recycling of the plus-end-directed kinesins back to the inner segment appears to require retrograde IFT (see Figure 5). Another feature of axoneme organization is that its microtubules can assemble or disassemble only at the plus end, which is located distally. Since the axoneme can both elongate and shorten, its building blocks are likely cargo in both directions. It is important to point out that since the axoneme provides the principle cytoskeletal scaffold for the OS, IFT would be of great significance if its main purpose were to maintain axoneme structure. Recent evidence, however, suggests that IFT transports membrane components of the OS as well. The photoreceptor OS provides a special challenge in that the components of phototransduction are present in high abundance and turn over rapidly. The question is: Which of these components is moved to the OS by IFT? One reasonably clear conclusion is that not all OS components require IFT. For example, some phototransduction proteins, such as arrestin and transducin, move between inner and OS in relationship to light and dark adaptation and a strong case can be made for free diffusion as the underlying mechanism for movement of abundant freely soluble proteins. Current evidence favors the idea that many membrane and membrane-associated proteins may require IFT. In C. rheinhartii and C. elegans, where real time imaging is feasible, movement of specific membrane proteins has been detected along the cilium. Rigorous proof that a particular protein is IFT cargo requires direct demonstration that it is linked to the IFT machinery and moves with IFT components, and there are no examples of this type of evidence outside of C. rheinhartii and C. elegans. Nonetheless, mutation of the KIF3A subunit of the kinesin II motor, and mutation of the IFT88 subunit of the IFT particle both result in rhodopsin mislocalization, suggesting that rhodopsin is carried as IFT cargo. In support of the specificity of this effect, we recently demonstrated that expression of a dominant-negative


form of the KIF3B subunit of kinesin II in zebrafish cones results in cone opsin mislocalization, but dominantnegative KIF17 blocks OS elongation without causing opsin mislocalization. Since studies of protein mislocalization in cells carrying mutations is an inherently indirect measure, we have recently used a variety of pull-down assays to isolate IFT protein complexes containing the two kinesin motors as well as rhodopsin and another OS membrane protein, retinal guanylyl cyclase 1 (RetGC1 also called GUCY2E). The work with GC1 is particularly interesting because we have identified a small chaperone protein (DNAJB6) that binds specifically to IFT88 in the IFT particle and to the kinase homology domain in the cytoplasmic tail region of GC1. We have proposed that this is the key linkage required for association of membrane GC1 with the IFT machinery and that the binding can be regulated directly through the ATPase activity of heat-shock cognate protein 70 (HSC70). This represents the first specific model for the molecular linkage of a cargo protein with the IFT machinery.

Summary and Perspective The fact that photoreceptor OSs are sensory cilia has been known for many years, but recent advances in understanding the mechanisms underlying ciliogenesis, including the IFT pathway, are likely to have a large impact on our understanding of the cell biology of photoreceptors. Although mutations in genes involved in ciliogenesis often cause embryonic lethal phenotypes and will not frequently appear among the causes of photoreceptorspecific degenerative disease, our understanding of disease mechanisms are likely to improve through understanding which photoreceptor proteins are IFT cargo. For example, human GC1 contains three human disease causing mutations in the domain that we have recently shown to bind a linker chaperone that couples GC1 to the IFT machinery. This has led us to propose that those human mutations lead to abnormal ciliary trafficking. The importance of photoreceptor cilia also provides a basis for understanding syndromic diseases such a Bardet–Biedl syndrome, which includes RP among a complement of other abnormalities. Such diseases are now referred to as ciliopathies because cilia provide a common basis for understanding pathology in the different tissues that are affected. Finally, placement of photoreceptor OSs in their appropriate niche as a special type of sensory cilium provides evolutionary perspective on pathways common to all cilia that emerged early in eukaryote evolution. See also: Circadian Photoreception; Genetic Dissection of Invertebrate Phototransduction; Limulus Eyes and Their Circadian Regulation; Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors; Microvillar and Ciliary Photoreceptors in Molluskan Eyes;

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The Photoresponse in Squid; Phototransduction: Adaptation in Rods; Phototransduction in Limulus Photoreceptors; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Phototransduction: The Visual Cycle; Retinal Degeneration through the Eye of the Fly; Rod and Cone Photoreceptor Cells: Outer Segment Membrane Renewal.

Further Reading Baker, S. A., Freeman, K., Luby-Phelps, K., Pazour, G. J., and Besharse, J. C. (2003). IFT20 links kinesin II with a mammalian intraflagellar transport complex that is conserved in motile flagella and sensory cilia. Journal of Biological Chemistry 278: 34211–34218. Besharse, J. C. and Horst, C. J. (1990). The photoreceptor connecting cilium. A model for the transition zone. In Bloodgood, R. A. (ed.) Ciliary and Flagellar Membranes, pp. 389–417. New York: Plenum. Bhowmick, R., Li, M., Sun, J., et al. (2009). Photoreceptor IFT complexes containing chaperones, guanylyl cyclase 1, and rhodopsin. Traffic 10(6): 648–663. De Robertis, E. (1960). Some observations on the ultrastructure and morphogenesis of photoreceptors. Journal of General Physiology 43(6): supplement, 1–13. Eckmiller, M. S. (1996). Renewal of the ciliary axoneme in cone outer segments of the retina of Xenopus laevis. Cell Tissue Research 285: 165–169. Hong, D. H., Yue, G. H., Adamian, M., and Li, T. S. (2001). Retinitis pigmentosa GTPase regulator (RPGR)-interacting protein is stably associated with the photoreceptor ciliary axoneme and anchors RPGR to the connecting cilium. Journal of Biological Chemistry 276: 12091–12099. Inglis, P. N., Ou, G., Leroux, M. R., and Scholey, J. M. (2007). The sensory cilia of Caenorhabditis elegans. WormBook March 8: 1–22. Insinna, C. and Besharse, J. C. (2008). Intraflagellar transport and the sensory outer segment of vertebrate photoreceptors. Developmental Dynamics 237: 1982–1992. Insinna, C., Pathak, N., Perkins, B., Drummond, I., and Besharse, J. C. (2008). The homodimeric kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment development. Developmental Biology 316: 160–170. Maerker, T., van Wijk, E., Overlack, N., et al. (2008). A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells. Human Molecular Genetics 17: 71–86. Marszalek, J. R., Liu, X., Roberts, E. A., et al. (2000). Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors. Cell 102: 175–187. Pazour, G. J., Baker, S. A., Deane, J. A., et al. (2002). The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance. Journal of Cell Biology 157: 103–113. Rohlich, P. (1975). The sensory cilium of retinal rods is analogous to the transitional zone of motile cilia. Cell Tissue Research 161: 421–430. Tokuyasu, K. and Yamada, E. (1959). The fine structure of the retina studied with the electron microscope. IV. Morphogenesis of outer segments of retinal rods. Journal of Biophysical and Biochemical Cytology 6: 225–230. Young, R. W. (1967). The renewal of photoreceptor cell outer segments. Journal of Cell Biology 33: 61–72. Young, R. W. and Bok, D. (1969). Participation of the retinal pigment epithelium in rod outer segment renewal process. Journal of Cell Biology 42: 392–403.

The Photoresponse in Squid J Mitchell and W Swardfager, University of Toronto, Toronto, ON, Canada ã 2010 Elsevier Ltd. All rights reserved.

Glossary Arhabdomeral lobe – The proximal segment of the photoreceptor cell containing the nucleus and other organelles. Arrestin – A protein that binds to metarhodopsin and arrests phototransduction by inhibiting metarhodopsin activation of its target gaunine nucleotide-binding protein. Calpain-like protease – An enzyme isolated from squid photoreceptors that, like calpain, requires millimolar concentrations of calcium for its proteolytic activity. Metaretinochrome – The conformation of retinochrome when retinal has been photoisomerized to 11-cis-retinal. Metarhodopsin – The light-activated conformation of opsin when bound to all-trans retinal. Retinochrome – A photosensitive protein that binds all-trans retinal. Rhabdomeral lobe – The distal segment of the photoreceptor cell containing the molecular machinery of phototransduction. Rhabdomere – The collective surfaces of the distal segment composed of densely packed microvilli. Rhodopsin kinase – An enzyme that adds phosphates onto serine or threonine residues in the carboxyl-terminus of metarhodopsin.

Introduction Squid, like most invertebrates, have light-sensing organs. The visual systems of squid are composed of camera-type eyes. Incoming light passes through a single lens and an image is formed on the light-sensing cells of the retina in the anterior chamber of the eye. The retina consists of a single layer of photoreceptive neurons that are segmented in structure. The outer segment, also known as the rhabdomeral lobe, contains the protein machinery of phototransduction. The inner segment, or the arhabdomeral lobe, contains the cellular organelles involved in protein synthesis and the soma contains the cell nucleus. Axons arising from the soma of the photoreceptors comprise the optic nerve that projects to the squid brain (Figure 1).

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The photoreceptor outer-segment membrane forms densely packed microvilli that greatly increase the surface area of the membrane available to capture incoming photons of light (see enlargement in Figure 1). Embedded in the membrane are the rhodopsin receptors containing light-sensitive chromophores. Light activation of rhodopsin sets off a cascade of molecular interactions that culminate in depolarization of the photoreceptor membrane (Figure 2). In recent years, many of the molecular components of the light-activated signaling system have been identified and a review of our current knowledge of these components is outlined here. Equally important to vision are the molecular mechanisms that inhibit signal transduction after transmission of the light signal (Figure 3). These mechanisms are not well understood; however, many of the components of the inactivation pathway have been revealed and these will also be discussed.

Molecular Components of Squid Visual Signal Transduction The squid visual signal transduction system is composed of a light-sensitive receptor, rhodopsin, that transduces its activation signal via a heterotrimeric G protein (Gq) to a phospholipase C (PLC) enzyme. Activated phospholipases hydrolyze membrane phospholipids liberating soluble inositiol 1,4,5-trisphosphate (IP3) and membranebound diacylglycerol (DAG). While it is still not clear how these second messengers stimulate membrane depolarization, it probably involves release of calcium from the submicrovillar tubules as a result of IP3 stimulation of receptors on these organelles and perhaps direct stimulation of transient receptor potential (TRP)-like channels in the membrane by DAG. Together, these mechanisms raise intracellular calcium and increase membrane depolarization.

Squid Rhodopsin Squid rhodopsin consists of a guanine nucleotide-binding protein-coupled receptor (GPCR) or opsin bound to a light-absorbing retinoid chromophore. Squid opsin genes have been cloned from Loligo forbesi, Loligo pealei, and Todarodes pacificus, and they share 45% sequence identity with other invertebrate and vertebrate opsins. However,


Outer segments

Iris

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Inner segments

Lens Retina Microvilli Pigment granules

Nucleus

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Figure 1 Structure of the squid eye and photoreceptors. Squid have camera-type eyes in which incident light enters the eye and an image is focused through a lens onto the retina in the anterior chamber of the eye. The enlargement on the right shows the orientation of the photoreceptors in the retina. The outer (rhabdomeral) segments contain the microvillar membranes in which rhodopsin and all of the photoreceptor proteins are embedded or associated. The inner (arhabdomeral) segments contain the cell organelles while the photoreceptor cell axons compose the optic nerve that extends to the brain.

h(500 nm)

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(b)

Depolarizing current

Intracellular release of

2+

Ca

Figure 2 Activation of the squid visual system. (a) In the dark state, rhodopsin is likely coupled to an inactive (GDP-bound) invertebrate Gq (iGq) protein. On absorption of a photon, 11-cis-retinal isomerizes to all-trans-retinal changing rhodopsin to metarhodopsin, which stimulates GDP–GTP exchange on the iGqa subunit. (b) Activated iGqa-GTP changes conformation to interact with PLC stimulating the enzyme to hydrolyze phosphoinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and leaving DAG in the membrane. IP3 stimulates the release of calcium (Ca2+) from submicrovillar stores and, together with DAG, stimulates opening of an ion channel in the membrane. Membrane depolarization is transmitted to the optic lobe of the squid brain through the photoreceptor cell axons.

squid opsins are about 100 residues larger than those of vertebrates, primarily owing to the addition of a prolinerich carboxyl-terminal tail. Squid rhodopsins bear structural hallmarks of the GPCR superfamily, including

amino-terminal sites of N-linked glycosylation, carboxylterminal sites of palmitoylation, a disulfide bridge between two extracellular loops, proline residues in the a-helices of the transmembrane domains, and a (D/E)R(Y/W)

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h(493 nm)

h(500 nm)

(a)

(b)

(c)

(d)

Figure 3 Inactivation of the squid visual system. (a) Rhodopsin (R) stimulation by light (h 500 nm) stimulates a conformational change to metarhodopsin (M). (b) SQRK and arrestin (Arr) both bind with high affinity to metarhodopsin. Arrestin binding obstructs further interaction of metarhodopsin with iGq, uncoupling metarhodopsin from further stimulation of the signal transduction system. (c) SQRK phosphorylates metarhodopsin and following an increase in intracellular Ca2+ concentration, SQRK also phosphorylates arrestin. (d) A second light stimulus (h 493 nm) isomerizes all-trans-retinal back to 11-cis-retinal and converts metarhodopsin back to rhodopsin. Phosphorylation of arrestin and rhodopsin by SQRK may facilitate dissociation of the two proteins to return the system back to the dark state, which is primed to receive subsequent stimulation by light.

sequence in the third helix which is involved in G protein interactions. The presence of a proline-rich carboxyl-terminal tail is unique to cephalopod rhodopsins. This motif consists of 9–10 repeats of the pentapeptide Pro–Pro–Gln–Gly–Tyr that may facilitate receptor trafficking and morphogenesis. Though unique among rhodopsins, tandem repeats of proline-rich sequences are found in other protein families, where they are often associated with protein–protein interactions. Rhodopsin–rhodopsin interactions may be of structural importance in the cephalopod rhabdomere, since rhodopsin networks form in the microvilli of the rhabdomeral lobe. In native membranes, electron microscopy has revealed both poorly ordered rhodopsin clusters of 4–10 molecules and ordered rhodopsin pentameres. Intermolecular interaction is mediated in part by the rhodopsin carboxyl-termini, which aggregate and extend intracellularly from the membrane surface. In contrast, when the carboxyl-terminal is cleaved, crystalline lattice formation is observed both in reconstituted membranes and in crystallized rhodopsin. These more highly ordered crystalline arrays may be favored by interactions between transmembrane domains of adjacent molecules. It has therefore been suggested that the proline-rich region may function, in part, to limit crystalline array formation in native membranes, contributing instead to the formation of less-ordered rhodopsin clusters that confer membrane superstructure. Unfortunately, technical considerations prevent the observation of the squid rhodopsin crystal structure with an intact proline-rich tail, precluding definitive structural analysis.

The majority of opsins are covalently linked to an 11-cis-retinal chromophore and squid rhodopsin employs the 4-hydroxy-retinal derivative. In the squid, retinal is attached via a protonated Schiff ’s base linkage to a lysine residue in transmembrane domain 7 (residue 303 in Loligo and 305 in Todarodes). In contrast to the mammalian opsins, however, where the Schiff ’s base is stabilized using a glutamic acid residue of transmembrane domain 3 as a counterion, the crystal structure of Todarodes reveals the counterion to be a glutamic acid at residue 180, which is located between transmembrane helices 3 and 4. On absorption of a photon, 11-cis-retinal isomerizes to all-trans-retinal in a few hundred femtoseconds, which induces conformational changes in the opsin component. In invertebrates, the product of photoexcitation is an active metarhodopsin that is comparable in stability to inactivated rhodopsin. This contrasts the situation in vertebrates, where rhodopsin undergoes rapid sequential transitions between several unstable intermediate states. In vertebrates, all-trans-retinal is released during thermal relaxation of photoexcitation products and rhodopsin must be regenerated through subsequent recombination of opsin with another molecule of 11-cis-retinal. In the squid, however, the retinoid chromophore can remain attached to opsin throughout the rhodopsin activation cycle, and in a subsequent deactivating photoconversion event, invertebrate metarhodopsin can be converted to inactive rhodopsin by photon absorption. Thus, photobleaching may not be inevitable in the squid retina. The spectral sensitivities of squid rhodopsin and metarhodopsin differ by only a few nanometers (493 and 500 nm,


respectively) suggesting that the same visual stimulus could be both activating and inactivating. Due to the comparable stabilities and interconverting wavelengths of rhodopsin and metarhodopsin, it has been suggested that substantial populations of each could exist at steady state in the squid eye. However, retinas obtained from freshly caught squid are found to contain almost exclusively rhodopsin, a finding that suggests a highly efficient inactivation pathway. In addition to rhodopsin, cephalopod photoreceptor cells contain retinochrome, a second photosensitive retinal-binding protein implicated in rhodopsin regeneration. Squid retinochrome from Todarodes pacificus has been cloned, revealing a 301-amino-acid protein. The structure of retinochrome resembles that of rhodopsin, but its retinoid occupancy is reversed; whereas rhodopsin binds 11-cis-retinal and produces all-trans-retinal on photoisomerization to metarhodopsin, retinochrome binds and photoisomerises all-trans-retinal to 11-cis-retinal in its conversion to metaretinochrome. Metaretinochrome then releases 11-cis-retinal, providing it to rhodopsin via a shuttling protein known as retinal-binding protein (RALBP). In the dark, metaretinochrome is localized in the arhabdomeral lobe, where it releases 11-cis-retinal and, subsequently, binds all-trans-retinal released from metarhodopsin. Soluble RALBP then shuttles 11-cis-retinal to the rhabdomeral microvilli, where it binds retinalfree opsin, which may have arisen as a photoproduct or by synthesis de novo, in order to generate rhodopsin. Lightdependent translocation of both rhodopsin and retinochrome has been documented. In the dark, rhodopsin and retinochrome colocalize at the base of the microvilli, while in the light rhodopsin redistributes along the entire area of the microvillar membrane, and retinochrome becomes more plentiful in the rhabdomeral lobe. Dynamic control of the availability of rhodopsin (and other lightabsorbing pigments and signaling proteins) in signaling compartments may modulate the cascade and rhodopsin regeneration. Thus, the eye can adjust to dim or bright ambient light conditions, accurately perceive objects in each, and regenerate rhodopsin when necessary.

Squid Visual Guanine Nucleotide-Binding Protein, Gq Squid rhodopsin couples to its effector enzyme, a PLC, via a heterotrimeric G protein belonging to the Gq subfamily. Like all G proteins in this family, squid Gq is composed of three nonidentical subunits, a, b, and g. These subunits are associated with each other in the inactive state with GDP bound to the a subunit. In this state, the G protein is tightly bound to the rhabdomeric membrane, likely in association with rhodopsin. Upon 11-cis-retinal isomerization by light, a conformational change in the receptor causes a

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conformational change in the G protein subunits, which opens up the gunanine nucleotide-binding site on the a subunit allowing GDP to be exchanged for GTP. In the GTP-bound state, Gqa has a lower affinity for rhodopsin and its bg partners, but gains a higher affinity for its effector, PLC. PLC is recruited to the rhabdomeric membrane to interact with activated Gqa and its substrate phosphotidylinositol 4,5-bisphosphate. Gqa binding activates the PLC enzyme and at the same time the PLC stimulates the GTPase activity of Gqa resulting in hydrolysis of the terminal phosphate group on the bound GTP, thus rendering the G protein once again inactive in the GDP-bound state. Reassociation of the G protein subunits completes the cycle of activation and inactivation back to the basal state. The G protein is then ready to receive the next signal from rhodopsin. Gq protein subunits have been purified and cDNA sequences encoding the proteins have been reported from L. forbesi and L. pealei. Loligo Gqa is similar in amino acid sequence to Gqa proteins of other species with a conserved guanine nucleotide-binding domain and three switch regions that are the major sites of conformational change in the protein when GDP is exchanged for GTP on the protein. The sites of interaction between a G protein a subunit and its receptor are primarily in the two ends of the protein. The carboxyl-terminus of squid Gqa is identical to that found in Gq proteins from other species; however, the amino-terminus of the protein in all invertebrates lacks a six-amino-acid extension found in mammalian Gqa. Studies using L. pealei Gqa expressed in mammalian cells suggest that the modified amino-terminus found in invertebrate proteins increases the efficacy of G protein activation by receptors. The amino-terminus is also the site of posttranslational addition of palmitic acid on one or more of the two cysteine residues at position 3 and 4 of the Gqa subunit. This lipid modification helps maintain membrane association of the Gq protein and is particularly important for keeping Gqa attached to the membrane following activation by the receptor when Gqa dissociates from the receptor and Gqbg subunits. The Gqbg subunits have not been examined extensively. They appear to have similar functions to other G protein bg subunits in that they associate with the Gqa subunit when it is bound to GDP and dissociate when Gqa is bound to GTP. The bg subunits are tightly associated with the retinal membranes at all times and lipid modifications of the g subunit may contribute to this localization. Loligo Gb has a similar sequence to that of all other G protein b subunits, whereas Loligo Gg is quite distinct from Gg subunits of other species. Gqbg subunits do not activate purified PLC from squid eyes. Further studies will be required to determine if squid G protein bg subunits have any additional roles in visual signal transduction.

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Squid Visual PLC The protein stimulated by activated Gq in the squid visual system is PLC. The protein has been purified and the amino acid sequence determined from L. pealei. Immunoblot analysis of many squid tissues showed that the visual PLC is uniquely expressed in the photoreceptor membranes. Squid visual PLC is a 140 kDa protein that has significant sequence similarity and a domain structure that is common to phospholipase b enzymes; a PH domain that helps the protein bind to membranes, X and Y catalytic domains, and a C2 domain that is likely the site of calcium binding to the enzyme. In the absence of light stimulus to the photoreceptors, intracellular calcium concentrations are low and the PLC has very little catalytic activity. Upon activation of rhodopsin, the catalytic activity of the phospholipase is highly stimulated by the binding of activated Gqa to domains in the carboxyl-terminal end of the protein known as P and G boxes. The enzyme hydrolyzes membrane phospholipids with preference for phosphatidylinositol 4,5-bisphosphate, which is converted into inositol 1,4,5-trisphosphate (IP3) and DAG. A network of submicrovillar tubules has been observed beneath the microvilli that may express IP3 receptors and release stored calcium in response to IP3 activation. A rapid rise in intracellular calcium may help to maintain PLC activity as these enzymes are stimulated in the presence of elevated calcium concentrations. In addition to the role that calcium plays in visual signal transduction, high (millimolar) calcium concentrations can activate a calpain-like protease found in the squid retina. This protease can cleave several proteins in the visual signaling pathway. Calpain cleaves PLC near the carboxyl-terminus and renders the protein insensitive to Gqa activation. The protease can also cleave rhodopsin near its carboxyl-terminus removing the proline-rich repeat sequence from the rest of the molecule. Calcium activation of this protease may therefore play a role in freeing rhodopsin and Gq to allow for greater membrane turnover following prolonged light exposure.

Light-Activated Ion Channel The final component in the visual signal transduction system is the channel regulated by PLC hydrolysis of membrane lipids. This component of the squid visual system has been characterized in only one report; an electrophysiological recording made from an isolated L. pealei photoreceptor. Light stimulation of the squid photoreceptor evoked an inward current with a peak amplitude greater than 1000 pA and channel activity was most frequently recorded when the patch electrode was placed near the apical tip of the cell where microvilli are present.

A squid photoreceptor channel has been identified and cloned from L. forbesi and it was found to be most homologous to that of the visual transient receptor potential (TRP) ion channels identified in Drosophila. TRP and an additional TRP-like channel have been shown to constitute the light-activated response in Drosophila. Analysis of the amino acid sequence of the squid channel suggests a protein with six or eight membrane spanning segments. The amino-terminus contains an ankyrin-like repeat sequence similar to that found in Drosophila TRP channels, and may account for the association of the protein with the cytoskeleton during purification. The carboxyl-terminus of the squid channel is considerably shorter than that found in the Drosophila TRP channel and lacks the proline-rich repeat sequence suggested to link TRP to intracellular Ca2+ stores. Expression of a peptide composed of the squid channel carboxyl-terminus bound to calciumcalmodulin in vitro. These studies suggest that the squid channel may be regulated differently from that of Drosophila, however, since the squid channel has not yet been characterized, its properties and the mechanisms by which it is regulated by the PLC signaling cascade remain speculative.

Desensitization of Visual Signal Transduction Once rhodopsin has been activated, a series of protein–protein interactions occur within the photoreceptor cell to terminate signal transduction and restore the photoreceptor to its inactive state. Like most GPCRs, activated squid rhodopsin is phosphorylated by a G-proteincoupled receptor kinase (GRK) also called rhodopsin kinase. Light-activated rhodopsin also binds arrestin and biochemical studies using purified arrestin have demonstrated that this uncouples rhodopsin from activation of Gq.

Squid Rhodopsin Kinase The squid visual system expresses a kinase that has sequence and functional similarity with other GRKs. The most extensively studied GRKs are the mammalian rhodopsin kinases (GRK1 and GRK7) and b-adrenergic receptor kinases (GRK2 and GRK3). Interestingly, molecular cloning of squid rhodopsin kinase (SQRK) revealed much higher sequence similarity to the mammalian b-adrenergic receptor kinase GRK2 (66%) than to GRK1 (33%), which terminates signaling in the mammalian visual system. This is a common theme among invertebrate rhodopsin kinases, as eye-specific GRK1 cloned from Drosophila and octopus rhodopsin kinase (ORK) also bear higher sequence identity to GRK2 than to GRK1. The structural similarities between SQRK and GRK2 include a central


serine/threonine kinase catalytic domain, a structurally conserved amino-terminal domain bearing an RGS domain, a conserved carboxyl-terminal sequence and a PH domain in the carboxyl-terminal. In GRK2, it has been established that a carboxyl-terminal region partially overlapping the PH domain associates with the G protein bg subunits hence facilitating membrane localization in a stimulus-dependent manner. These structural similarities suggest that the phosphorylation of squid rhodopsin may more closely resemble the phosphorylation event of the mammalian b-adrenergic receptor than that of mammalian rhodopsin. SQRK structural motifs bearing high homology to the GRK2 Ca2+/CaM-binding domain and the GRK2 clathrin box motif suggest that Ca2+ may have a role in regulating SQRK activity and that SQRK may also bind to the clathrin heavy chain and play a role in endocytosis. To date, functional characterization of SQRK has confirmed that purified SQRK is able to phosphorylate squid rhodopsin in rhabdomeric membranes in a light-dependent manner. SQRK phosphorylation of rhodopsin requires GTP and Mg2+ ion cofactors that may relate to the need to activate the Gq protein to allow SQRK access to rhodopsin.

Squid Visual Arrestin Squid visual arrestin (sArr) has been cloned, purified, and characterized with respect to its functional interactions with rhabdomeric membranes. Squid arrestin from L. pealei is a 400-amino-acid protein with an estimated mass of 55 kDa that is expressed exclusively in eye tissue. Sequence identity between sArr and those from Drosophila and Limulus are 42% and 37%, respectively (sequence similarity including conservative substitutions is considerably higher, at 61% and 60%, respectively). Of the mammalian arrestins, visual arrestin from L. pealei shares highest identity with b-arrestins (44% identity and 64% similarity to b-arrestin1; 42% identity and 63% similarity to b-arrestin2). Squid visual arrestin is only 32% identical to mammalian visual arrestin, conservatively substituted to 49% similarity. This pattern of similarity parallels that between invertebrate rhodopsin kinase and mammalian b-adrenergic receptor kinases, and suggests that the functional interactions with invertebrate rhodopsin resemble those of the mammalian b-adrenergic receptor more closely than those of mammalian rhodopsin. For many GPCRs, including mammalian rhodopsin, it has been demonstrated that receptor phosphorylation enhances high-affinity binding of arrestin. Accordingly, the primary structure of squid arrestin contains both conserved residues associated with both high- and lowaffinity phosphate interactions. However, purified sArr does not seem to require rhodopsin phosphorylation to

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bind light-activated rhodopsin. This parallels biochemical studies in Drosophila, where purified Arr2 can bind to phosphorylated and unphosphorylated light-activated rhodopsin with comparable affinity. Further, light-dependent binding of Arr2 was equivocal in wild type and mutants where rhodopsin cannot be phosphorylated (both a truncation mutation lacking the phosphorylation site, and a serine to alanine point mutation that retains a similar structure but which cannot be phosphorylated). These findings are consistent with observations that the phosphorylation-deficient rhodopsin mutants display similar deactivation kinetics. Thus for invertebrates, the role of rhodopsin phosphorylation in arrestin binding is unclear, and light activation of rhodopsin may be sufficient. Squid visual arrestin associates with the rhabdomeric membrane in a light-dependent manner and inhibits light-activated GTPase activity. This is consistent with the notion that recruitment and stoichiometric binding to the intracellular surface can uncouple Gq from the receptor and terminate signaling by a competitive mechanism. In the dark, squid arrestin also has appreciable affinity for the rhabdomeric membrane, an interaction which can be abrogated by inositol 1,2,3,4,5,6-hexakisphosphate (IP6), a soluble analog of the membrane lipid phosphatidylinositol 3,4,5-triphosphate. This suggests that arrestin can also bind to membrane phospholipids, an interaction that studies in Drosophila suggest may mediate arrestin trafficking along an elaborate system of cytoplasmic structural components. The primary structure of sArr includes five fingerprint motifs that correspond with domains of distinct functional importance conserved among the arrestin family; a region that recognizes receptor activation, a domain rich in hydrophobic interactions, a domain that recognizes receptor phosphorylation, a carboxyl-terminal regulatory domain, and a conserved amino-terminal domain. Overall, the arrestin molecule adopts a concave saddle-like conformation consisting of amino- and carboxyl-terminal domains rich in antiparallel b-sheets that hinge on a central polar core region of buried salt bridges that are disrupted when the molecule encounters the activated receptor. The primary sequence of sArr contains 26Asp, 169Arg, 293Asn, 300 Asp, and 381Arg, which are identical to bovine visual arrestin (except 293Asn, which is a conservative substitution). These five essential conserved residues are contained within consensus sequences of five to eight identical or conservatively substituted amino acids that form the salt bridges buried in the polar core. Both the amino- and carboxyl-terminal domains mediate high-affinity binding to rhodopsin intracellular loops and these regions are located exclusively on the concave side of arrestin. Arrestin binding to rhodopsin thus occludes the binding of the G protein, preventing catalysis of GTP exchange, and uncoupling the receptor from its effector.

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Interestingly, sArr can be phosphorylated by SQRK, a novel function among the GRK family. This contrasts the phosphorylation of Drosophila and Limulus visual arrestins, which are phosphorylated by Ca2+/calmodulin-dependent kinase II. Phosphorylation of purified sArr requires SQRK, membranes, light-activated rhodopsin, and the presence of Ca2+. Though the kinase is different, Ca2+ dependence is common to Limulus and Drosophila. Studies in Drosophila show that the kinetics of invertebrate visual arrestin phosphorylation are fast (seconds after illumination, 43% of Drosophila Arr2 is phosphorylated) and that arrestin phosphorylation can facilitate its release from rhodopsin once arrestin-bound metarhodopsin is photoconverted back to its inactive state. A similar role for squid arrestin phosphorylation has been found, as phosphorylated arrestin dissociates from dark-adapted membranes more readily than unphosphorylated arrestin.

Conclusion Many of the molecular components of the squid visual system have been identified and characterized. Studies have suggested many similarities between the squid and other invertebrate visual systems. The strength of the squid system has been in the abundance of retinal tissue that makes protein purification and characterization feasible. These biochemical studies have complemented the power of genetic manipulations in the Drosophila system and the ease of electrophysiology in Limulus. Further studies are still required to determine the roles of rhodopsin and arrestin phosphorylations as well as identification of the many proteins involved in resetting the signaling components in the dark. We still know very little about how the activation of PLC results in membrane depolarization and the characteristics of the ion channels in the microvillar membranes. Future studies may eventually reveal all the molecular machinery of this fascinating visual system.

See also: Circadian Photoreception; Genetic Dissection of Invertebrate Phototransduction; Microvillar and Ciliary Photoreceptors in Molluskan Eyes; Phototransduction in Limulus Photoreceptors; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Evolution of Opsins; The Photoreceptor Outer Segment as a Sensory Cilium.

Further Reading Go, L. and Mitchell, J. (2007). Receptor coupling properties of the invertebrate visual guanine nucleotide-binding protein iGqa. Cellular Signalling 19: 1919–1927. Mayeenuddin, L. H., Bamsey, C., and Mitchell, J. (2001). Retinal phospholipase C from squid is a regulator of Gq alpha GTPase activity. Journal of Neurochemistry 78: 1350–1358. Mayeenuddin, L. H. and Mitchell, J. (2001). cDNA cloning and characterization of a novel squid rhodopsin kinase encoding multiple modular domains. Visual Neuroscience 18: 907–915. Monk, P. D., Carne, A., Liu, S.-H., et al. (1996). Isolation, cloning and characterization of a trp homologue from squid (Loligo forbesi) photoreceptor membranes. Journal of Neurochemistry 67: 2227–2235. Murakami, M. and Kouyama, T. (2008). Crustal structure of squid rhodopsin. Nature 453: 363–367. Nasi, E. and Gomez, M. (1992). Electrophysiological recordings in solitary photoreceptors from the retina of squid, Loligo pealei. Visual Neuroscience 8: 349–358. Ryba, N. J., Findlay, J. B., and Reid, J. D. (1993). The molecular cloning of the squid (Loligo forbesi) visual Gq-alpha subunit and its expression in Saccharomyces cerevisiae. Biochemical Journal 292: 333–341. Swardfager, W. and Mitchell, J. (2007). Purification of visual arrestin from squid photoreceptors and characterization of arrestin interaction with rhodopsin and rhodopsin kinase. Journal of Neurochemistry 101: 223–231. Venien-Bryan, C., Davies, A., Langmack, K., et al. (1995). Effect of C-terminal proline repeats in ordered packing of squid rhodopsin and its mobility in membranes. FEBS Letters 359: 45–49. Walrond, J. P. and Szuts, E. Z. (1992). Submicrovillar tubules in distal segments of squid photoreceptors detected by rapid freezing. Journal of Neuroscience 12: 1490–1501.

Phototransduction: Adaptation in Cones T D Lamb, The Australian National University, Canberra, ACT, Australia ã 2010 Elsevier Ltd. All rights reserved.

Glossary Avoidance of saturation – The ability of cones (but not rods) to continue functioning in steady background illumination of arbitrarily high intensity. Light adaptation – The rapid adjustment of sensitivity and kinetics (of the entire visual system, or of the photoreceptors) that occurs in response to altered ambient light intensity. The adjustment is rapid irrespective of whether the change is an increase or decrease in intensity, provided that the change is not too great. Saturation – The failure of the photoreceptors (or of the visual system) to respond to incremental illumination in the presence of appropriately bright illumination. The rod photoreceptors saturate at a relatively low background intensity; in contrast, the cone photoreceptors avoid saturation by steady backgrounds, no matter how bright the light is. Weber’s law – The reduction in visual sensitivity that occurs in inverse proportion to the intensity of the ambient background illumination. This corresponds to a rise in visual threshold in direct proportion to the ambient illumination.

Performance of the Photopic (Cone) System Workhorse of Vision For human vision, the photopic cone system can be considered the workhorse of vision, because it is operational under almost all of the conditions that we (in a modern world) experience. Thus, it is our photopic system that provides our sense of vision under all lighting conditions, apart from exceptionally low levels such as starlight conditions. Under moonlight conditions, our scotopic and photopic systems are both functional, over an intensity range that is termed mesopic. If you are ever in doubt as to whether you are using your photopic system under twilight or nighttime conditions, there is a simple test: if you are able to detect any color in the scene, then your cones are active; your rods may also be active, but this is one test of whether there is any cone activity at that level of intensities. In addition, the photopic system remains functional at all higher intensities, up to the brightest sunlit conditions than we ever experience.

It is interesting to consider that, despite their enormous importance to our vision, cones make up perhaps only 5% of the population of photoreceptors over most of our peripheral retina. This relatively low proportion of cone photoreceptors in the peripheral retina is entirely adequate for our normal peripheral vision, which requires only relatively low spatial acuity. Even though the great majority of peripheral photoreceptors are rods, they are simply not used under most of the circumstances that we think of as vision – thus, they are only used at exceedingly low ambient lighting levels. The reason for the great numerical preponderance of rods is to be able to capture every available photon at those very low light intensities. Rapid Response and Moderate Sensitivity In survival terms, one of the greatest advantages of cones over rods is their much faster speed of response. The responses of our rods, even when they are light adapted, are much too slow to allow us to function visually at the speeds that are required to escape predators and to capture prey. Cones, instead, are specialized so as to permit extremely rapid signaling of visual stimuli to the brain. Cones are often described as having much lower sensitivity than rods, but this view is misleading, especially when considered in terms of the rapidly changing visual stimuli that the cones are specialized for signaling. Although the peak sensitivity to a brief flash of light may be perhaps 30-fold lower in a cone than in a rod, the sensitivity to rapidly fluctuating stimuli is considerably higher in cones than in rods; thus, the slow response of the rods makes them quite insensitive to rapidly changing stimuli. When expressed in terms of the efficacy of activation within the G-protein cascade of phototransduction, the amplification in cones and rods appears to be essentially indistinguishable – the real difference is in the speed of inactivation. Avoidance of Saturation The second crucial feature of cones, in terms of survival advantage, is their amazing ability to avoid saturation no matter how intense the steady background illumination becomes. This property stands in stark contrast to the situation in rods, which are completely incapable of responding once the background exceeds a relatively low level (corresponding roughly to twilight illumination). One of the major challenges in photoreceptor research is

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to provide a clear understanding of how it is that cones are able to avoid saturation at arbitrarily high light intensities, whereas rods succumb to saturation at very low light intensities. As will be described below, considerable advances have recently been made toward providing this understanding.

Light Adaptation of the Cones Cone photoreceptors undergo light adaptation over an enormously wide range of intensities, and it is likely that almost all of the adaptation that is observed in the overall photopic visual system is mediated by these changes occurring at the level of the receptor cells. This section describes the adaptational effects that occur in the cone photoreceptors.

A

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Flashes on Backgrounds: Desensitization and Acceleration Figure 1 illustrates the responses of a cone photoreceptor to the same set of flashes presented under three different conditions; the flashes A–F were of progressively greater intensity from left to right, but were exactly the same in each of the three panels. In (a), the flashes were presented in darkness, and represent a standard dark-adapted flash family; in (b), the same flashes were presented shortly after a dim steady background had been turned on; and in (c), the same flashes were presented after the onset of a brighter background. In the presence of the background illumination, the amplitude of the responses to dim flashes was smaller. For example, for the second flash intensity, B, the response amplitude becomes markedly smaller from (a) to (b) to (c). In other words, backgrounds of increasing intensity progressively desensitized the cone’s incremental

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Figure 1 Circulating current of a salamander cone in response to flashes and steps of illumination. Timing of illumination is indicated by the marker trace at the top; flashes A–F increased in intensity by factors of 4, and were the same intensity in each of (a)–(c). In (a), these flashes were presented in darkness; in (b) and (c) the same flashes were presented on steady backgrounds that had been switched on at time zero; the background in (c) was 4 times brighter than in (b). Reproduced from Matthews, H. R., Fain, G. L., Murphy, R. L. W., and Lamb, T. D. (1990). Light adaptation in cones of the salamander: A role for cytoplasmic calcium concentration. Journal of Physiology 420: 447–469.


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Figure 2 Incremental responses of a salamander cone to test flashes presented on backgrounds of increasing intensity. The largest trace is for a dim flash presented in darkness, while the other traces correspond to the same test flash presented on backgrounds. In fact, in the presence of brighter backgrounds, the test flash intensity was increased in order to obtain measurable responses, and the plotted traces have therefore been scaled as response divided by test flash intensity, so as to provide a direct measure of sensitivity. Reproduced with permission from Matthews, H. R., Fain, G. L., Murphy, R. L. W., and Lamb, T. D. (1990). Light adaptation in cones of the salamander: A role for cytoplasmic calcium concentration. Journal of Physiology 420: 447–469.

response. Such behavior is very characteristic of photoreceptors, and these responses from cones are qualitatively similar to those obtained from rods. The manner in which the response to a dim flash is modified by the presence of backgrounds of different intensity is illustrated in Figure 2. The largest trace is the response to a dim flash presented under fully darkadapted conditions, while the other traces are for the same flash presented on backgrounds of progressively brighter intensity. (In fact, in order to maintain responses of measurable amplitude, the flash intensity was increased in the presence of backgrounds, and the traces actually plot response divided by flash intensity; i.e., the response sensitivity.) The traces in Figure 2 demonstrate that the effect of backgrounds of increasing intensity is to both desensitize and accelerate the response to an incremental dim flash. This behavior of cones is very similar to that exhibited by rods.

−8 −9 −10 −2 −1 0 1 2 3 4 5 6 7 8 9 10 11 Log background intensity (photons s−1 μm−2) Figure 3 Cone sensitivity as a function of background intensity. Data are plotted in double logarithmic coordinates, and were obtained from intracellular measurements averaged from 15 cones in the turtle eyecup preparation. The experiments used laser illumination to achieve very high background intensities, and monitored step sensitivity rather than the more conventional flash sensitivity. The smooth curve plots Weber’s law, given by eqn [1]. Reproduced from Burkhardt, D. A. (1994). Light adaptation and photopigment bleaching in cone photoreceptors in situ in the retina of the turtle. Journal of Neuroscience 14: 1091–1105.

turtle), so that the photoreceptors remained in contact with the retinal pigment epithelium (RPE) and thereby experienced normal regeneration of visual pigment, in order that meaningful results could be obtained even at very high background intensities. (One methodological difference between the results plotted in Figure 3 and the results that are more usually plotted is that step sensitivities rather than flash sensitivities are plotted; however, this does not, in practice, make much of a difference.) For background intensities from 103 to 1011 photons mm2 s1, the relationship between log sensitivity and log background intensity is a straight line with a slope of 1; in other words, over roughly 8 log units of background, the turtle cone’s sensitivity declines inversely with background intensity. The curve plotted near the points in Figure 3 represents Weber’s law, described by: S 1 ¼ SD 1 þ ðI =I0 Þ

Dependence of Sensitivity on Background Intensity: Weber’s Law By plotting the peak amplitude of each of the traces in Figure 2 as a function of the background intensity on which it was measured, one obtains a sensitivity versus background plot of the type illustrated in Figure 3. The results plotted in Figure 3 were obtained over an extremely wide range of background intensities by Dwight Burkhardt using a laser source of illumination. Importantly, the preparation was the intact eyecup (of the

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where S is flash sensitivity, SD is its dark-adapted value, I is the background intensity, and I0 is the half-desensitizing intensity, also known as the dark-adapted equivalent background intensity. The good fit of the Weber’s law expression shows, very importantly, that cone photoreceptors in the intact eyecup are able to completely avoid saturation, even at enormously high intensities of steady illumination. This feature represents a crucial distinction between the properties of cones and rods. The circulating current of rods is shut off at quite low background intensities, so that

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the rods are unresponsive to superimposed stimuli, in the presence of background illumination of moderate intensity. In the same set of experiments, Burkhardt measured the intensity at which 90% of the pigment was in the bleached state, and found this to be around 106 photons mm2 s1. For all intensities above that level, the observed Weber’s law behavior can be accounted for in terms of pigment bleaching. For each additional 10-fold increase in intensity, there will be a 10-fold reduction in the amount of pigment remaining available to absorb light, and hence there will necessarily be a 10-fold reduction in sensitivity in the absence of any other change of parameters of transduction in the outer segment. In other words, if the photoreceptor is able to avoid saturation up to intensities that cause substantial bleaches, then it will be able to exhibit Weber’s law desensitization at higher intensities purely by means of pigment bleaching. Cones are able to function up to this critical intensity, whereas rods saturate at much lower intensities. Extremely Rapid Recovery of Cone Photocurrent In order to measure the performance of mammalian cones in the presence of extremely bright background illumination, it is necessary to use a preparation in which the cones

are in contact with the RPE (as was the case in the experiments above with turtle cones). Accordingly, it is not appropriate to use suction-pipette experiments at very high intensities. On the other hand, experiments measuring the electroretinogram (ERG) in the intact eye are very suitable. Results from an experiment designed to measure the kinetics of recovery of the circulating current of human cone photoreceptors, upon extinction of steady illumination that bleached 90% of the visual pigment, are illustrated in Figure 4. This Figure shows recordings of the a-wave of the human ERG, which monitors primarily the response of photoreceptors; and at these incredibly high intensities, only the cone photoreceptors are responding. The left panel shows the response to a bright flash superimposed on the intense steady background, while the right panel shows the response obtained at extinction of that background. As indicated by the right-hand pair of vertical scales, the bright flash responses established the zero level of circulating current, as well as the level of circulating current during the intense background (i.e., unity on the inner scale). Separate measurements (not shown) established the dark current (i.e., unity on the outer right-hand scale). Even during the presence of the intense steady background, the cone circulating current was roughly 50% of its original level in darkness.

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Figure 4 Extremely rapid recovery of human cone photocurrent upon extinction of intense illumination, measured with the ERG. The four colored traces plot ERG responses from two subjects, at two different flash intensities. The light stimulus is monitored by the black traces at the top. Left panel is for an intense flash presented on the intense steady background; right panel is for extinction of that background. The ON a-wave and b-wave elicited by the bright flash are indicated by the red arrows; the OFF a-wave and b-wave elicited by extinction of the background are indicated by the blue arrows. The b-wave is roughly similar in the two cases, and arises from postreceptoral activity. The OFF a-wave represents recovery of the cone circulating current, and begins around 7 ms after the intense background is turned off. Dashed horizontal lines represent the following levels of cone circulating current (from bottom): zero level, steady level during intense background, dark level, as indicated by the two normalized scales on the right. Reproduced from Kenkre, J. S., Moran, N. A., Lamb, T. D., and Mahroo, O. A. R. (2005). Extremely rapid recovery of human cone circulating current at the extinction of bleaching exposures. Journal of Physiology 567: 95–112.


The traces on the right show the ERG a-wave upon extinction of the intense background. Little change occurs for the first 7 ms, but thereafter a substantial upward response occurs, the OFF a-wave indicated by the blue arrow, until about 15 ms after extinction of the background; at this point, the a-wave is obscured by spikelike activity of the b-wave. There is compelling evidence that the a-wave traces for these subjects monitor the recovery of the cone circulating current. On this basis, the cone circulating current is essentially fully recovered within about 15 ms after extinction of illumination so intense that it bleaches 90% of the cone pigment. This is extremely rapid recovery, at least when compared with the time course of recovery following intense flashes delivered from darkness. The next section considers the speed that is required for the shut-off reactions of phototransduction, in order to be able to account for recovery of the circulating current as fast as is shown in Figure 4. The smooth gray curve near the measured traces in the righthand panel of Figure 4 was calculated from the model presented in the next section, using the short time constants listed in Table 1. Extremely rapid recovery of cone circulating current, as inferred from the results of Figure 4, is also required in order to account for classical experiments on the flickerfusion frequency of human subjects. Even at quite low photopic intensities, human subjects are able to detect square-wave flicker at a frequency of around 50 Hz using peripheral vision. However, at higher intensities, the flicker-fusion frequency increases to 100 Hz or more. At a frequency of 100 Hz, the illumination is being switched on and off at intervals of 5 ms each. Thus, in Table 1

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order for the flicker to be detectable, some degree of recovery of cone circulating current must have occurred within 5 ms. Hence, this finding is broadly consistent with the time course inferred from Figure 4.

Molecular Basis of Cone Light Adaptation Reaction Steps Underlying Rapid Recovery of the Cone’s Light Response Figure 5 presents a schematic of the reaction steps underlying phototransduction in cones, where shut-off reactions and the lifetimes (or turnover times) of important intermediates are indicated in red. For mammalian cones, the speed of the various shut-off reactions shown in the schematic of Figure 5 has been estimated in a number of recent studies using intact preparations. In the case of monkey cones, the parameters were extracted through theoretical modeling of results obtained from intracellular recordings of horizontal cells in the retina–RPE–choroid preparation. In the case of human cones, the parameters were extracted through theoretical modeling of ERG results, including those of the type illustrated in Figure 4. The shut-off reactions have been found to be extremely rapid, and the parameters that have been reported are summarized in Table 1. The collected estimates in Table 1 are consistent with the notion that all four of the shut-off time constants in human cones are extremely short, with values in the range of 3–18 ms; in fact, it appears that three of the time constants could be around 5 ms or less, and one around

Shut-off time constants estimated for mammalian cones and rods tR ms

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9-log-unit range, since the pupil diameter changes from a maximum of 8 mm to a minimum of 2.5 mm, corresponding to a 10-fold reduction in area. Instead, the great bulk of the operational range is achieved by the combination of, first, a switch between the rod (scotopic) and cone (photopic) pathways in our duplex visual system and, second, the ability of each of these photoreceptor systems to operate over a range of 5 log units (100 000-fold) or more.

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This ability of the visual system (or of any of its component parts, such as the photoreceptors) to adjust its performance to the ambient level of illumination is known as light adaptation; the adjustment typically occurs very rapidly (within seconds), whether the light intensity is increasing or decreasing. The term dark adaptation is reserved for the special case of recovery in darkness, following exposure of the eye to extremely bright and/ or prolonged illumination that activates (bleaches) a substantial fraction of the visual pigment, rhodopsin. Dark adaptation occurs slowly, and the full recovery of the scotopic visual system after a very large bleach can take as much as an hour. The changes that accompany light adaptation are beneficial to the possessor of the eye. At very low intensities, the sensitivity is increased to the utmost that is possible so that the rod photoreceptors reliably signal the arrival of individual photons and the scotopic visual system operates in a photon-counting mode. The ability of the scotopic system to operate at incredibly low intensities is enhanced by two deliberate trade-offs – of reduced spatial resolution (increased spatial summation) and reduced temporal resolution (increased temporal integration) – that permit more reliable detection of small signals in the presence of noise. Similar trade-offs are used in the photopic system so that as the ambient illumination decreases from daylight levels toward twilight levels, one’s spatial and temporal resolution deteriorate; this is why, in cricket, bad light stops play. In contrast, the changes that characterize dark adaptation are disadvantageous. To be essentially blind to dim stimuli, for some considerable time following intense light exposure, cannot in any way be useful to an organism. Indeed, for a caveman, entering a cave from bright sunshine, it may have been a serious handicap to have been unable to see well for tens of minutes. Why should such an apparently unsatisfactory situation have persisted? A possible reason could be because it represents an unfortunate downside that has somehow resulted from the enhancements that were needed in order to enable the scotopic system to detect individual photons, and thereby be able to function at incredibly low light levels.

Performance of the Scotopic (Rod) System For the rod pathway, the dominant mechanisms of scotopic light adaptation result from alterations of signal processing at postsynaptic stages within the retina, and the


rods themselves adapt over only a modest range of intensities before being driven into saturation. This is illustrated in Figure 1, which compares the changes in sensitivity of the rod photoreceptors and of the overall visual system during scotopic light adaptation. The blue symbols and curve plot the relative sensitivity of the overall visual system, measured psychophysically, while the red symbols and curve plot the relative sensitivity of primate rod photoreceptors. Importantly, the overall scotopic visual system begins desensitizing at intensities around 1000 times lower than those required to begin desensitizing the rod photoreceptors. This occurs because the postreceptoral scotopic system is able to integrate photon signals from large numbers of rod photoreceptors, thereby gaining increased sensitivity, while

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introducing the need to begin desensitizing at much lower background intensities in order to avoid saturation. Hence, the rod photoreceptors maintain their maximal sensitivity over several log units of the lowest intensity regime (up to 10 isomerizations per second) where the visual system needs to exhibit gradual desensitization. When the background intensity is reduced from relatively high scotopic intensities (moving from right to the left along the x-axis in Figure 1), the sensitivity of rods, and of the scotopic visual system, steadily rises. However, below the intensities indicated by the blue and red arrows, the sensitivity of, first, the rods and, second, the visual system fails to continue increasing, as if the respective mechanism were experiencing a phenomenon equivalent to light. Accordingly, the arrowed intensities for the rods and for the scotopic visual system have been referred to as equivalent background intensities. Clearly, the equivalent background for the scotopic system (around 0.016 photoisomerizations per second) is several log units lower than the equivalent background intensity for the rods (around 50 isomerizations per second). The curves in Figure 1 plot desensitization according to the combination of Weber’s law with saturation at high intensities, as described by S 1 expðI =Isat Þ ¼ SD 1 þ ðI =I0 Þ

Isat

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Log background (isoms/s) Figure 1 Sensitivity of the human scotopic visual system (blue) and of monkey rod photoreceptors (red) as functions of background intensity in double logarithmic coordinates. The blue symbols are from human psychophysical measurements and the blue curve plots Weber law desensitization in conjunction with saturation, as described by eqn [1], with parameters I0 ¼ 0.016 photoisomerizations per second (blue arrow) and Isat ¼ 2500 photoisomerizations per second (black arrow). The red symbols are from suction pipette measurements from isolated rod photoreceptors of monkeys (Macaca fascicularis); these symbols have been shifted vertically to align with the blue symbols in the upper intensity range. The red curve also plots eqn [1] with the same value of Isat, but with I0 ¼ 50 photoisomerizations per second (red arrow). Data for the blue symbols are from Figure 3 of Aguilar, M. and Stiles, W. S. (1954). Saturation of the rod mechanism of the retina at high levels of stimulation. Optica Acta 1: 59–65. Their troland values were converted using a factor of K ¼ 8.6 photoisomerizations per second per troland. Data for the filled symbols are from Figure 9A and Table III of Tamura, T., Nakatani, K., and Yau, K.-W. (1991). Calcium feedback and sensitivity regulation in primate rods. Journal of General Physiology 98: 95–130.

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½1

where S is flash sensitivity, SD is its dark-adapted value, and I is the background intensity. The first term on the right-hand side expresses Weber’s law, where I0 is the equivalent background intensity mentioned above. This first term indicates that, at low background intensities (when I > I0) the sensitivity declines inversely with background intensity. At higher scotopic intensities, both the rods and the overall scotopic system exhibit saturation, characterized by a steep decline in sensitivity with increasing background intensity. This behavior is described by the second term on the right-hand side in eqn [1], where Isat is termed the saturation intensity of around 2500 isomerizations per second. It is almost certain that saturation of the overall scotopic system results directly from saturation of the rods. The span of intensities from I0 (the equivalent background) to Isat (the saturation intensity) is known as the Weber region and, in this range of background intensities, the sensitivity declines inversely with background intensity; that is, S / 1/I. Since the contrast in a visual stimulus is, likewise, inversely proportional to background intensity (i.e., contrast = △I/I ), this Weber region is characterized by a fixed level of contrast sensitivity; that is, a given level of contrast elicits a fixed size of response. Thus, an important feature of Weber’s law light adaptation is that it provides automatic extraction of visual contrast.

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For mammalian rods, the Weber region encompasses only 1–2 log units of intensity, though for the larger rods of lower vertebrates, it may encompass a slightly wider range of about 3 log units. On the other hand, for the overall scotopic system, the Weber region covers a much wider range of at least 5 log units (i.e., over 100 000-fold). In addition, for cone photoreceptors, it extends over an even wider range.

The Purpose of Light Adaptation: Optimization of Performance The purpose of light adaptation is to permit the visual system (or any neuron within it) to provide the best performance possible at that particular level of illumination. However, it is not always clear what constitutes best. For example, for the rod photoreceptors, it is clear that at very low ambient levels of illumination, their sensitivity should be as high as possible. However, we cannot readily anticipate the time course of their response that will be optimal. Avoidance of Saturation: Range Extension As the ambient light intensity increases, it is important that the rod (or any other cell) should avoid saturating, or else it will be unable to signal. By preventing saturation, light adaptation permits a photoreceptor to extend the range of intensities over which it operates. Although the rods achieve light adaptation over a limited range of intensities, the cones excel, and are able to avoid saturation no matter how bright the steady illumination becomes. Why has evolution permitted the rods to be driven into saturation by relatively low intensities? In part, it is because the photopic (cone) system is functional at these intensities, so that there is no disadvantage if the rods saturate. Not only is there no disadvantage – in fact, there is a distinct advantage when the rods saturate, in conserving energy during daylight conditions. Maintenance of the rod circulating current, in darkness and at low light levels, represents an extremely high metabolic load on the cells, and the elimination of this load when the cones are functional provides a major benefit to retinal metabolism. From this perspective, the limited range of rod light adaptation is beneficial, whereas an extended range (as occurs in cones) would be detrimental. Extraction of Contrast Information and Optimization of Response Kinetics In addition to the very important function of extending the operating range of the photoreceptor, there are two other ways in which photoreceptor light adaptation

optimizes the cell’s response. First, as described above in relation to eqn [1], it permits the extraction of contrast in the visual scene, independent of the absolute level of illumination. Second, it provides real-time adjustment of the time course of the response to an incremental flash of light, in a manner that is presumed to be optimal for the visual system. Thus, at very low background intensities, the response is sluggish, and postreceptoral elements are able to integrate visual signals over relatively long times. At progressively higher background intensities, the response becomes progressively accelerated, thereby improving the time resolution of the system. However, we do not have sufficient information yet to be able to describe exactly how it is that kinetic changes of this kind are actually optimal for the visual system.

Light Adaptation of the Rod Photoreceptors: Range Extension, Desensitization, and Acceleration In the presence of background illumination, it is not only the overall visual system that adapts, but also the rod photoreceptors themselves display light adaptation, characterized by an extension of their operating range and by desensitization and acceleration of the incremental flash response.

Prevention of Rod Photoreceptor Saturation: Range Extension The response of a salamander rod to the onset of steady illumination at different intensities is illustrated in Figure 2. At the beginning of the step of light, the rod’s response begins rising according to what is predicted from the time integral of the flash response, but very soon deviates, falling well below the linear prediction (upper panel). Characteristically, the response to such a step of light typically exhibits an early peak followed by a sag. This deviation from the simplest linear prediction is a crucial aspect of light adaptation – if this deviation did not occur, then the rod would be driven into saturation by lights of very low intensity. Such saturation can be induced by exposing the rod to a solution that clamps the cytoplasmic calcium concentration; in the presence of calcium-clamping solution (lower panel), the responses of the rod follow the predictions of the smooth theoretical curves, and a very low intensity (labeled 2) saturates the rod. This result shows that at least a part of the rod’s ability to continue operating in backgrounds of moderate intensity (i.e., the extension of its operating range) is a consequence of changes in cytoplasmic calcium concentration; the molecular mechanisms that contribute to this will be discussed below.


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Desensitization and Acceleration The manner in which background illumination modifies the rod’s response to a dim test flash is illustrated in Figure 3. The uppermost trace is for a dim flash presented in darkness, while the remaining traces are for exactly the same flash presented on backgrounds of successively higher intensity. Characteristically, the flash response becomes progressively more desensitized and accelerated with backgrounds of higher intensity. Thus, the peak of the incremental flash response moves downward and leftward as the background intensity increases. By plotting the peak amplitude of the flash response as a function of the background intensity upon which it was elicited, one obtains a plot of the kind indicated by the red symbols in Figure 1, where sensitivity declines according to Weber’s law, given above in eqn [1].

Unaltered Rising Phase, but Accelerated Recovery For the incremental flash responses in Figure 3, the vertical scale has been adjusted to take account of changes

Fractional response per photoisomerization

Figure 2 Responses of a salamander rod to onset of steps of light of different intensity. Upper panel: under control conditions (Ringer solution). Lower panel: in the presence of Ca2+-clamping solution. The step intensities increased by factors of 4 for traces labeled 1–4; D, darkness. The smooth curves are predictions obtained by integrating the measured dim flash response (not shown), and represent the step responses that are predicted in the absence of any adaptation. Reproduced from Fain, G. L., Lamb, T. D., Matthews, H. R., and Murphy, R. L. W. (1989). Cytoplasmic calcium as the messenger for light adaptation in salamander rods. Journal of Physiology 416: 215–243.

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Figure 3 Dim flash responses of a salamander rod obtained in the dark (top trace) or in the presence of backgrounds of progressively higher intensity. Each trace was obtained by taking the raw response and dividing by the circulating current, and then dividing by the flash intensity. Reproduced from Pugh, E. N., Jr., Nikonov, S., and Lamb, T. D. (1999). Molecular mechanisms of vertebrate photoreceptor light adaptation. Current Opinion in Neurobiology 9: 410–418.

in the level of circulating current remaining in the presence of the different background intensities. Thus, rather than plotting raw sensitivity (response per photoisomerization), Figure 3 instead plots the fractional response

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(i.e., the incremental response as a fraction of the circulating current at that background) per photoisomerization. This has been done in order to provide a direct measure of the level of activation of the guanine nucleotide-binding protein (G protein) cascade of phototransduction; thus, it can be shown that the level of cascade activation is best measured by the fractional channel opening, which in turn is measured by the incremental response expressed as a fraction of the existing circulating current. When plotted in this manner, the incremental responses in Figure 3 demonstrate the remarkable property that the onset phase of the response is invariant; that is, the traces for different background intensities exhibit a common rise at early times, indicated by the smooth gray trace. This behavior indicates that the amplification parameter describing the activation steps in phototransduction is unaltered during light adaptation; in other words, light adaptation causes no change in the efficacy of the activation steps in phototransduction. Instead, it is clear that light adaptation causes a marked speeding up of the shut-off steps in the transduction cascade. The molecular identity of the steps that are accelerated is analyzed below.

Saturation of the Rod Photocurrent at Higher Background Intensities At higher background intensities, the rod circulating current is completely suppressed. Thus, in the upper panel of Figure 2, intensities higher than those labeled 4 cause the response simply to rise to its maximum level (corresponding to the closure of all cyclic guanosine monophosphate (cGMP)-gated channels in the outer segment) and, as a result, incremental stimuli are unable to elicit any incremental response so that the cell’s response is saturated. Typically, such saturation sets in exponentially with increasing background intensity, as described by the second term on the right of eqn [1].

Calcium-Dependent Mechanisms of Rapid Light Adaptation in Rod Photoreceptors The mechanisms that contribute to light adaptation in photoreceptors (i.e., to the alteration in response properties of the photoreceptors upon exposure to background illumination) are closely associated with the mechanisms of response recovery. These mechanisms of adaptation can be classified broadly as (1) those that are calcium dependent and (2) those that do not involve calcium. Both categories are important; yet, the noncalcium-dependent mechanisms have frequently been overlooked.

Role of Calcium: Resensitization through Prevention of Saturation When cGMP-gated ion channels in the outer segment are closed in response to light, the cytoplasmic concentration of calcium drops. This drop in Ca2þ concentration is vitally important to light adaptation, though it is crucial to emphasize that it does not cause the desensitization that characterizes photoreceptor light adaptation. Quite the contrary: the drop in Ca2þ concentration actually rescues the rod from the saturation that would otherwise be induced by light, and thereby prevents the onset of massive desensitization at relatively low intensities of background illumination. Thus, the light-induced drop in Ca2þ acts to increase the rod’s sensitivity above the drastically reduced level that would occur either if the Ca2þ concentration did not alter or if the rod’s calciumdependent mechanisms were inoperative.

Powerful Negative-Feedback Loop Mediated by Calcium Calcium is the cytoplasmic messenger for a very powerful negative-feedback loop that tends to stabilize the rod’s circulating current. If ever the Ca2þ concentration drops (e.g., in response to light, or as a result of some other perturbation), then, as described below, a number of changes occur very rapidly. These changes are stimulated by the unbinding of Ca2þ from at least three classes of calcium-sensitive protein: (1) guanylyl cyclase activator proteins (GCAPs) 1 and 2, which activate guanylyl cyclase; (2) recoverin, which regulates the lifetime of activated rhodopsin; and (3) calmodulin, which modulates the opening of cGMP-gated channels. Calcium’s action via each of these pathways leads to the opening of cGMPgated channels, thereby increasing the circulating current and admitting Ca2þ ions from the extracellular medium. This influx of Ca2þ ions tends to counteract the initial reduction in Ca2þ concentration, thereby completing a negative-feedback loop. Each of these molecular mechanisms contributing to the calcium negative-feedback loop contributes toward extending the rod’s operational range of light intensities by helping prevent saturation of the circulating current. Thus, each of these three molecular mechanisms assists in rescuing the rod from saturation and hence increasing, rather than decreasing, the rod’s sensitivity compared with the case that would exist if the mechanism were absent. Each of the three mechanisms is most effective over some range of calcium levels, and a corresponding range of light intensities. Overall, the most powerful of the three (at least in rods) is the GCAPs’ activation of guanylyl cyclase. Since the various components of the calcium negativefeedback loop act quite rapidly, they contribute to determining not only the photoreceptor’s sensitivity in the


presence of background illumination, but also the kinetics of its response to an incremental flash presented on the background. The importance of altered Ca2þ concentration in setting the incremental flash response kinetics can be demonstrated by incorporating a calcium buffer (such as 1,2-bis(o-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA)) into the outer segment. Although the flash response begins rising exactly as in control conditions, it does not begin recovering as soon and, instead, rises to a substantially larger and later peak with slower final recovery (see also Figure 4). Three Calcium-Sensitive Molecular Pathways Guanylyl cyclase activation

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In response to a drop in calcium concentration, Ca will unbind from the GCAP proteins (GCAP1 and GCAP2), thereby activating guanylyl cyclase and stimulating the production of cGMP at a greatly increased rate, leading to the opening of cGMP-gated channels. The cyclase activity increases roughly as the fourth power of the drop in Ca2þ concentration, and furthermore (as also applies for the other two routes considered below), the number of channels open increases approximately as the cube of the cGMP concentration. Because of the cascading of two such steep dependencies, any small fractional change in Ca2þ concentration stimulates a large and opposite fractional change in channel opening; that is, the fractional change in channel opening is opposite in sign to, and up to 12 the magnitude of, the originating fractional change in Ca2þ concentration. As a result, this molecular mechanism is the most potent of the three that contribute to the calcium negative-feedback loop and, hence, to setting the adaptational state in rods. It is especially dominant at relatively bright background intensities, corresponding to low Ca2þ concentrations, and is

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Figure 4 Single-photon responses from rods of wild-type (WT) and GCAPs knock-out (GCAPs/) mice. Suction pipette recordings from single rods, analyzed to extract the mean response to a single photoisomerization. Circulating current in rods of both strains averaged 12 pA. Reproduced from Burns, M. E., Mendez, A., Chen, J., and Baylor, D. A. (2002). Dynamics of cyclic GMP synthesis in retinal rods. Neuron 36: 81–91.

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therefore the most important in extending the rod’s operating range to high intensities. The role of the GCAPs/guanylyl cyclase component of the Ca2þ feedback loop in setting the waveform of the incremental flash response is illustrated in Figure 4, where averaged responses are shown for two classes of rod: rods from wild-type (WT) mice and rods from GCAPs knock-out mice. In a manner very similar to that seen in rods containing the calcium buffer BAPTA, the response in the GCAPs knockout case begins rising exactly as for the control (WT) case, but it does not recover as soon; therefore, the response continues rising and reaches a larger and later peak. Shortened R* lifetime Activated rhodopsin (R*) is inactivated by multiple phosphorylation steps mediated by rhodopsin kinase (GRK1) followed by binding of arrestin. It is generally assumed that the decline in R* activity follows exponential kinetics, and can therefore be described by a characteristic lifetime, tR; however, it is worth bearing in mind that there is no direct evidence for this assumption. It was established by Satoru Kawamura that GRK1’s phosphorylation of R* is calcium dependent and that the effect is mediated by the calcium-binding protein recoverin. The molecular mechanism of this dependence is not entirely clear; however, some evidence suggests that the calcium-bound form of recoverin binds to GRK1, thereby preventing it from interacting with R*. In any case, it is proposed that a reduction in Ca2þ concentration leads to a shortened R* lifetime, tR. The slowest time constant in the phototransduction cascade (the so-called dominant time constant, tdom) can be estimated from the steepness of the relationship between the duration that the rod is held in saturation by a bright flash and the flash intensity. Over the years, there has been considerable debate as to whether this dominant time constant is set by the R* lifetime, tR, or, instead, by the lifetime tE of the transducin–phosphodiesterase (PDE) complex (the effector). The situation may be species dependent; however, in mouse rods, it has now been clearly established by Marie Burns’ group that, under dark resting conditions, the dominant time constant is that of transducin–PDE, with tE 200 ms, while the R* lifetime is shorter, with tR 80 ms. In the scenario where the R* lifetime is shorter than the transducin–PDE lifetime, further light-induced shortening of tR is likely to have very little effect on the response kinetics, but will instead cause a reduction in sensitivity because fewer molecules of transducin will be activated during the R* lifetime. Although it remains difficult to establish the effectiveness of any individual mechanism in an intact rod with a functional calcium feedback loop, it appears that the recoverinmediated reduction in R* lifetime plays a moderate role, especially at relatively low background intensities.

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Channel reactivation

In response to a drop in calcium concentration, Ca2þ unbinds from calmodulin (in the case of the rods), leading to a lowered dissociation constant (K½) for the binding of cGMP to the channels. The effect of the lowered K½ is that any given concentration of cGMP will cause the opening of a larger fraction of the cGMP-gated channels, leading to an increase in circulating current and the influx of more calcium. However, the potency of this effect is low in rods, and the mechanism contributes only weakly to rod adaptation. In contrast, cones possess a much more powerful mechanism, mediated by a different calciumsensitive protein.

Rod Photoreceptor Light Adaptation Independent of Calcium There are at least three classes of noncalcium-dependent phenomena that represent mechanisms of light adaptation in rod photoreceptors, insofar as the properties of the response to light are altered in comparison with the dark-adapted state. First, there is response compression, whereby the reduced level of circulating current in the presence of steady background illumination reduces the size of the flash response. This phenomenon will not be discussed here, in part because it is both very well known and very simple and also because (in philosophical terms) it can be viewed as a failure of light adaptation; in comparison, cones cope much better and effectively avoid response compression by feedback mechanisms that maintain the circulating current. Second, there is pigment depletion. However, this is never relevant in rod light adaptation because the rods are driven into saturation even by very low levels of bleached pigment (see section titled ‘Dark adaptation of the rods: Very slow recovery from bleaching’). Third, there is a direct effect of PDE activation, which is now considered. Accelerated Turnover of cGMP In a rod outer segment in darkness, the activity of the PDE is low; therefore, the turnover rate constant for cGMP (denoted b) is low, with a correspondingly long turnover time constant for cGMP, tcGMP ¼ 1/b, of around 1 s in amphibian rods and around 200 ms in mammalian rods. The magnitude of this parameter has a major effect on both the sensitivity and the kinetics of the rod’s response to a flash. Thus, when the PDE activity increases in steady illumination, the shorter turnover time for cGMP contributes to both desensitization and acceleration of the photoresponse (compared with the case that would have applied, had the steady level of PDE activity not increased). To provide an intuitive understanding of this mechanism, it is helpful to consider what we have referred to

previously as the bathtub analogy. Imagine a container of water, such as a tall cylinder, and let the height of water in the cylinder represent the level (concentration) of cGMP in the outer segment. The rate at which water runs out of the cylinder, through a drain hole at the base, is proportional both to the height of water and to the size of the opening, representing the cGMP level and the PDE activity, b, respectively. Likewise, the rate at which water flows in to the cylinder through a tap at the top represents the activity of guanylyl cyclase, a. When a steady state is reached, the height of water will equal the rate of influx divided by the size of the drain hole; that is, cGMP ¼ a/b. Importantly, whenever the water level is perturbed from this steady-state level (e.g., upon a brief opening of an additional drain hole), the level will re-equilibrate with a time constant tcGMP ¼ 1/b (provided that the rate of influx through the tap remains constant). Hence, if the drain hole is small (and the inflow via the tap correspondingly small), then any perturbation in water level elicited by a transient additional outflow will be corrected only slowly; if the drain hole is large (and the influx correspondingly large), then any perturbation will be rapidly corrected. Furthermore, though perhaps less intuitively, it can be shown that for a noninstantaneous perturbation, corresponding to the normal flash response, not only will the kinetics of recovery be faster, but the peak also will be smaller. Hence, the effect of the increased PDE activity during steady illumination is both to accelerate the response kinetics and to reduce the peak amplitude (i.e., reduce the sensitivity) to an incremental flash. Calculations show that in rods the 20-fold increase in b during steady illumination provides the primary mechanism underlying the measured shortening of the time to peak and the decrease in flash sensitivity.

Slow Changes in Rods: Light Adaptation or Dark Adaptation? In addition to the conventional features of rod photoreceptor light adaptation that occur extremely rapidly (on a subsecond time scale), other changes have been reported to occur over a time frame of minutes of exposure, in response to lights that saturate the cell’s response. As the effects of these changes are very slow, and can only be observed in darkness when the adapting exposure is extinguished, there is a semantic issue as to whether these phenomena should be thought of as light adaptation or as dark adaptation. Light-Induced Change in the Dominant Time Constant It has recently been shown by Marie Burns’ group that exposure of mouse rods to a just-saturating intensity of


around 1000 photoisomerizations per second, for 1 min or more, leads to a persistent speeding of the bright-flash response upon extinction of the background. The change did not involve any reduction in the activation phase of transduction, but instead involved a reduction in the dominant time constant of response recovery; typically, the dominant time constant tdom dropped from around 200 ms under dark-adapted conditions to around 100 ms immediately after extinction of the saturating light. The adaptational effect developed relatively slowly, building up over 60 s or so, and it required a rhodopsin bleach level of around 2% for full effect. The effect was relatively long lasting, declining with a time constant of around 80 s. The molecular mechanism giving rise to this adaptational effect is not known, though some evidence suggests that it corresponds to a reduction in lifetime of the activated transducin–PDE complex. If so, it represents a phenomenon distinct from the actions of dimmer adapting lights. Light-Induced Translocation of Proteins The light-induced translocation of transducin, recoverin, and arrestin in photoreceptors is dealt with in detail elsewhere in this encyclopedia and, therefore, mentioned only briefly here. Movements of protein are elicited only at quite bright intensities (generally in the saturating range) and occur over a time scale of many minutes. In mouse rods, intensities above 3000 photoisomerizations per second for 30 min (which bleach a substantial fraction of the rhodopsin) trigger the movement of transducin from the outer segment to the inner segment, while slightly lower intensities of 1000 photoisomerizations per second or more trigger the movement of arrestin in the opposite direction; recoverin also leaves the outer segment in bright light. Protein movements of these kinds may well affect the adaptational state of the rod, though this is yet to be established clearly. Since the movements are triggered only by saturating light intensities, the electrical effects cannot readily be observed during the illumination because the circulating current is completely suppressed. One possibility is that the protein translocation contributes to some form of conservation – for example, lowering the guanosine 50 -triphosphate (GTP) consumption involved in the continual (and maximal) activation of transducin during daylight conditions. Alternatively, it may be that the changes help prepare the rod for its return to lower intensities, as occurs around dusk. Interestingly, in one attempt that was made to detect any change in the amplification constant of human rods (using the electroretinogram (ERG)) following exposures to intensities that elicit transducin translocation in mouse rods, no change in amplification was detectable.

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Dark Adaptation of the Rods: Very Slow Recovery from Bleaching Following exposure of our eye to very intense illumination, our visual threshold is greatly elevated and may take tens of minutes to recover fully. Closely comparable effects can be measured in the overall visual system and at the level of the rod photoreceptors or the rod bipolar cells. The slow recovery of sensitivity is referred to as dark adaptation or bleaching adaptation; however, it should be noted that this use of the term adaptation is something of a misnomer. Adaptation normally refers to beneficial adjustments; yet, the changes that accompany intense illumination are distinctly disadvantageous – thus, there can be no advantage in being almost blind following exposure to intense light. The recovery of visual threshold for a human subject is plotted in Figure 5, following the cessation of nine light exposures that bleached from 0.5% to 98% of the rhodopsin. For a bleach of 20%, the visual threshold was initially elevated by 3.5 log units. This indicates that the elevation of threshold is out of all proportion to the fraction of pigment remaining unbleached; even though 80% of the rhodopsin remained functional, the threshold was raised 3000-fold. Instead, there is overwhelming evidence that the phenomenon arises from the presence within the outer segment of unregenerated opsin (i.e., the presence of the protein part of the visual pigment, prior to its recombination with the regenerated 11-cis retinal). Remarkably, the recovery of scotopic (rod-mediated) threshold exhibits a region of common slope across all the bleach levels, as indicated by the parallel red lines in Figure 5. This region is termed the S2 component of recovery, and has a slope CS2 = 0.24 log unit min1 that is characteristic of dark adaptation recovery in normal (young adult) human eyes; also characteristic is the nature of the rightward shift of the recovery traces as a function of increasing bleach level – the form of this shift is as expected for a rate-limited (zero-order) recovery process, as distinct from an exponential (first-order) recovery process. From a detailed analysis of results of this kind, in combination with knowledge of the retinoid cycle, Trevor Lamb and Edward Pugh developed a cellular model that can account for human dark adaptation behavior. They postulated that (1) the presence of opsin (without chromophore) gives rise to a phenomenon closely equivalent to light, through activation of the G protein cascade of transduction and (2) the elimination of opsin via its reconversion to rhodopsin follows rate-limited kinetics because of a limitation in the supply of 11-cis retinal that results from the movement of this substance from a pool in the retinal pigment epithelium. Application of this cellular model has provided an accurate account of (1) the regeneration of visual pigment

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Figure 5 Human psychophysical dark adaptation. Recovery of log threshold elevation in a normal human observer is plotted as a function of time in darkness, after a wide range of bleaching exposures (from 0.5% to 98%). Parallel red lines represent component S2, with a slope of –0.24 decades min1 (see text). The lateral shift between the lines is consistent with the rate-limited delivery of 11-cis retinal from the RPE to opsin in the outer segments. Reproduced from Lamb, T. D. and Pugh, E. N., Jr. (2006). Phototransduction, dark adaptation, and rhodopsin regeneration. The Proctor Lecture. Investigative Ophthalmology and Visual Science 47: 5138–5152, with permission of the Association for Research in Vision and Ophthalmology.

in humans and other mammals, measured by retinal densitometry; (2) normal human dark adaptation behavior (as in Figure 5); and (3) the slowed regeneration of pigment and the slowed dark adaptation that is characteristic of a number of diseases that affect the photoreceptors and/or retinal pigment epithelium. See also: Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors; Phototransduction: Adaptation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Rods; Phototransduction: The Visual Cycle.

Further Reading Cameron, A. M., Mahroo, O. A. R., and Lamb, T. D. (2006). Dark adaptation of human rod bipolar cells measured from the bwave of the scotopic electroretinogram. Journal of Physiology 575: 507–526.

Krispel, C. M., Chen, C-K., Simon, M. I., and Burns, M. E. (2003). Novel form of adaptation in mouse retinal rods speeds recovery of phototransduction. Journal of General Physiology 122: 703–712. Lamb, T. D. and Pugh, E. N., Jr. (2004). Dark adaptation and the retinoid cycle of vision. Progress in Retinal and Eye Research 23: 307–380. Lamb, T. D. and Pugh, E. N., Jr. (2006). Phototransduction, dark adaptation, and rhodopsin regeneration. The Proctor Lecture. Investigative Ophthalmology and Visual Science 47: 5138–5152. Nikonov, S., Lamb, T. D., and Pugh, E. N., Jr. (2000). The role of steady phosphodiesterase activity in the kinetics and sensitivity of the lightadapted salamander rod photoresponse. Journal of General Physiology 116: 795–824. Pugh, E. N., Jr. and Lamb, T. D. (2000). Phototransduction in vertebrate rods and cones: Molecular mechanisms of amplification, recovery and light adaptation. In: Stavenga, D. G., de Grip, W. J., and Pugh, E. N., Jr (eds.) Handbook of Biological Physics, Vol. 3, Molecular Mechanisms of Visual Transduction, ch. 5, pp. 183–255. Amsterdam: Elsevier. Pugh, E. N., Jr., Nikonov, S., and Lamb, T. D. (1999). Molecular mechanisms of vertebrate photoreceptor light adaptation. Current Opinion in Neurobiology 9: 410–418. Tamura, T., Nakatani, K., and Yau, K-W. (1991). Calcium feedback and sensitivity regulation in primate rods. Journal of General Physiology 98: 95–130.

Phototransduction: Inactivation in Cones V V Gurevich and E V Gurevich, Vanderbilt University, Nashville, TN, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Arrestin – A protein that selectively binds light-activated phosphorylated photopigment and blocks further signal transduction. Cones express two subtypes, arrestin1 and arrestin4 (often termed rod and cone arrestins, respectively). Cone opsins – Light receptors, consisting of the protein part (opsin) and 11-cis-retinal covalently attached via Schiff base to a lysine in the seventh transmembrane domain. All opsins are members of superfamily of G-protein-coupled receptors (GPCRs), the largest family of signaling proteins in animals (mammals have 1000 different GPCRs). GCAP – Guanylyl cyclase activating protein is a member of the superfamily of EF-hand-containing calcium-binding proteins. Cones express two homologs, GCAP1 and GCAP2, which in the calcium-liganded form inhibit and in magnesium-liganded form enhance the activity of retinal guanylyl cyclase (retGC). GRKs – G-protein-coupled receptor kinases that specifically phosphorylate active forms of their cognate receptors. Cones express rhodopsin kinase (systematic name: GRK1) and a cone-specific form GRK7. However, mice do not have GRK7; therefore, photopigments in mouse cones and rods are phosphorylated by a single isoform, GRK1. Phosphodiesterase (PDE) – The photoreceptorspecific cyclic guanosine monophosphate (cGMP) PDE, PDE6. Cone PDE6 is a heterotetramer, consisting of two identical catalytic a0 -subunits and two inhibitory g-subunits. PDE rapidly hydrolyzes cGMP upon its activation by transducin, when its catalytic activity approaches the theoretical limit set by the rate of cGMP diffusion. RetGC – Retinal guanylyl cyclase is structurally related to receptor guanylyl cyclases. Cones predominantly express RetGC1, in contrast to rods that express RetGC1 and RetGC2 at comparable levels. RGS9-1 – Photoreceptor-specific short isoform of the regulator of G-protein signaling 9 expressed in both rods and cones. It interacts with the complex of the guanosine triphosphate (GTP)-liganded active a-subunit of transducin with PDEg and facilitates its intrinsic GTPase activity, thereby directly inactivating

transducin and indirectly PDE. Cones express much more RGS9-1 than rods. Transducin – Photoreceptor-specific heterotrimeric G protein that couples to light-activated opsins. Its a-subunit belongs to Gi/o family. All types of cones express the same a-subunit that is different from the rod variant.

Rod photoreceptors are often described as a marvel of molecular engineering, which creates an impression that cones are just noisier and less-sensitive rods. In fact, as light sensors, cones are just as amazing: their adaptability gives cones a much wider dynamic range covering more than seven orders of magnitude of light intensity without saturation. Cones begin to function in the light of the full moon reflected from objects in the night and are still adequate for a direct look at the sun. Mostly for technical reasons, the biochemistry of cone photoreceptors, particularly the molecular mechanisms underlying adaptation, is not as well studied as the signaling in rods. The assumption that the signaling and shutoff mechanisms in cones and rods are qualitatively similar is often used to fill the gaps in our knowledge of cone biochemistry. To avoid repetition, here we emphasize known differences between the cone and rod inactivation mechanisms.

Cone Signaling Cascade Cone opsins are closely related to rhodopsin and belong to the same branch of the G-protein-coupled receptor superfamily. Gene duplication events in early vertebrate evolution produced five groups of light receptors: rhodopsins and four classes of cone opsins. Mammals lost half of cone opsin classes, retaining only two. Light activates cone opsins via induced isomerization of 11-cis-retinal covalently attached to a lysine in the seventh transmembrane domain. Cone opsins use the same 11-cis-retinal as rhodopsin, but have very different spectral sensitivity, with maxima ranging from 360 nm (ultraviolet) to 575 nm (red). Spectral tuning of covalently linked retinal is achieved by changing its environment in the retinalbinding pocket of opsin. Light-activated cone opsins couple to cone transducin, which, in turn, activates the cone subtype of phosphodiesterase 6 (PDE6). Subsequently,

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a decrease of cytoplasmic cyclic guanosine monophosphate (cGMP) reduces the influx of Naþ and Ca2þ through the cone variant of cGMP-gated channels, resulting in cell hyperpolarization. Similar to rods, the decrease in Ca2þ concentration and consequent replacement of bound Ca2þ with Mg2þ converts guanylate cyclase activating proteins (GCAPs) from inhibitors to activators of retinal guanylate cyclase (RetGC). The latter replenishes cGMP lost during the light response, which opens the channels, thereby restoring cytoplasmic Ca2þ to the original levels. Thus, the activation and deactivation mechanisms employed by rods and cones are quite similar. However, subtle differences at every step of the pathway, including differences in the subtypes of signaling proteins involved, their expression levels, and the geometry of the cell, result in striking functional specialization of the two types of photoreceptors.

Shutoff of the Light-Activated Cone Opsins As far as signaling is concerned, the key difference between rhodopsin and cone opsins is lower thermal stability of the latter. Because cone opsins spontaneously activate with much higher probability than rhodopsin and readily release retinal even in the dark, cones generate noise that is orders of magnitude higher than in rods. This rules out the detection of signals below the noise level (e.g., a few photons) and makes even completely dark-adapted cones pre-desensitized and ready to operate at light levels they can detect. Loose attachment of the retinal to opsin also results in a significantly faster spontaneous decay of the light-activated cone opsin. For example, mouse cone S-opsin transgenically expressed in rods lacking arrestin was estimated to decay 40 times faster than rhodopsin coexpressed in the same cell. However, this spontaneous decay with a time constant of 1.3 s is still much slower than the rate of recovery in cones. Evolution equipped cones with a more elaborate (and presumably more efficient) photopigment shutoff machinery than that found in rods. The opsin inactivation is accelerated by pigment phosphorylation followed by arrestin binding. In most species, cones express two G-protein receptor kinase (GRK) subtypes: GRK1 (shared with rods) and cone-specific GRK7. It is likely that the co-expression of GRK7 with higher enzymatic activity accelerates opsin phosphorylation in cones. However, it should be noted that mice and rats are rare exceptions: these nocturnal rodents have only GRK1 in both types of photoreceptors. Cones also express two arrestin subtypes, arrestin1 and cone-specific arrestin4 (formerly known as rod and cone arrestins, respectively). Arrestin1 is present in cones at 50-fold molar excess over arrestin4. A recent study in knock-out animals shows

that both arrestins contribute comparably to the shutoff of the photopigment in cones. It is not entirely clear why cones express two arrestin subtypes, rather than a higher level of one subtype, especially considering that the cone opsin transgenically expressed in mouse rods is rapidly and efficiently deactivated by rod arrestin1. Two functional differences between these arrestins provide some clues. Arrestin1 has high propensity to selfassociate, cooperatively forming dimers and tetramers at physiological concentrations. Even in the dark-adapted rod, where the outer segment contains a small fraction of the total arrestin1, most of arrestin1 is a tetramer. It has been unambiguously shown that only monomeric arrestin1 is an active rhodopsin-binding species; therefore, oligomers appear to be storage forms. In contrast, cone-specific arrestin4 does not self-associate at physiologically relevant concentrations; therefore, the whole complement of arrestin4 present in cones is an active monomer. Recent estimates of their expression and arrestin1 self-association constants suggest that dark-adapted cones have in the outer segment 60 mM of arrestin4 and 30 mM (5% of the total) of monomeric arrestin1 ready to bind phosphorylated opsin at any time, in addition to a huge backup supply of arrestin1 oligomers. The second important difference lies in the stability of the arrestin complex with phosphorylated opsin. Arrestin1 forms very stable complexes that take the bound molecule of phosphopigment out of the game for a long time. This is important in the rod to ensure the fidelity of the shutoff. In order to release completely inactive rhodopsin upon dissociation, arrestin1 must stay bound until metarhodopsin II (Meta II) slowly decays and likely until it is regenerated with 11-cis-retinal. In contrast, arrestin4 forms fairly transient complexes with phosphorylated cone opsins, likely to ensure the quick return of the opsin back into the active pool. This is important for cone photoreceptors that function at a high rate of pigment bleaching. Although we do not know with certainty why cones express both arrestin subtypes, one scenario appears to provide a plausible explanation. Given the concentrations of the two arrestins in cones, in moderately bright light arrestin4 likely has an advantage, so that the majority of phosphorylated cone opsin would be rapidly recycled to the signalingcompetent pool. Increasing levels of illumination inducing massive pigment bleaching would force the cell to draw increasingly on the virtually inexhaustible supply of arrestin1, which forms long-lived complexes with the opsin. The recent finding that arrestin1 plays a more prominent role in cone recovery after very bright flashes is consistent with this model. The formation of arrestin1opsin complexes would take larger and larger fraction of the pigment out of action for a relatively long time, possibly serving as one of the mechanisms of light adaptation. Even though a cone-specific visual cycle involving


Mu¨ller glia provides 11-cis-retinal faster than the canonical retinal pigment epithelium-based visual cycle supplying rods, very bright light bleaches cone pigment faster than it can be regenerated. This loss of functional opsin was proposed to reduce light capture, acting as a mechanism of adaptation. It is entirely possible that in bright light, both incomplete regeneration of opsin and its binding by arrestin1 cooperate to limit the active pool, thereby reducing light sensitivity of cones. Overall, cones combine less-stable photopigment with more sophisticated machinery of its inactivation (Figure 1). These factors apparently contribute to faster shutoff at the opsin level and likely provide cone-specific mechanisms for light adaptation.

Inactivation of Transducin and PDE It is generally accepted that the activation of cone transducin by cone opsins and that of cone PDE6 by the guanosine triphosphate (GTP)-liganded a-subunit of cone transducin proceeds in similar ways to corresponding processes in rods. All three subunits of cone transducin differ from their rod counterparts, but the significance of this specialization is uncertain. In fact, cone S-opsin transgenically expressed in mouse rods efficiently activates the signaling cascade coupling to rod transducin. Cone PDE6 is an a0 2g2 heterotetramer, in contrast to the abg2 version in rods, but the functional significance of the use of different catalytic subunits remains to be elucidated. There is one biochemical difference that undoubtedly contributes to the much faster inactivation of transducinPDE6 complex in cones: 10-fold higher level of the regulator of G-protein signaling 9-1 (RGS9-1) expression. It has been convincingly shown that the deactivation at this step rate limits the recovery kinetics in rods, and that the level of RGS9-1, which accelerates self-inactivating GTPase of transducin a-subunit, sets the speed of transducin-PDE6 inactivation. Thus, the shutoff at the opsin and transducin-PDE6 level in cones is much faster than corresponding processes in rods; however, it is still not clear which step is rate limiting in cone recovery.

Restoration of cGMP and Intracellular Calcium Level Similar to the situation in rods, cone activation results in a drop in the intracellular Ca2þ concentration due to the closure of the cGMP-gated channels mediating the bulk of Ca2þ entry. In order to return to the initial state after opsin and PDE6 are fully inactivated, cones need to restore cytoplasmic cGMP hydrolyzed by PDE6. Cones and rods use the same negative-feedback mechanism that

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translates the drop in Ca2þ resulting from the reduction in the cGMP level into a signal to make more cGMP. Ca2þ dissociates from GCAPs when its cytoplasmic concentration drops in the light. The replacement of lost Ca2þ by Mg2þ converts GCAPs from inhibitors to activators of RetGC. The generated cGMP opens the channels, and the consequent increase in cytoplasmic Ca2þ stops further cGMP synthesis. Cones apparently express the same combination of GCAP1 and GCAP2 (which differ in their Ca2þ sensitivity) as rods. The functional significance of the predominance of the RetGC1 isoform in cones (in contrast to similar levels of RetGC1 and RetGC2 in rods) is not clear. Several important differences between rods and cones are known to be responsible for much faster cone recovery. First, the rate of recovery depends on the absolute amounts of cGMP and Ca2þ that need to be replenished. Here cones hold an obvious advantage due to the much smaller volume of their outer segments: the hydrolysis or synthesis of the same absolute amount of cGMP leads to a more significant change in its concentration. Similarly, the closure of the same fraction of cGMP-gated channels leads to a more profound drop in intracellular Ca2þ in cones. However, geometry is only part of the story. The channel expressed in cones has a different subunit composition and ion preference. About 35% of the inward current via the cone cGMP-gated channel is carried by Ca2þ, whereas in rods this fraction is only 20%. Thus, the closure of the same fraction of channels upon PDE6 activation results in a substantially greater change in the absolute number of Ca2þ ions entering the cell. Increased Ca2þ influx in cones is balanced by its accelerated extrusion via Naþ/Kþ–Ca2þ-exchanger, so that the turnover of Ca2þ in cone outer segments is more rapid. The combination of faster constitutive extrusion, larger fraction of the current carried by Ca2þ through cGMP-gated channels, and much smaller outer segment volume greatly increases the rate of Ca2þ drop in response to light stimulus, speeding up RetGC activation and cGMP resynthesis. High intracellular Ca2þ reduces the sensitivity of the channels to cGMP, so that when the intracellular Ca2þ drops, the channels become more sensitive to the cytoplasmic cGMP and therefore reopen faster. This mechanism operates in both types of photoreceptors, but it is more powerful in cones, further contributing to accelerated recovery.

Conclusions Cone photoreceptors use essentially the same molecular mechanisms of signal shutoff at the opsin level as rods. At this step, cones achieve much higher speed of inactivation by employing, in addition to GRK1 and arrestin1

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Figure 1 Biochemical mechanisms of rapid inactivation in cones. (a) Cone opsins are phosphorylated by both GRK1 and GRK7 coexpressed in cones of most vertebrates, including humans. At moderate light levels, the signaling by phosphorylated photopigment is largely quenched by constitutively monomeric arrestin4, which forms transient complexes with the receptor. (b) During massive opsin activation in very bright light, the amount of expressed arrestin4 becomes insufficient to quench all active opsins; therefore, cones increasingly use coexpressed arrestin1, which forms longer-lived complexes with phosphorylated cone opsins. The consumption of monomeric arrestin1 by the photopigment shifts its monomer–dimer–tetramer equilibrium toward dissociation of oligomers, which generates virtually inexhaustible supply of binding-competent monomer. (c) RGS9-1 is expressed at 10-fold-higher level in cones than in rods, ensuring much faster inactivation of transducin and PDE. Cone opsin is shown as a bundle of seven transmembrane domains; opsin-attached phosphates are shown as spheres; lipid modifications anchoring recoverin, GRK1, GRK7, a-subunit of transducin, and catalytic a0 -subunits of PDE are shown as membrane-imbedded arrows. Rec: recoverin, Arr1: arrestin1, and arr4: arrestin4.

used by rods, cone-specific GRK7 and arrestin4. The presence of two GRKs speeds up the phosphorylation of light-activated opsin, whereas the expression of two arrestin subtypes with very different functional characteristics

likely results in a gradual switch from rapidly reversible arrestin4 interaction with phospho-opsin at moderate light levels to semi-irreversible binding of arrestin1 in very bright light. Inactivation at the transducin/PDE


level is accelerated by a 10-fold higher expression of RGS9-1 in cones. Two key features ensure faster recovery in cones than in rods. Faster Ca2þ turnover due to higher influx through cone-specific cGMP-gated channels and efflux via Naþ/Kþ–Ca2þ-exchanger generate greater net changes in the number of Ca2þ ions in the outer segment when the same fraction of the channels is closed. Due to much smaller outer-segment volume, the same net change in the number of cGMP molecules or Ca2þ ions produces greater changes in the concentration of these second messengers. Rapid response and recovery gives cones better temporal resolution than rods. The high speed of activation and inactivation in combination with more powerful adaptation mechanisms (many of which still need to be elucidated at the molecular level) allows cones to function in a broad range of light levels without saturation. See also: Phototransduction: Adaptation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin.

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Further Reading Cote, R. H. (2006). Photoreceptor phosphodiesterase (PDE6): A G-protein-activated PDE regulating visual excitation in rod and cone photoreceptor cells. In: Beavo, J. A., Francis, S. H., and Houslay, M. D. (eds.) Cyclic Nucleotide Phosphodiesterases in Health and Disease, pp 165–193. Boca Raton, FL: CRC Press. Dizhoor, A. M., Olshevskaya, E. V., and Peshenko, V I. (2006). Calcium sensitivity of photoreceptor guanylyl cyclase (RetGC) and congenital photoreceptor degeneration: Modeling in vitro and in vivo. In: Philippov, P. P. and Koch, K.-W. (eds.) Neuronal Calcium Sensor Proteins, pp 203–219. New York: Nova Science Publishers, Inc. Gurevich, V. V., Hanson, S. M., Gurevich, E. V., and Vishnivetskiy, S. A. (2007). How rod arrestin achieved perfection: Regulation of its availability and binding selectivity. In: Kisselev, O. and Fliesler, S. J. (eds.) Signal Transduction in the Retina. Methods in Signal Transduction Series, pp 55–88. Boca Raton, FL: CRC Press. Hanson, S. M., Van Eps, N., Francis, D. J., et al. (2007). Structure and function of the visual arrestin oligomer. European Molecular Biology Organization Journal 26: 1726–1736. Knox, B. E. and Solessio, E. (2006). Shedding light on cones. The Journal of General Physiology 127: 355–358. Korenbrot, J. I. and Rebrik, T. I. (2002). Tuning outer segment Ca2þ homeostasis to phototransduction in rods and cones. Advances in Experimental Medicine and Biology 514: 179–203. Nikonov, S. S., Brown, B. M., Davis, J. A., et al. (2008). Mouse cones require an arrestin for normal inactivation of phototransduction. Neuron 59: 462–474.

Phototransduction: Inactivation in Rods V V Gurevich and E V Gurevich, Vanderbilt University, Nashville, TN, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Arrestin (also known as S-antigen, 48-kDa protein, and rod or visual arrestin; systematic name: arrestin1) – A protein that selectively binds light-activated phosphorylated rhodopsin and blocks further signal transduction. Guanylyl cyclase activating protein (GCAP) – A member of the superfamily of EF-hand-containing calcium-binding proteins. Rods express two homologs, GCAP1 and GCAP2, which in calciumliganded form inhibit and in magnesium-liganded form enhance the activity of retinal guanylyl cyclase (retGC). Phosphodiesterase (PDE) – Photoreceptorspecific cGMP phosphodiesterase, PDE6. Rod PDE6 is a heterotetramer, consisting of two nonidentical catalytic subunits (a- and b-) and two inhibitory g-subunits. PDE6 rapidly hydrolyzes cyclic guanosine monophosphate (cGMP) upon its activation by transducin. In fully activated state, its catalytic activity approaches the limit set by the rate of cGMP diffusion. RetGC – It is structurally related to receptor guanylyl cyclases. Rods express comparable levels of two homologs, RetGC1 and RetGC2. Regulator of G-protein signaling 9 (RGS9-1) – Photoreceptor-specific short isoform of the regulator of G-protein signaling 9 expressed in both rods and cones. It interacts with the complex of the guanosine triphosphate (GTP)-liganded active a-subunit of transducin with PDEg and facilitates its intrinsic GTPase activity, thereby directly inactivating transducin and indirectly PDE. Rhodopsin – Light receptor, consisting of the protein part (opsin) and 11-cis-retinal covalently attached via Schiff base to a lysine in the seventh transmembrane domain. A member of the superfamily of G-proteincoupled receptors (GPCRs), also known as seven transmembrane domain receptors (7TMRs), the largest family of signaling proteins in animals (mammals have 1000 different GPCRs). Rhodopsin kinase (RK) (systematic name: GRK1) – It is a member of the G-protein-coupled receptor kinase (GRK) family expressed in both rods and cones.

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Transducin – Photoreceptor-specific heterotrimeric G protein that couples to light-activated rhodopsin. Its a-subunit belongs to Gi/o family.

As light sensors, vertebrate rod photoreceptors are a remarkable evolutionary achievement: rods yield amazingly low noise despite the presence of 108–109 molecules of the light receptor rhodopsin, and demonstrate singlephoton sensitivity and a dynamic range of seven orders of magnitude of light intensity. This level of perfection is achieved through several unique structural and biochemical adaptations. The rod outer segment (OS) is a specialized signaling compartment containing rhodopsin molecules tightly packed in disks. It is separated from the inner segment (IS), which is a mitochondria-rich power station providing huge amounts of energy. Several soluble signaling proteins move between the two compartments depending on the illumination, ensuring their on-demand delivery to the OS. The OS concentrations of transducin (Td) and arrestin, the proteins that transmit and shut down rhodopsin signaling, respectively, change by at least 10-fold. An important functional feature of the rod is that every biochemical step in the pathway between photon capture and the change in synaptic output has its own dedicated shutoff mechanism.

What Needs to Be Inactivated: Overview of the Signaling Cascade Rhodopsin activation by a photon of light is the first step in visual signaling. Due to extremely high concentration of its cognate G protein, Td, and rapid diffusion of both active rhodopsin (Rh*) and Td in the plane of the disk membrane, Rh* activates a molecule of Td every few milliseconds, generating 50–100 active Td (Td*) during its lifetime. These events occur in the two-dimensional space on the cytoplasmic surface of disk membranes. Each Td* binds the inhibitory g-subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6), turning the enzyme on. Each molecule of active PDE6 hydrolyzes several cGMP molecules per millisecond, producing a rapid drop in the cGMP concentration in the three-dimensional cytoplasmic space. This results in closure of cGMP-gated Na+/Ca2+ channels on the


plasma membrane. In rods, light activation of a single rhodopsin translates into the hydrolysis of 100 000 cGMP molecules. The channels are heterotetramers, with each subunit carrying a cGMP-binding site in its C-terminal domain. Highly cooperative cGMP binding to the four sites in the channel greatly increases its response to the change in cGMP concentration. The decrease of the inward current hyperpolarizes the rod, reducing neurotransmitter release in its output synapse. Several features of the rod signaling machinery bring its light sensitivity within the range of its physical limit: the detection of single photons. First, the concentration of signaling molecules in the rod OS is orders of magnitude higher than in normal cells: 3 mM rhodopsin (compared to low nanomolar concentrations of related receptors elsewhere), 0.3 mM Td, 60 mM PDE, and so on. Second, all three signaling proteins involved have much lower basal activity than their counterparts in other cells. This results in an incredibly low noise level, making signal-to-noise ratio favorable for the detection of even an extremely weak signal. Third, very efficient shutoff mechanisms at every step of the pathway rapidly terminate the signaling, allowing for an exquisite subsecond temporal resolution of mammalian rods.

Shutoff of the Light-Activated Rhodopsin Rhodopsin is a prototypical G-protein-coupled receptor. In contrast to 1000 other members of this superfamily, it has virtually no basal activity, because it is effectively suppressed by the covalently attached inverse agonist, 11-cis-retinal. Retinal is converted by light into the alltrans form, which is a potent agonist of rhodopsin. The fact that it remains covalently attached to the receptor (in contrast to other GPCRs where bound and free agonists are in dynamic equilibrium) ensures a powerful burst of signaling. Through a series of short-lived photoproducts, light-activated rhodopsin reaches the Metarhodopsin II (Meta II) state, which is an active form (Rh*) that couples to Td. Meta II is in equilibrium with the two other states, Meta I and Meta III, which are believed to be inactive, or at least considerably less active than Meta II. Ultimately, all-trans-retinal dissociates, yielding empty protein opsin, which has orders of magnitude lower ability to activate Td than Meta II. However, this spontaneous deactivation of rhodopsin is inadequate as a shutoff mechanism for two reasons. At physiological temperatures, rhodopsin decay takes about a minute, which would greatly compromise temporal resolution. Moreover, the activity of opsin, which is much higher than that of dark rhodopsin, would generate considerable noise, compromising rod sensitivity. Therefore, rods use a sophisticated two-step mechanism to achieve rapid and complete rhodopsin deactivation.

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First, light-activated rhodopsin is phosphorylated by rhodopsin kinase (RK). Similar to Td, RK is activated by binding to Rh*. Therefore, it selectively phosphorylates the active form of rhodopsin. It should be noted that at low light levels, RK was reported to phosphorylate multiple rhodopsin molecules for each light-activated one, likely by targeting neighboring inactive rhodopsins in the crowded disk membrane. In mammals, RK activity is believed to be held in check by its interaction with Ca2+loaded recoverin. As a result, RK is fully unleashed only after a brief delay, which allows Td activation to continue until the Ca2+ concentration in the rod actually drops. However, low affinity of recoverin for Ca2+ (KD 5 mM) is the weak point of this model. Current estimates of the Ca2+ concentrations in the dark- and light-adapted mouse OS are 250 nM and 25 nM, respectively, so that only a very small fraction of recoverin would be Ca2+-loaded in either. In addition, unlike RK, recoverin predominantly localizes in the inner segment. Still, rods express 50 molecules of recoverin for each RK, so a relatively small change in the Ca2+ occupancy of a fraction of recoverin present in the OS could conceivably play a role in RK regulation. Phosphorylation per se reduces, but does not abolish the ability of rhodopsin to activate Td. In the next step, arrestin binds active phosphorylated rhodopsin (P-Rh*), shielding its cytoplasmic tip and precluding further Td interaction. Arrestin apparently remains bound until rhodopsin decays to opsin, and very likely even longer, until opsin is regenerated with 11-cis-retinal to the truly inactive dark rhodopsin. Arrestin has several dedicated phosphate-binding residues and other elements that specifically interact with light-activated rhodopsin independently of its phosphorylation state. These partial interactions mediate relatively low-affinity binding to dark P-Rh and unphosphorylated Rh*, respectively. Arrestin elements participating in these interactions also serve as sensors, allowing arrestin to test the functional state of the rhodopsin molecule it encounters and then quickly dissociate from its low-affinity targets, dark Rh, dark P-Rh, or Rh*. In contrast to all other forms, P-Rh* simultaneously engages both sets of elements. This turns the two sensors on at the same time, allowing the arrestin transition into a high-affinity rhodopsin-binding state. This transition involves a global conformational change in arrestin, which mobilizes additional arrestin elements for the interaction. Thus, arrestin works as a molecular coincidence detector, swinging into action only when the rhodopsin molecule it encounters is both active and phosphorylated. The model of sequential multisite interaction readily explains exquisite arrestin selectivity, that is, manifold difference in arrestin binding to Rh* and dark P-Rh on the one hand, and to its preferred target P-Rh* on the other. The salt bridge between positively charged Arg175 and negatively charged Asp296, which is one of the

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intramolecular interactions holding arrestin in its basal state, was identified as the main phosphate sensor in arrestin. Rhodopsin-attached phosphates bind Arg175 and neutralize its charge, thereby breaking the salt bridge and facilitating arrestin transition into its active conformation. The reversal of either charge by targeted mutagenesis yields mutants with reduced need for rhodopsinattached phosphates that bind Rh* with much higher affinity than wild-type protein. Rhodopsin has multiple phosphorylation sites in its Cterminus. The issue of the number of rhodopsin-attached phosphates necessary for high-affinity arrestin binding was resolved only recently. Studies performed in vitro with rhodopsin carrying defined number of phosphates and in vivo with mice expressing rhodopsin mutants with different number of sites show that rhodopsin multi-phosphorylation is required. A single rhodopsin-attached phosphate does not appreciably increase arrestin affinity, two somewhat enhance the binding, and three phosphates are necessary for high-affinity interaction in vitro and for the rapid shutoff of photoresponse in vivo. Whereas arrestin binding does not further increase when Rh* has more than three phosphates, the presence of additional phosphorylation sites on Rh accelerates the shutoff of the photoresponse in vivo. This likely reflects the kinetic effect of the abundance of sites that remain available to RK on partially phosphorylated rhodopsin. For example, rhodopsin with three sites would have only one possible RK target left after the incorporation of two phosphates, whereas rhodopsin with six sites would still have four available targets at the same level of phosphorylation. Thus, supernumerary sites would ensure that the magic number of three phosphates per rhodopsin is achieved faster.

Inactivation of Td and PDE Td is a prototypical heterotrimeric G protein consisting of a-, b-, and g-subunits. In the inactive state, the abgtrimer has guanosine diphosphate (GDP) in the nucleotide-binding site of the a-subunit. In this state, lipid modifications of both a-(N-terminal myristoyl) and g(C-terminal farnesyl) subunits provide a fairly strong membrane anchor. This restricts the Td diffusion to the plane of the disk membrane and enforces the orientation favorable for Rh* interaction, thereby maximizing its chances of encountering active rhodopsin and being activated by it. The Td interaction with Rh* opens its nucleotide-binding pocket, whereupon GDP promptly falls out and is immediately replaced by GTP simply because the latter is much more abundant in the cytoplasm. The GTP-liganded a-subunit dissociates from Rh* and bgdimer. Tda-GTP binds the inhibitory g-subunit of cGMP PDE, greatly increasing PDE activity by relieving

the inhibition. Importantly, the separation of the two parts of Td heterotrimer dramatically weakens their membrane anchoring, so that active Tda-GTP can jump off the disk where it was generated by Rh* and activate PDE on neighboring discs, spreading the signaling in three dimensions. Due to its very high catalytic activity (kcat 2000 s 1 per subunit), active PDE rapidly reduces cGMP concentration in its vicinity, which leads to the closure of cGMP-gated channels and hyperpolarization of the rod within milliseconds of rhodopsin activation by light (Figure 1). Similar to other heterotrimeric G proteins, Tda has GTPase activity, which serves as a built-in self-inactivation mechanism. However, the intrinsic GTPase of free Tda is very slow. Interaction of Tda with PDE g-subunit increases the activity of its GTPase. The interaction of Tda–GTP–PDEg complex with rod-specific GTPase activating protein (GAP) increases the GTPase activity even further. GAP consists of the short isoform of RGS9, Gb5 (homolog of G-protein b-subunits), and another protein that provides membrane anchor for the complex, RGS9 anchoring protein (R9AP). Low basal GTPase of Tda gives it time to diffuse around searching for PDE to activate without losing the signal in the transmission. The dramatic acceleration of the GTPase activity of Tda by the PDE and GAP ensures that the signal is terminated quickly after it is received by PDE, improving the temporal resolution of the photoreceptor cell. The recent finding that the expression of the GAP complex in rods increases the rate of the response shutoff in a dose-dependent manner convincingly demonstrated that the inactivation of Tda–GTP– PDEg complex is the rate-limiting step in this process. These elegant experiments also revealed that when this step is maximally accelerated, the recovery kinetics becomes dominated by some other process with the time constant of 80 ms. This number sets the upper limit for the next slowest step, which could be one of the following: the average lifetime of active Rh*; the release of PDEg from Tda-GDP; reassociation of PDEg with PDE catalytic subunits; or even the time free Tda-GTP spends searching for PDE and/or docking to it.

Resynthesis of cGMP and Restoration of Calcium Level Obviously, to return to its initial state and become ready to respond to the next photon with the same vigor, the rod photoreceptor needs to do more than just turn off Rh* and all Td and PDE molecules activated by it. The response leaves, in its wake, substantially reduced cytoplasmic cGMP concentration and very low intracellular calcium due to the closure of the cGMP-gated channels that are responsible for the bulk of Ca2+ entry into the OS. Photoreceptors are equipped with an


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Figure 1 Biochemical mechanisms of signal inactivation in rods. (a) Visual amplification cascade. Light-activated rhodopsin (Rh*) catalyzes GDP/GTP exchange on visual G protein, Td, sequentially activating dozens of Td molecules. Inactive Td is an abgheterotrimer, whereas upon activation the GTP-liganded a-subunit dissociates from the bg-dimer and binds the inhibitory g-subunit of rod PDE (which is an abg2 heterotetramer). This activates PDE, which hydrolyzes massive amounts of cGMP (over 100000 molecules per one Rh*). The decrease in cytoplasmic cGMP closes cGMP-gated cation channels on the plasma membrane (right panel). Channel closure reduces the influx of Na+ and Ca2+, hyperpolarizing the cell up to 1 mV per one Rh*. (b) Rh* is phosphorylated by the rhodopsin kinase (RK, systematic name GRK1), which is expressed in rods and cones of all vertebrates. In the dark, RK may be kept away from rhodopsin via its interaction with Ca2+-liganded recoverin (Rec). Multiphosphorylation prepares Rh* for arrestin (Arr) binding. Arrestin shields the cytoplasmic tip of rhodopsin, sterically blocking its interactions with transducin, thereby completing rhodopsin inactivation. (c) The intrinsic GTPase activity of the Td a-subunit serves as a built-in inactivation mechanism. Its interaction with PDEg and RGS9-1 (which exists in constitutive complex with Gb5 and membrane anchoring protein R9AP) greatly facilitates GTP hydrolysis, ensuring rapid inactivation of Td and PDE. (d) In the dark, retinal guanylyl cyclase (GC) is inhibited by Ca2+-liganded GCAPs. Light-induced closure of the cGMP-gated channels results in the drop in cytoplasmic Ca2+. Its replacement with Mg2+ on the metal-binding sites of GCAPs converts them into GC activators. GC replenishes the cytoplasmic cGMP and consequent opening of the channels restores cytoplasmic Ca2+, thereby turning off GC. Rhodopsin is shown as a bundle of seven transmembrane domains; Rhodopsin-attached phosphates are shown as spheres; lipid modifications anchoring recoverin, GRK1, a- and g-subunits of Td, and catalytic a- and b-subunits of rod PDE are shown as membrane-imbedded arrows. Arr, arrestin1; GC, gyanylyl cyclase; Rec, recoverin; Rh*, light-activated rhodopsin; RK, rhodopsin kinase.

ingenious negative-feedback mechanism that translates the drop in Ca2+ resulting from the reduction in cGMP level into a signal to replenish it. In photoreceptors, cGMP is synthesized by retinal guanylyl cyclases (retGCs). RetGCs are related to a family of hormoneregulated guanylyl cyclases, such as atrial natriuretic factor receptor, which have extracellular hormone-binding domain connected via a single transmembrane helix to the intracellular guanylyl cyclase domain. Similar to these receptors, retGCs are dimeric, with each monomer

equipped with a catalytic domain and an extracellular domain. Interestingly, Mg2+ and GTP are bound by two different subunits forming the active catalytic site. As far as we know, the extracellular domain of retGCs neither binds any ligands nor participates in the enzyme regulation. Instead, the activity of retGCs is tightly regulated by their interaction via intracellular elements with GCAPs. GCAPs, as well as recoverin, are members of the neuronal calcium sensor protein branch of the superfamily of calcium-binding proteins containing EF hands (that includes calmodulin).

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Similar to other members of this family, GCAPs have four EF hands, three of which actually bind divalent cations. Strictly speaking, GCAPs are Ca2+–Mg2+-binding proteins. The word activating in their name is a bit misleading: Mg2+liganded GCAPs activate retGCs, whereas Ca2+-liganded forms actually inhibit cGMP synthesis. Thus, in darkadapted rods with high free-Ca2+ concentrations (estimates range from 250 to 600 nM in different species), GCAPs keep the retGC activity at low level. This makes perfect sense, because high Ca2+ indicates that there is enough free cGMP (2–5 mM) to keep the channels open. Light-induced decrease of intracellular Ca2+ (to 5–50 nM, based on different estimates) is the direct result of channel closure, reflecting reduced cGMP in need of replenishing. The loss of bound Ca2+ and its replacement by Mg2+ (which is always 1 mM in the cytoplasm) switches GCAPs from the inhibitory to the activating mode exactly when rapid cGMP synthesis is necessary to restore its level. Increasing cGMP opens more channels, thereby gradually restoring Ca2+. Rising Ca2+ displaces Mg2+ on GCAPs, progressively reducing retGC activity, so that the cell returns to the initial state. After a dim flash, this process often overshoots, leading to a transient increase in the cGMP and Ca2+ concentration, likely because PDE is inactivated faster than retGC. The absence of GCAPs slows down cGMP resynthesis, so that light-induced PDE activity results in a more profound decrease of cGMP than in the normal rod. This results in closure of more channels and greatly increases the amplitude of single-photon response. This compromises temporal resolution, prolonging the rising and falling phase of the light response, and limits the working range of rods to lower light levels. Interestingly, vertebrate photoreceptors express two isoforms of retGC, retGC1 and retGC2, and at least two GCAPs, GCAP1 and GCAP2. The presence of two isoforms of each protein in rods of all vertebrates, including fish, clearly indicates that the different isoforms have nonredundant functions. RetGCs are membrane proteins, suggesting that retGC1 and retGC2 may be localized to different membranes within the OS. RetGC1 was reliably detected in disks, and the possibility that a fraction may also be present in the plasma membrane remains open. The localization of RetGC2 was not studied with sufficient spatial resolution. Since retGC is a dimer, two isoforms of RetGC could give rise to three types of dimers, two homo- and one heterodimer. The fact that each subunit interacts with either GCAP1 or GCAP2 further expands the number of combinatorial possibilities. Definitive experiments, such as knockouts of individual isoforms of either protein, singly and in different combinations, are needed to fully elucidate the biochemistry of the Ca2+ feedback mechanism. A recent study of GCAP2 knockout mice revealed that although each GCAP is responsible for about half of the total retGC activation, the functions of the two proteins are quite

distinct. Due to lower affinity for Ca2+, GCAP1 switches to the activation mode as soon as the concentration of Ca2+ begins to fall, whereas GCAP2 responds later, when Ca2+ levels drop further. Thus, together the two GCAPs ensure graded increase in retGC activity in a wider range of Ca2+ concentrations than either one could have covered alone. Another issue in need of clarification is the physiological role of a remarkable buffering capacity of the OS cytoplasm for both second messengers. According to current estimates, total cGMP in the OS is as high as 50 mM, with only 2–5 mM of it being free and the rest bound to the noncatalytic sites on PDE a- and b-subunits. The polycationic region of PDEg appears to stabilize the interaction of cGMP with these sites. Reciprocally, the presence of cGMP in noncatalytic sites enhances the interaction of the a- and b-subunits with PDEg. This mechanism implies that PDE activation by Td would release cGMP from noncatalytic sites. The role of this event in the photoresponse remains unclear. Similarly, free Ca2+ represents only a small fraction of the total Ca2+ in the OS cytoplasm, the rest being bound to several abundant proteins, such as recoverin, GCAPs, and calmodulin. Ca2+ binding by these proteins is a two-way street: on the one hand, it critically regulates their function, on the other hand, by soaking up Ca2+, they significantly change its concentration, modulating the input they respond to. Obviously, Ca2+ and cGMP buffering cannot be separated by purely experimental means from other functional modalities of the proteins involved. Therefore, rigorous experimentation must be supplemented with detailed biochemically realistic mathematical modeling to distinguish between the effects of binding on protein activity and on the concentration of free second messenger in the cytoplasm, which is necessary to elucidate the exact biological roles of both.

Light-Dependent Protein Translocation and Rod Signaling Arrestin localization to the OS in the light and to the IS in the dark was first described in 1985, before the role of this protein in signal termination was established. The subsequent discovery that Td also translocates in a light-dependent fashion, moving in the opposite direction, suggested an idea that translocation may underlie well-known adaptation of rods to different light levels. Preferential localization in the dark-adapted rod of a signal transducer to the rhodopsin-rich OS and a signal terminator to the IS could increase light sensitivity by slowing down shutoff. Conversely, the removal of Td from the OS and accumulation of arrestin in this compartment in the light could significantly reduce it by decreasing the number of Td molecules activated by


Rh* and speeding up rhodopsin inactivation. Subsequent studies showed that the fraction of recoverin, which presumably slows down rhodopsin phosphorylation by keeping RK away from Rh*, in the OS decreases dramatically from 12% in the dark to less than 2% in the light (the bulk of recoverin localizes to the IS in both conditions). The amplification constant in light-adapted rods was found to decrease 10-fold, in line with the reduction of Td concentration in the OS. While these results support the idea that Td translocation plays a role in rod adaptation, by fully explaining the changes in sensitivity by Td movement alone, they effectively rule out any significant contribution of the translocation of other proteins. The recent finding that phosducin, which interacts with free bg-dimer released upon Td activation and demonstrates robust translocation from the OS in the light, does not contribute to rod adaptation, supports this notion. The movement of Td, arrestin, phosducin, and recoverin in both directions is a relatively slow process that takes many minutes, which does not seem adequate to explain much faster photoreceptor adaptation. The translocation of arrestin in both directions is energy independent. It is driven in the dark by its low-affinity interactions with microtubules, particularly abundant in IS, and in the light by its binding to light-activated forms of rhodopsin. These findings suggest that the translocation of arrestin and other proteins is more likely to play a role in rod survival during daytime than in relatively fast light/dark adaptation. However, the translocation of different proteins may have distinct functions, which to a large extent remain to be elucidated.

Why Rods Do Not Have an Action Potential In most neurons, extracellular Ca2+ enters presynaptic terminals during an action potential. A brief increase in its local concentration triggers transient exocytosis of neurotrasmitter-containing vesicles. In contrast, vertebrate rod photoreceptors work backward. In the dark, rods are partially depolarized (OS membrane potential is about –35 mV) due to massive influx of Na+ and Ca2+ ions through cGMPgated channels. This results in continuous release of the neurotransmitter (L-glutamate) from ribbon synapses. By virtue of closing cGMP-gated channels, light of increasing intensity induces progressive hyperpolarization of rods up to –60 mV (a change of 25 mV). The activation of a single rhodopsin changes the membrane potential by as much as 1 mV. Thus, light intensity is encoded in the extent of hyperpolarization, which determines the magnitude of the decrease of neurotransmitter release. This mechanism makes the signaling graded, in contrast to the all-or-nothing type in neurons with a conventional action potential. It

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directly couples the change in membrane potential with synaptic activity, so that both the closure of the cGMPgated channels upon light stimulation and their reopening upon signal termination described above immediately translate into corresponding changes in neurotransmitter release. The absence of thresholds ensures that the information is not lost in transmission, so that the brain can take full advantage of the single-photon sensitivity of the rod photoreceptors. In addition, this mechanism creates a natural ceiling: a full stop of neurotransmitter release is the maximum possible effect of the illumination of any intensity.

Conclusions In many respects, rod photoreceptors are virtually perfect light sensors that combine single-photon sensitivity with a surprisingly wide dynamic range. Exquisitely timed and extremely efficient inactivation at every step of the signaling cascade between light absorption by rhodopsin and changes in the membrane potential plays an important role in their function. Not surprisingly, molecular errors in this complex multistep inactivation mechanism due to mutations in key proteins underlie a variety of congenital visual disorders in humans, ranging in severity from night blindness to retinal degeneration. See also: Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors; Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin.

Further Reading Burns, M. E. and Arshavsky, V. Y. (2005). Beyond counting photons: Trials and trends in vertebrate visual transduction. Neuron 48: 387–401. Cote, R. H. (2006). Photoreceptor phosphodiesterase (PDE6): A G-protein-activated PDE regulating visual excitation in rod and cone photoreceptor cells. In: Beavo, J. A., Francis, S. H., and Houslay, M. D. (eds.) Cyclic Nucleotide Phosphodiesterases in Health and Disease, pp. 165–193. Boca Raton, FL: CRC Press. Dizhoor, A. M., Olshevskaya, E. V., and Peshenko, I. V. (2006). Calcium sensitivity of photoreceptor guanylyl cyclase (RetGC) and congenital photoreceptor degeneration: Modeling in vitro and in vivo. In: Philippov, P. P. and Koch, K.-W. (eds.) Neuronal Calcium Sensor Proteins, pp. 203–219. New York: Nova Science Publishers, Inc. Gurevich, V. V. and Gurevich, E. V. (2004). The molecular acrobatics of arrestin activation. Trends in Pharmacological Science 25: 105–111. Gurevich, V. V., Hanson, S. M., Gurevich, E. V., and Vishnivetskiy, S. A. (2007). How rod arrestin achieved perfection: Regulation of its availability and binding selectivity. In: Kisselev, O. and Fliesler, S. J. (eds.) Signal Transduction in the Retina, Methods in Signal Transduction Series, pp. 55–88. Boca Raton, FL: CRC Press.

Phototransduction in Limulus Photoreceptors R Payne and Y Wang, University of Maryland, College Park, MD, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Confocal fluorescence microscopy – An optical microscopy method that uses a focused laser spot to measure fluorescence within an extremely small volume. Cytosol – The liquid portion of a cell’s contents, or cytoplasm. Depolarization – A positive-going change in the membrane potential of a cell. Diacylglycerol (DAG) – A glyceride consisting of two fatty acid chains covalently bound to a glycerol molecule through ester linkages. d-myo-Inositol (1,4,5) trisphosphate (IP3) – An inositol derivative having three phosphate groups covalently bound to the inositol ring. Guanine-nucleotide-binding protein (G protein) – A family of signaling proteins that are activated by the exchange of guanine triphosphate for guanine diphosphate bound to the protein. On-cell patch clamp – An electrophysiological recording technique that uses a polished glass micropipette to measure currents flowing through a small patch of cellular plasma membrane containing ion channels. Phosphoinositide – A phospholipid containing a polar inositol headgroup. Smooth endoplasmic reticulum (SER) – A network of membrane sacs found in animal cells that, among other things, functions as a store of calcium ions. Transient receptor potential (TRP) channels – A widespread family of ion channels. The lightactivated channels of Drosophila are founding members of the family.

Arrangement of Eyes in Limulus American horseshoe crabs (Limulus polyphemus) have 10 eyes. They have two large lateral compound eyes, each containing about 1000 clusters of photoreceptors or ommatidia. A small lens within each ommatidium focuses light from a small patch of visual space onto each photoreceptor cluster, which transmits information about local changes in light intensity to the brain through a nerve fiber. There are five additional eyes on the top side of its first (anterior) major

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body section, two median eyes, one endoparietal eye, and two rudimentary lateral eyes. Two ventral eyes are located on the underside of the animal above the mouth (Figure 1). Photoreceptors located on the telson (tail) constitute the 10th eye. Of these eyes, the lateral compound, median ocellar and ventral eyes have been extensively studied. All have microvillar photoreceptors, in which the visual pigment, rhodopsin, is embedded in the membrane of fingerlike projections of the plasma membrane called microvilli (Figure 2). Each eye has allowed for a study of different aspects of invertebrate vision. The large compound eyes have been favorite preparations for the study of image processing by compound eyes, the biochemistry of visual transduction, and the circadian control of visual sensitivity. The medial eye has been studied for its sensitivity to ultraviolet (UV) light which has allowed the elucidation of the physiological consequences of the reversible photoisomerization of invertebrate rhodopsin. Lastly, the ventral eyes have played a role in the understanding of phototransduction in invertebrate photoreceptors.

The Microvillus is the Cellular Structure Mediating Visual Transduction Central to the performance of each photoreceptor cell is the rhabdomere – an array of photoreceptive microvilli positioned so as to maximally absorb light entering the eye. Each microvillus is a cylindrical outgrowth of the plasma membrane, 50–80 nm in diameter and 0.5–2 mm in length (Figure 2). Electron micrographs often show an axial filament within each microvillus. The axial filament contains a bundle of actin filaments with their þ ends directed toward the tip of the microvillus. The actin filaments appear to extend through the bottom of the microvillus into the cytoplasm, through fenestrations in submicrovillar cisternae (SMC) of smooth endoplasmic reticulum (SER). The actin cytoskeleton is responsible for the presence of an unconventional motor protein, myosin III, in the microvilli of Limulus lateral eye photoreceptors. The membrane of a typical microvillus contains 1000 or more particles, which are presumed to be mostly molecules of the visual pigment, rhodopsin, which absorbs light and initiates the physiological response of the photoreceptor. A photoreceptor might possess 105 microvilli, resulting in a total rhodopsin content of 108 molecules, comparable to that of vertebrate retinal photoreceptors.


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Figure 1 (a) Underside and top side (inset) of the Atlantic horseshoe crab (Limulus polyphemus). The green box shows the area enlarged in (b). (b) Enlarged view of the underside, just above the mouthparts. The drawn overlay shows the arrangement of the ventral nerves (green box, enlarged in (c)) and ventral eyes under the skin. The ventral nerves lead from the brain ( just above the mouthparts at the bottom of the figure) to the ventral eyes above. (c) Diagram of ventral nerve axons and attached photoreceptor cell bodies (green box). Drawings in (b) and (c) are adapted from Calman, B. G. and Chamberlain, S. C. (1982). Journal of General Physiology 80: 839–862.

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Figure 2 Electron micrograph of a section through the R-lobe of a ventral photoreceptor, showing the microvilli (MV) and submicrovillar cisternae of smooth endoplasmic reticulum (SER; arrow). Adapted from Dabdoub, A., Payne, R., and Jinks, R. N. (2002). Journal of Comparative Neurology 442: 217–225. Copyright 2009 Wiley-Liss, Inc.

Studies of Visual Transduction Using Limulus Ventral Photoreceptors The Limulus ventral photoreceptor (Figure 3) is a highly polarized cell divided into two lobes, analogous to the inner and outer segments of vertebrate retinal photoreceptors. The rhabdomeral (R) lobe bears microvilli on its plasma membrane and is therefore light sensitive. The light-insensitive arhabdomeral (A) lobe contains the cell’s

nucleus. An axon projects from the A lobe toward the animal’s central nervous system. Ventral photoreceptors were originally chosen as a model for invertebrate phototransduction because of their large size (>200 mm). This facilitates insertion of multiple electrodes and makes it possible to clamp the membrane potential of the cells, measure electrical current flow across the plasma membrane, and inject compounds of interest into the cytoplasm (Figure 4). The essential electrical response to illumination is the activation of a very large flow of current into the cell, carried mostly by sodium ions (Figure 4). The result is a depolarization (positive-going change) of the cell membrane which is graded with light intensity, of up to 60 mV. The reversal potential of the light-sensitive current is between þ10 mV and þ20 mV and its dependence on extracellular ion concentrations indicates that this conductance is sodium- and potassium-, but not Ca2+-permeable. This is in contrast to the light-activated transient receptor potential (TRP) channel conductance in the photoreceptors of the fruit fly, Drosophila, which is highly 2+ Ca permeable. The study of ventral photoreceptors has revealed that they have remarkable performance characteristics, most notably the very large amplification of the transduction process. Amplification refers to the amount of charge that is carried across the plasma membrane as a result of excitation by a single photon. In ventral photoreceptors, this gain can be directly measured because the single-photon response, termed a quantum bump, is easily recorded using glass micropipettes. In response to very dim illumination,

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Figure 3 (a) Light micrograph of a ventral photoreceptor. R-lobe, rhabdomeral lobe; A-lobe, arhabdomeral lobe. (b) Immunolocalization (blue) of rhodopsin within the same photoreceptor cell. The ventral photoreceptor has two lobes, a light-sensitive rhabdomeral lobe (R-lobe), which bears rhodopsin-containing microvilli on its plasma membrane and is analogous to the outer segments of vertebrate photoreceptors, and a light-insensitive arhabdomeral lobe (A-lobe), which is analogous to the inner segment of vertebrate photoreceptors. From Battelle, B. A. et al. (2001). Journal of Comparative Neurology 435: 211–225. Copyright 2009 WileyLiss, Inc.

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Figure 4 Experimental arrangement for recording electrical activity (membrane potential and voltage clamp currents, right) from ventral photoreceptors impaled with a glass micropipette, left.

quantum bumps over 10 mV in amplitude occur randomly as individual photons are effectively absorbed by rhodopsin molecules. Under voltage clamp, the peak current across the membrane generated by an effectively absorbed photon can exceed 1 nA and appears to be generated over several square microns of membrane surface, containing hundreds of microvilli. The comparatively large currents flowing indicate that quantum bumps are caused by the passage across the plasma membrane of hundreds of millions of cations through ion channels. By contrast, in the smaller Drosophila photoreceptors or amphibian rods, a singlephoton event involves a maximum current of less than 10 pA. Limulus photoreceptors achieve this large amplification in only 100–200 ms, faster than amphibian rods. A further remarkable feature of Limulus photoreceptors is their broad dynamic range. Whereas most vertebrate rods

work over about a 4-log-unit range of light intensity before saturating, Limulus photoreceptors work over 8. They achieve this range through a strong adaptation process that reduces amplification at high light intensity. The large dynamic range of these cells obviates the need for the dual system of photoreceptors (rods and cones) used to achieve a large dynamic range in the vertebrate eye.

The Light-Sensitive Conductance Consists of the Summed Effect of Conventional Ion Channels The glial cells that surround individual ventral photoreceptors can be removed, exposing the plasma membrane surface. This preparation allows On-cell patch-clamp


recording of single ion channels in the microvillar membrane. As expected, channels whose opening was triggered by light were recorded by this method, with singlechannel conductance ranging from 18 to 50 pS. Identification of these channels as the light-activated conductance requires establishing a close correspondence between the properties of individual channel currents and those of photocurrents recorded from the whole cell. Depolarizing potentials applied in the dark did not open the channels; therefore the light-activated opening was not just a secondary consequence of the depolarizing receptor potential. Light-evoked single-channel activity was graded with light intensity, reduced by light adaptation, and the singlechannel currents reversed in the same membrane potential range as the macroscopic photocurrent. While these characteristics are consistent with the recorded channels being those that carry the light-activated current, definitive molecular or pharmacological proof is still needed.

The Response of the Ventral Photoreceptor is Mediated by the Phosphoinositide Cascade A large body of evidence now demonstrates that the phosphoinositide (PI) pathway links the absorption of light to the activation of ion channels in the plasma membrane of invertebrate microvillar photoreceptors (Figure 5). The PI pathway is a mechanism for releasing intracellular messengers upon the activation of a receptor

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protein, using inositol phospholipids as a substrate. In microvillar photoreceptor cells, the receptor protein is rhodopsin and the cascade is localized to the membrane of the microvilli that cover the plasma membrane of the light-sensitive R-lobe. Activated rhodopsin catalyzes the exchange of guanine triphosphate (GTP) for guanine diphosphate (GDP) bound to the alpha subunit of a heterotrimeric GTP-binding protein of the Gq subfamily (Gqa), which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5 bisphosphate (PIP2), a minor membrane phospholipid, into a lipid messenger, diacylglycerol (DAG), and the water-soluble messenger, d-myo-inositol 1,4,5 trisphosphate (IP3). In support of this biochemical pathway, light-activated PIP2 hydrolysis and/ or IP3 production has been reported in ventral photoreceptors of Limulus. Gqa has been amplified and sequenced from Limulus ventral eye tissue and immunolocalized to the rhabdomeral microvilli. Pharmacological agents that inhibit PLC, neomycin and U-73122, dramatically desensitize the light response of Limulus photoreceptors.

The PI Cascade Generates at Least Two Intracellular Messenger Molecules The PI cascade generates two messenger molecules with very different properties (Figure 5) DAG is essentially confined to the plasma membrane, while IP3 can diffuse into the surrounding cytoplasm. In addition, the decline of the precursor, PIP2 may act as an additional signal within

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Cytoplasm Figure 5 Diagram of mechanisms proposed to mediate phototransduction within a microvillus of the Limulus photoreceptor. cGMP, cyclic guanosine monophosphate; DAG, diacylglycerol; ER, endoplasmic reticulum; Gqa, alpha subunit of a heterotrimeric GTP-binding protein of the Gq subfamily; b,g, beta and gamma subunits of a heterotrimeric GTP binding protein of the Gq sub-family; GC, guanylate cyclase; IP3, d-myo-inositol (1,4,5) trisphosphate; IP3R, d-myo-inositol (1,4,5) trisphosphate receptor; PLC, phospholipase C; Rh, rhodopsin; TRPC, transient receptor channel C.


the membrane. The difficult task of assessing the relative roles of these messengers in activating the light-sensitive ion channels has dominated research on the invertebrate phototransduction cascade.

Roles of IP3 and Intracellular Ca2+ Ions in Excitation of Limulus Ventral Photoreceptors There is no evidence that IP3 can itself activate the lightsensitive channels of ventral photoreceptors. The bestcharacterized alternative target of IP3 is a Ca2+ channel, the IP3 receptor protein (IP3R), located in the membrane of endoplasmic reticulum (ER). IP3 therefore releases Ca2+ from intracellular stores within the ER. In Limulus photoreceptors, suitable calcium stores are located close to the base of the rhodopsin-containing microvilli – the subrhabdomeral cisternae (SMC) of SER. Extensions of the SMC are juxtaposed less than 100 nm from the bases of the microvilli. The cytoplasm of both the R lobe containing the SER and, to a lesser extent, the A lobe are immunoreactive when probed with an anti-IP3R antibody. In darkness, ventral photoreceptors, like most cells, maintain a low free cytosolic Ca2+ concentration ([Ca2+]i) of 200–600 nM. Release of calcium from the SMC results in a very large elevation of [Ca2+]i during the first few hundred milliseconds of the light response. Confocal fluorescent light microscopy has enabled the measurement of the light-induced elevation of [Ca2+]i in ventral photoreceptors at spots within 4 mm of the microvillar membrane in the R lobe (Figures 6 and 7). Photomultiplier (PMT)

Following a very bright flash, [Ca2+]i begins to rise after a latent period of approximately 20 ms. Thereafter, [Ca2+]i rises at an initial rate of 1–2 mM s–1 to reach a peak of 100–200 mM within 200 ms. For less-intense flashes, the peak elevation of [Ca2+]i is graded with flash intensity, increasing from 2 mM to more than 140 mM as light intensity increases from 10 effective photons to 10 000 effective photons. For the dimmest flashes so far investigated, these concentrations represent 600 free Ca2+ ions per effective photon generated within the confocal measurement volume. Very high levels of [Ca2+]i are reached only transiently upon illumination. Following a dim or moderate-intensity light flash, [Ca2+]i falls close to its resting value within 1 s, although a small lingering elevation may persist for up to a minute afterward. Even during sustained intense illumination, [Ca2+]i falls within 5 s to a sustained plateau elevation of less than 20 mM. As expected, if calcium release from stores occurs, the light-induced rise in [Ca2+]i is unaltered by removal and chelation of extracellular Ca2+, but is severely reduced by pharmacological agents expected to deplete Ca2+ stores.

IP3 Can Release Ca2+ from the SER Microinjections of IP3, or photolysis of caged IP3, rapidly release Ca2+ from the R lobe in darkness (Figure 8). Photolysis of caged IP3 by UV light delivered to a spot beneath the microvillar membrane results in local elevations of Ca2+ that are comparable in magnitude and rate

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Figure 7 (a) Receptor potential (solid line) and reconstructed elevation of [Ca2+]i (red symbols) recorded following a flash from an attenuated laser beam that delivered 50 effective photons to a photoreceptor. The bar beneath the trace indicates the timing of the flash. (b) The responses to the dim flash used in (b) are shown on an expanded timescale on the right. On the left is shown the rising edge of the responses to a much brighter step of light produced by the unattenuated laser beam, delivering 108 effective photons per second. The bars below the traces indicate the onset and duration of the stimuli. Adapted from Payne, R. and Demas, J. (2000). Journal of General Physiology 115: 735–748.

of rise to those elicited by bright visible light. However, the latency of the Ca2+ signal that follows illumination by visible light is 30 ms longer than that of the response to the release of caged IP3 (compare Figures 8(a) with 8(b)), the difference being presumably the time required for light to activate the PI pathway. There is therefore convincing evidence that the light-induced release of Ca2+ from internal stores is mediated by the PI pathway acting on IP3Rs in SER that are closely juxtaposed to the microvillar membrane.

Released Ca2+ Ions can Activate an Inward Current Pulsed pressure injections of solutions containing 1–2 mM Ca2+ into the R lobe of ventral photoreceptors activates a current in the plasma membrane of up to 20 nA, with a similar reversal potential, sodium dependence, and outward rectification to that activated by light. Release of Ca2+

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Figure 8 Timing of Ca2+ release by caged InsP3. Photoreceptors were loaded with caged IP3 and the Ca2+ indicator dye fluo-3; 488 nm and UV laser beams were focused onto the edge of the R-lobe. As an indicator of [Ca2+]i, uncalibrated dye fluorescence is shown, expressed as a fraction of the background fluorescence during the latent period of the response (F/F0). (a) Membrane potential (solid line) and fluo-3 fluorescence (red dots) recorded during illumination by the 488-nm laser alone, stimulating Ca2+ release and depolarization through the photoisomerization of rhodopsin, that is, through the natural phototransduction pathway. Laser stimulation began at the beginning of the fluorescence trace. (b) Effect of superimposing a 20-ms duration UV flash and so releasing caged InsP3 into the cytoplasm of the cell, stimulating an earlier release of Ca2+ directly from the SER. Adapted from Ukhanov, K. and Payne, R. (1997). Journal of Neuroscience 17: 1701–1709.

ions through IP3 activates the same conductance, indicating that the [Ca2+]i generated through the endogenous Ca2+ release pathway is sufficient. The current, typically 5–20 nA in amplitude following a pulse of 100 mM IP3, generates a transient depolarization of the photoreceptor lasting for less than 1 s. The coupling between the elevation of [Ca2+]i and the depolarization of the photoreceptor is rapid. Depolarization follows caged IP3-induced Ca2+ release after 2.5 3.3 ms (Figure 8), while photolysis of caged Ca2+ (O-nitrophenyl ethylene glycol tetraacetic acid (EGTA)) at the edge of the R lobe activates current within 1.8 0.7 ms.

Light-Induced Ca2+ Release can be Detected before the Electrical Response The above experiments indicate that micromolar [Ca2+]i, released from internal stores by IP3, can activate an

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inward current through the plasma membrane within a few milliseconds. It follows that if light-induced Ca2+ release is to act similarly, then a component of the photocurrent must be initiated a few milliseconds after Ca2+ is released. Certainly, the onset of the two signals is highly correlated if measured confocally beneath the microvillar membrane. The time for [Ca2+]i to exceed 2 mM is approximately equal to that for the receptor potential to exceed 8 mV (mean difference: 2.2 6.4 ms). However, the question of which event occurs first is difficult to address, since two signals with different noise levels are compared (Figure 7). The detection of the Ca2+ signal lead the electrical response by up to 5 ms within about one-third of cells examined. The lag in other cells could indicate the presence of an early Ca2+-independent component of the response present in some cells, but it is difficult to be sure of this because the placement of the confocal measuring spot relative to the microvillar membrane is critical. However, in any case, given the rapidity with which Ca2+ can elicit an inward current (1–3 ms; see above) and the fact that it takes the photocurrent 50 ms to rise to peak, this timing appears to be sufficient for released Ca2+ ions to contribute to the activation of the photocurrent during the rising edge of the response to light.

How Does IP3-Induced Ca2+ Release Activate Inward Current and is this Current Flowing through the Light-Sensitive Conductance? Given the detailed evidence above, it seems reasonable to propose that IP3-induced Ca2+ release can activate the light-activated conductance in ventral photoreceptors (Figure 5). However, the molecular nature of the channel and the site of calcium’s action are not yet known. An ideal preparation for electrophysiology, ventral photoreceptors do not have the advantage of molecular genetic approaches that have allowed the identification of the light-sensitive channels in Drosophila as members of the TRP family. Still, two hypotheses have been developed for the nature of the light-activated ion channels based on physiological and molecular evidence. The first hypothesis is that the channels are not Ca2+gated, but are cyclic guanosine monophosphate (cGMP) gated. This seems a remote possibility at first, given the evidence that IP3-induced Ca2+ release can rapidly activate a plasma membrane conductance in intact cells. However, the proposal that a further messenger exists downstream from Ca2+ is driven by the experimental inability of Ca2+ to directly activate ion channels when applied to the inside of patches of plasma membrane excised from the rhabdomeral lobe. Instead, application of cGMP activated channels in a minority of excised patches. The channel events activated during application

of cGMP had a similar conductance to light-activated channels, similar reversal potential when bathed in media mimicking intracellular and extracellular ion concentrations and a similar increase in open probability upon membrane depolarization. Since intracellular injections of cGMP or its analogs depolarize the photoreceptor, it was proposed that cGMP might be a terminal messenger in the visual cascade in ventral photoreceptors, as well as in vertebrate rods and cones. A putative cGMPgated channel has been sequenced from ventral photoreceptors and localized to the microvillar photoreceptors. However, to reconcile this hypothesis with the large body of evidence for the initiation of the light response by the PI pathway, some coupling mechanism must be found to link IP3-induced Ca2+ release to the production of cGMP. There is some pharmacological evidence that a Ca2+activated guanylate cyclase (GC) might provide this link (Figure 5), but no biochemical or molecular evidence has so far been obtained for this hypothesis. The second hypothesis is that Limulus channels are members of the TRP family which, unlike Drosophila TRP and TRPL, do not display a high Ca2+- permeability (Figure 5). TRP channel homologs have been cloned from ventral photoreceptor messenger RNA (mRNA), and an established activator of some TRP-family channels, the synthetic lipid, 1-oleoyl-2-acetyl-sn-glycerol (OAG), activates a conductance with reversal potential similar to that activated by light. Activation of this conductance by OAG apparently requires the presence of free Ca2+ ions in the injection pipette, which may explain why extracellularly applied OAG has no effect and why there is a complete dependence of the light response on light-induced Ca2+ release from intracellular stores. The hypothesis of a Limulus homolog of the TRP channel provides a testable alternative to the proposed role of a cyclic nucleotide channel and may resolve the differences in phototransduction between Limulus and Drosophila photoreceptors.

Adaptation, a Decrease in the Sensitivity of the Visual Cascade, is Mediated by Small, Lingering Elevations of Ca2+ The onset of prolonged illumination of ventral photoreceptors, or the huge elevations of [Ca2+]i that occur following flashes of light are not sustained but fall back to the micromolar range within seconds. Concurrently, the photocurrent also falls from tens or hundreds of nA to a few nA. These declines are the result of a decrease in the photoreceptor’s sensitivity that prevents saturation of the photoreceptors in bright light and so extends its dynamic range. The initial light-induced elevation of [Ca2+]i therefore appears to function as a feedback signal that subsequently reduces the sensitivity of the visual cascade.


In support of this concept, slow injection of Ca2+ ions into the cytosol of ventral photoreceptors diminishes the sensitivity and latency of the light response, mimicking the effect of an adapting light. Injection of Ca2+ chelators, such as EGTA or 1,2-bis(o-aminophenoxy)ethane-N,N, N0 ,N0 -tetraacetic acid (BAPTA), not only slows down and diminishes the initial photocurrent, but also blocks the decline of the light-induced current during prolonged illumination. The site of this feedback inhibition of the light response by Ca2+ could be at several points in the phototransduction cascade. For example, IP3-induced Ca2+ release is known to be inhibited by lingering elevations of [Ca2+]i in ventral photoreceptors. In addition, pharmacological activation of protein kinase C greatly reduces the sensitivity of the light-induced current, apparently acting upstream of IP3-induced Ca2+ release.

Drosophila and Limulus Photoreceptors Operate Differently and Illustrate Two General Mechanisms Coupling the PI Cascade to an Electrical Response The two most extensively studied microvillar photoreceptors, those of Limulus and Drosophila, have many aspects of their phototransduction mechanism in common. Both are thought to utilize the PI cascade to open non-selective cation channels and both exhibit large elevations of [Ca2+]i, which are necessary for signal amplification and speed, as well as light adaptation. However, there are also clear differences: Limulus photoreceptors utilize IP3-induced Ca2+ release to elevate intracellular free Ca2+ ion concentrations, while Drosophila photoreceptors are thought to utilize DAG or derived products to open Ca2+-permeable TRP channels that allow Ca2+ entry from the extracellular space. The photoreceptors of the two species are therefore specific examples of two general mechanisms in cells for coupling the PI pathway to [Ca2+]i elevation. Why is there this difference? One explanation is the need in Limulus for the extra amplification provided by IP3-induced Ca2+ release. One IP3 molecule can release hundreds of Ca2+ ions from the ER through the IP3R channel. These Ca2+ ions can then diffuse along the inner surface of the plasma membrane to activate downstream targets, such as the hundreds of channels required to open in order to produce a significant quantal event in these giant photoreceptors. The trade-off for this amplification is slower speed. The extra amplification step (Ca2+ release) required in the visual cascade may explain why Limulus photoreceptors are slower than fly photoreceptors to respond to light. This speed difference is entirely reasonable, given the animals’ differing demands on their visual systems. Horseshoe crabs do not fly; rather they use their lateral eyes to find mates near moonlit beaches.

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Extension of Phototransduction Mechanisms to other Microvillar Photoreceptor Types Detailed knowledge of the mechanisms of both Limulus and Drosophila photoreceptors may outline general approaches used by other cell types to the problem of tailoring the outcome of the PI cascade to the need for either speed or amplification. An outstanding question is the extent to which other photoreceptor types utilize these mechanisms. It is apparent that light-induced Ca2+ release from intracellular stores occurs in photoreceptors of the file clam, leech, and honeybee. Furthermore, the light-sensitive channels that mediate the immediate electrical response in the file clam are relatively impermeable to Ca2+ ions, like those of Limulus, but not Drosophila. Barnacle photoreceptors, on the other hand, like those of Drosophila, display prominent lightinduced Ca2+ influx. Thus, elements of the two mechanisms for coupling phototransduction to an elevation of Ca2+ seem to be scattered across the invertebrate phyla. Whether there is a functional or evolutionary rationale for these differences remains to be seen. Indeed, it is not known yet whether photoreceptors in the other eight eyes of Limulus function similarly to the much-studied ventral eye. See also: Circadian Rhythms in the Fly’s Visual System; Evolution of Opsins; Genetic Dissection of Invertebrate Phototransduction; Limulus Eyes and Their Circadian Regulation; Microvillar and Ciliary Photoreceptors in Molluskan Eyes; The Photoresponse in Squid.

Further Reading Bandyopadhyay, B. C. and Payne, R. (2004). Variants of TRP ion channel mRNA present in horseshoe crab ventral eye and brain. Journal of Neurochemistry 91: 825–835. Battelle, B. A. (2006). The eyes of Limulus polyphemus (Xiphosura, Chelicerata) and their afferent and efferent projections. Arthropod Structure and Development 35: 261–274. Brown, J. E. and Blinks, J. R. (1974). Changes in intracellular free calcium concentration during illumination of invertebrate photoreceptors. Journal of General Physiology 64: 643–665. Brown, J. E., Rubin, L. J., Ghalayini, A. J., et al. (1984). myo-Inositol polyphosphate may be a messenger for visual excitation in Limulus photoreceptors. Nature 311: 160–162. Chen, F. H., Baumann, A., Payne, R., and Lisman, J. E. (2001). A cGMP-gated channel subunit in Limulus photoreceptors. Visual Neuroscience 18: 517–526. Fein, A., Payne, R., Corson, D. W., Berridge, M. J., and Irvine, R. F. (1984). Photoreceptor excitation and adaptation by inositol 1,4,5 trisphosphate. Nature 311: 157–160. Hardie, R. C. and Minke, B. (1992). The trp gene is essential for a lightactivated Ca2+ channel in Drosophila photoreceptors. Neuron 8: 643. Nasi, E., Gomez, M., and Payne, R. (2000). Phototransduction mechanisms in microvillar and ciliary photoreceptors of invertebrates. In: Hoff, A. J., Stavenga, D. G., de Grip, W. J., and Pugh, E. N. (eds.) Molecular Mechanisms in Visual Transduction – Handbook of Biological Physics, vol. 3, pp. 389–448. Amsterdam: Elsevier Science.

Phototransduction: Phototransduction in Cones V J Kefalov, Washington University School of Medicine, Saint Louis, MO, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Dark adaptation – The mechanism that allows photoreceptors to recover their sensitivity to darkadapted levels following exposure to bright light. Light adaptation – The mechanism that allows photoreceptors to reduce their sensitivity in the presence of steady light. Phototransduction cascade – A series of reactions in the outer segments of photoreceptors through which the energy of a photon is converted into a change in the membrane potential of the cell. Visual cycle – A series of reactions initiated by the activation of the visual pigment by light and terminating in resetting the pigment to its inactive, ground state. It involves the decay of the photoactivated visual pigment to free opsin and all-trans retinal, the recycling of chromophore from all-trans to 11-cis outside of photoreceptors, and the regeneration of the visual pigment molecule. Visual pigment – A G-protein-coupled receptor consisting of protein, opsin, covalently linked to a chromophore, 11-cis retinal. The absorption of a photon by the visual pigment is the initial step in activating the phototransduction cascade.

Introduction Cone photoreceptors mediate our vision during the day and provide us with fine spatial and temporal resolution as well as color perception. In most species, cones are located mostly in the central area of the retina where the image directly in front of the eyes is projected. Unlike rods, where the signal from hundreds of photoreceptors is integrated for optimized photon detection in low light conditions, signals from individual cones are relayed to the brain. As a result, the spatial resolution of our central vision, driven primarily by the cones, is excellent, whereas that of our peripheral vision, driven by the rods, is significantly lower. Color discrimination is achieved as each cone typically expresses a single type of visual pigment which conveys different spectral sensitivity to different cone types. While single photoreceptors cannot discriminate colors as the degree of photoactivation depends not only on the wavelength of the stimulus but also on its intensity, the visual

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system extracts that information by comparing the signals coming from the different cone types. An interesting exception to the one cell–one pigment rule is the mouse retina where green and ultraviolet cone visual pigments are coexpressed in the same cells. The functional significance of that arrangement is not clear.

Functional Properties of Cones Cones use a phototransduction cascade, similar to the one well characterized in rods, to convert the energy of light into an electrical signal. In addition, cone phototransduction proteins are homologous, or sometimes even identical, to the ones found in rods. Yet, cones have functional properties that are distinct from those of rods and that are suited for their role as bright-light detectors. First, cones are significantly less sensitive than rods. The rod phototransduction cascade is tuned for high amplification which allows rods to achieve the maximal physically possible sensitivity and generate a detectable single photon response. As such enormous gain requires buildup of the reactions of the phototransduction cascade, the tradeoff is the slow kinetics of rod responses. Cones, on the other hand, are 30- to 100-fold less sensitive than rods (Figure 1) and require the simultaneous activation of tens to hundreds of visual pigment molecules to generate a detectable response. As a result of the low amplification of their phototransduction cascade, cones are not sensitive enough to function under low light conditions, depriving us of color vision in dim light. Instead, the low cone phototransduction gain shifts their dynamic range toward brighter light conditions and enables cones to function during the day. The low signal amplification in cones is made possible by the rapid inactivation of their phototransduction cascade. This results in the second notable difference from rods, namely, that cone responses are typically several fold faster than rod responses. The rapid activation and subsequent inactivation of the cone phototransduction cascade reactions provides the basis for the high temporal resolution of cone-mediated vision (Figure 1). The rapid activation of cones results in short latency of detection, whereas their rapid inactivation enables discrimination of stimuli spaced closely in time. In contrast, the slower rod responses limit the temporal resolution of rod-mediated vision. Third, following exposure to bright light, cones fully recover their sensitivity within a few minutes. Rods, in contrast, experience a long

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refractory period following exposure to bright light and can take up to an hour for a complete recovery of their sensitivity. This process, known as dark adaptation, prevents cones from becoming refractory and allows us to retain visual perception in a quickly changing light environment. Finally, cones have a remarkable ability to adjust their sensitivity over a very wide range and remain photosensitive even in extremely bright light. Rods, in contrast, saturate in even moderately bright light and remain nonfunctional during most of the day. This process, known as light adaptation, prevents cones from saturating in bright light and allows us to see throughout the day. With rods saturated, cones are responsible for most of the visual information reaching our brain during the day. In fact, with the introduction of artificial lighting, humans rely almost exclusively on cones both during the day and at night. This is why cone disorders, such as macular degeneration, the most common cause of blindness in the elderly, have a devastating effect on vision.

Obstacles for Studying Cone Phototransduction The last several decades have seen a tremendous advance in our understanding of the function of photoreceptors.

The development of electrophysiological tools for studying the function of single photoreceptors, together with biochemical and genetic tools have revealed the mechanism of phototransduction and provided quantitative description of the reactions involved in it. Unfortunately, these advances have been almost exclusively limited to rods. The great abundance of rods in most mammalian retinas (95% of all photoreceptors in human and 97% in mouse retinas) has facilitated the purification and biochemical study of rod phototransduction proteins. In contrast, the small fraction of cones and the homology between rod and cone phototransduction proteins have rendered comparable studies from cone proteins technically challenging. A further obstacle has been the fragility of mammalian cone photoreceptors, which has rendered physiological studies from cones also significantly more challenging than comparable rod studies. As a result, while mammalian rod phototransduction has been characterized in quantitative details, most of what we currently know about cone phototransduction is derived from studies of amphibian and fish photoreceptors. Based on the similarities in structure and transduction proteins between rods and cones, it has been assumed that phototransduction in cones follows the same set of reactions as phototransduction in rods. There exist, however, important quantitative phototransduction differences in

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rods and cones pertinent to their function in dim and bright light, respectively. The phototransduction cascade in cones will be discussed here in the context of the much better understood rod phototransduction cascade. In both, rods and cones, phototransduction takes place in specialized compartments, called outer segments, which consist of stacks of membrane disks, similar to a stack of coins. Unlike in rods, where these disks are surrounded by, but not connected to, the plasma membrane, in cones these disks are formed from invaginations of the plasma membrane. As a result, the plasma membrane of cone outer segment has significantly higher area, a factor possibly important for the rapid flow of molecules in and out of the cell. The transduction channels are cGMP-gated nonselective cation channels held open in darkness by the binding of free cGMP in the outer segment. Cone cGMP channels are homologous to those found in rods and in olfactory neurons and consist of two cyclic nucleotidegated alpha 3 (CNGA3) and two cyclic nucleotide-gated beta 3 (CNGB3) subunits. In darkness, the influx of Na+ and Ca2+ through these channels depolarizes the cells to about –40 mV, which results in the steady release of the neurotransmitter glutamate from the cone synaptic terminal. Photoactivation of the cell results in the hydrolysis of cGMP, closure of the transduction channels, hyperpolarization of the cell, and reduction in the release of neurotransmitter from the cone synaptic terminal.

Cone Visual Pigment and Phototransduction Phototransduction in cones is initiated by the activation of cone visual pigments by the absorption of a photon. The cone visual pigments, similar to rod pigments, consist of protein, opsin, covalently attached to a chromophore, typically 11-cis retinal. Cone opsins have a moderate level (50%) of homology to rod opsins. The visual chromophore is a derivative of vitamin A (all-trans retinol), which is converted in the pigment epithelium into 11-cis retinal and then transported to the photoreceptor’s outer segments where it combines with opsin to form the visual pigment. The visual pigment is expressed at very high levels in the disks of the outer segment (3.5 mM), so that a photon traveling along the outer segment has a 40% chance of activating a pigment molecule. Interestingly, the concentrations of rod and cone visual pigments in the outer segment as well as their extinction coefficients are similar. In addition, the probability that a pigment molecule will become activated once a photon has been absorbed (quantum efficiency) is also comparable between rod and cone pigments. Thus, with respect to the pigment distribution and optical properties, only the typically smaller size of the cone outer segment compared to that of the rod contributes to the lower sensitivity of cones.

Studies with amphibian photoreceptors indicate that the different stability of rod and cone pigments modulates their respective phototransduction cascades. First, studies of transgenic Xenopus rods expressing red cone opsin have allowed the direct observation of physiological responses to the activation of a single cone pigment molecule. This has made possible the determination of the rate of spontaneous thermal activation of red cone pigments, which produces a response identical to the activation by a photon. The molecular rate of thermal activation measured in this way is 10 000 times higher for red cone pigment than for rod pigment. As a result, amphibian red cones experience 200 pigment activations per second in darkness. This level of dark activity is comparable to the total dark noise measured from salamander red cones, indicating that most of the noise in these cells originates in the thermal activation of the pigment. This spontaneous activity acts as background light to induce adaptation and, therefore, desensitization and acceleration of the flash response. A second mechanism by which the stability of the visual pigment contributes to the differences between rods and cones is based on the covalent bond between opsin and retinal in their respective pigments. Both biochemical and physiological studies indicate that the formation of the covalent bond between opsin and chromophore is reversible in cones but not in rods. As a result, the visual pigment in cones, but not in rods, can spontaneously dissociate into free opsin and 11-cis retinal. The very low level of free 11-cis retinal in the outer segment (only 0.1% of the pigment content) shifts the equilibrium between free and chromophore-bound cone opsin so that even in dark-adapted cones, there is 10% free opsin. At this high level, the total catalytic activity of free opsin, though weak per single molecule, is sufficient to induce adaptation and further reduce the sensitivity and accelerate the kinetics of the cone flash responses. The effects of cone pigment properties on mammalian photoreceptor function have not been well characterized. Interestingly, studies from transgenic mouse rods expressing cone pigments indicate that, though still significantly higher than that of rod pigment, the rate of thermal activation of cone pigment is not high enough to affect cone photosensitivity significantly. A possible explanation for the relatively low thermal activity of cone pigments in mammalian species compared to amphibians might be that they use a slightly different chromophore (11-cis retinal or A1) than most amphibian photoreceptors (11cis 3-dehydroretinal or A2). The reversibility of cone pigment formation and its possible effect on cone function have not yet been examined in mammalian cones. Finally, differences in the properties of rod and cone visual pigment also contribute to the very different rates of dark adaptation in rods and cones.


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Once activated, the visual pigment binds to and activates a heterotrimeric G protein, called transducin (Gt). This triggers the exchange of GDP for GTP on the a-subunit of transducin (Gta) and the dissociation of GtaGTP from Gtbg. This represents the initial amplification step in phototransduction as one visual pigment molecule can activate multiple Gt molecules. Rod and cone transducins are closely related and the primary structures of their a-subunits are 80% identical, with even higher identity in the region of interaction with the visual pigment. Biochemical studies indicate that rod and cone pigments have comparable binding affinities for rod transducin and that they activate rod transducin with similar kinetics. Furthermore, studies with transgenic animals coexpressing rod and cone visual pigments in the same photoreceptor have shown that rod and cone pigments produce comparable responses. Thus, cone pigments expressed in rods produce a response with rod-like amplification and kinetics and, conversely, rod pigments expressed in cones produce a response with cone-like amplification and kinetics. These results indicate that the activation of the phototransduction cascade by the visual pigment and the inactivation of the visual pigment are not determined by its properties but rather, by the downstream transduction reactions, including the activation of transducin. Indeed, biochemical studies of fish photoreceptors have shown that the activation of transducin is 25 times less effective in cones compared to rods. This lower activation efficiency would contribute to the lower amplification of the signal and, therefore, to the lower sensitivity of cones. It is not clear yet whether the lower activation of transducin in cones is due to the properties of the cone isoform of transducin or due to the faster inactivation of pigment in cones compared to rods. An interesting recent observation is that exposure to bright light in rods triggers translocation of the subunits of activated transducin from the outer to the inner segment. In contrast, in cones, such translocation does not occur, possibly because transducin subunits are inactivated and re-form a trimer faster than in rods. The mechanism of this light-dependent translocation is still not well understood and is an active area of research. Once activated by the visual pigment, GtaGTP in turn activates cGMP phosphodiesterase (PDE) by binding to its inhibitory subunit PDEg and removing its inhibition on the catalytic PDEab. The resulting hydrolysis of cGMP by PDE leads to the closure of cGMPgated channels in the cone outer segment and the hyperpolarization of the photoreceptor to produce the light response. While cone PDE has 60% identity to rod PDE, biochemical studies of fish photoreceptors indicate that the activation of PDE by transducin might also be 10 times less effective in cones compared to rods, contributing further to the lower cone sensitivity.

Response termination is achieved as the visual pigment, transducin, and PDE are inactivated and the concentration of cGMP is restored to its dark, preflash level. Though these reactions in cones are not well characterized, it is clear that, similar to their activation, quantitative differences in the inactivation of phototransduction reactions in rods and cones contribute to the lower sensitivity and faster response kinetics of cones. The activity of the visual pigment is initially partially quenched when it is phosphorylated by a G-protein receptor kinase (GRK). Phosphorylation of activated visual pigment is 50 times faster in cones compared to rods. It appears that this faster phosphorylation is the result of two factors – higher expression of GRK in cones and higher efficiency of cone GRK (GRK7) compared to rod GRK (GRK1). While most species, including human, express GRK1 in rods and GRK7 in cones, the mouse retina is unusual as its rods and cones share the same kinase, GRK1. In this case, the faster pigment inactivation in cones is most likely due to the higher concentration of GRK1 and possibly also to differential modulation of that reaction by the calcium-binding protein recoverin. Following phosphorylation, complete inactivation of the phosphorylated visual pigment is achieved by the subsequent binding of a protein called arrestin. The cone isoform of arrestin (Arr4) has about 50% identity to rod arrestin (Arr1). The mouse retina again represents an unusual case, as in addition to Arr4, mouse cones also express Arr1. Interestingly, the ratio of arrestin to visual pigment is 7 times higher in cones compared to rods. In dark-adapted rods, most of arrestin is in the inner segment and does not, therefore, contribute to the inactivation of rod visual pigment. As a result, the quantity of arrestin in the outer segments of rods is only a few percent of their visual pigment. Exposure to bright light triggers the translocation of arrestin from the inner to the outer segment for more efficient pigment inactivation. While arrestin also transloactes in cones, the total quantity of arrestin in their outer segments in darkness is comparable to that of their visual pigment. Recent studies from mouse cones lacking both rod and cone arrestins reveal that either arrestin is capable of inactivating cone visual pigment though Arr1 is much more abundant than Arr4 in cones. Studies with transgenic rods expressing cone S-opsin and either rod or cone arrestin further demonstrate that rod arrestin is more efficient at inactivating cone pigment than cone arrestin. The relatively low expression of Arr4 in cones and its relative inefficiency suggest a possible additional role for this protein. The coexpression of two arrestins and their high concentration in cone outer segments would contribute to the rapid cone pigment inactivation and are consistent with the more rapid pigment inactivation and faster response termination in cones compared to rods.


Dark Adaptation of Cones Quantitative differences between the phototransduction cascades of rods and cones not only contribute to the difference in sensitivity and kinetics of photoresponses as discussed above, but also play a role for the very different adaptation properties of rods and cones. The ability to recover their sensitivity rapidly following exposure to bright light, or dark-adapt, is critical for the function of cones as daytime photoreceptors. The absorption of a photon by the visual pigment not only triggers its activation, but also results in its eventual decay into free opsin and all-trans retinal. Dark adaptation of both, rods and cones, after exposure to bright light requires regeneration of the visual pigment from opsin and 11-cis retinal. However, the speed of pigment regeneration, and hence sensitivity recovery, is very different in rods and cones, with full recovery requiring less than 5 min in cones and up to an hour in rods (see Figure 2).

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GtaGTP is inactivated as GTP is hydrolyzed into GDP. This reaction is catalyzed by PDEg as part of a GTPaseactivating protein (GAP) complex that consists, in addition, of regulator of G-protein signaling (RGS9), RGS9 anchoring protein (R9AP), and a Gb subunit (Gb5). The rod and cone PDEg have comparable potencies for inhibiting PDE and also for enhancing the hydrolysis of GTP by the GAP complex. In contrast, even though the identical RGS9 protein is present in rods and in cones, its concentration is more than 10 times higher in cones compared to rods. Deletion of RGS9 in the mouse greatly retards cone response inactivation, and mutations in RGS9 have been associated with slow cone deactivation in patients. Thus, while the extent to which the differences in GAP activity in rods and cones contribute to their functional differences is not well understood, RGS9 and the GAP complex clearly play an important role in the inactivation of cone phototransduction. The final step in photoresponse termination involves the upregulation of synthesis of cGMP by guanylyl cyclase (GC) to restore the concentration of free cGMP in the outer segment and reopen the cGMP-gated channels. While rods express two isoforms of GC, that is, GC1 and GC2, cones appear to express predominantly, if not exclusively, GC1. The role of GC2 in rods is not clear as its deletion produces only a mild change in rod physiology. It is also not understood how modulation of GC by the pair of GC-activating proteins (GCAP1 and GCAP2) contributes to the unique functional properties of cones. Although the distribution of GCAPs between rods and cones in different species is ambiguous, it appears that GCAP2 is prevalent in rods, while GCAP1 is expressed at high levels in cones. The possible role of GCAPs in mediating light adaptation in cones is discussed below in the context of light adaptation.

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Figure 2 Comparison of rod and cone dark adaptation. Recovery of the circulating (dark) current in salamander rod (a) and red cone (b) measured with a suction electrode. Cells were exposed to bright light that activated (bleached) 20% of the rod pigment and 90% of the cone pigment. The recording was done in the presence of exogenous 11-cis retinal to enable pigment regeneration in the isolated cells. Current recovery is fit by a single exponential decay function (solid line). Note the significantly faster recovery of the current in cone compared to the current in the rod.

Several factors contribute to the rapid pigment regeneration in cones. First, the decay of the photoactivated pigment to free opsin and all-trans retinal occurs in seconds for cone pigments compared to minutes for rod pigments. Second, the reduction of all-trans retinal into all-trans retinol, which takes place in the outer segment and is catalyzed by retinol dehydrogenase (RDH), is also 10–40 times faster in cones compared to rods. The reduction reaction requires the cofactor nicotinamide adenine dinucleotide phosphate oxidase (NADPH). While it is possible that the faster reduction of all-trans retinal in cones is due to the different properties of rod and cone RDH enzymes, a more likely hypothesis is that the reduction reaction is limited by the supply of NADPH from the inner segment. Third, single-cell measurements from amphibian photoreceptors indicate that the clearance of all-trans retinol from the outer segment is 25 times faster in cones compared to rods. However, the actual difference in these rates in the intact retina might be affected by factors such as the proximity to the pigment epithelium and the action of extracellular


chromophore-binding proteins such as interphotoreceptor retinoid-binding protein (IRBP). Finally, the formation of the covalent bond between opsin and 11-cis retinal during pigment regeneration occurs in seconds in cones and minutes in rods. Together, these factors contribute to the faster turnover of cone visual pigment and the faster dark adaptation of cones compared to rods. In addition to the effects of faster visual pigment decay and regeneration, cone dark adaptation is accelerated by the noncovalent interaction between opsin and 11-cis retinal. Pigment regeneration requires the initial binding of 11-cis retinal in the chromophore pocket of free opsin. While in rods, the noncovalent binding of retinal activates the opsin molecule and desensitizes the rods, in cones, this reaction has the opposite effect and inactivates cone opsin. As a result, the noncovalent binding of 11-cis retinal to opsin delays dark adaptation in rods but accelerates it in cones, as it allows cones to substantially recover their sensitivity even before the regeneration of their visual pigment. Recent biochemical studies indicate that another mechanism contributing to the faster dark adaptation of cones compared to rods is based on the supply of recycled chromophore for pigment regeneration. The canonical visual cycle involves the pigment epithelium, where alltrans retinol is converted into 11-cis retinal via a series of enzymatic reactions and then transported back to the photoreceptors for incorporation into opsin. The rapid dark adaptation of cones and their ability to maintain adequate levels of pigment and remain light sensitive even in steady bright light require rapid pigment regeneration, hence rapid recycling of chromophore for cones. However, the slow rate of chromophore turnover in the pigment epithelium and the competition for recycled chromophore between cone opsin and overwhelming levels of rod opsin in most rod-dominant species indicate that the canonical pigment epithelium visual cycle might not be sufficient to meet the chromophore demand of cones. Indeed, recent biochemical studies from conedominant species have brought up the idea of a second, cone-specific pathway for recycling of chromophore located within the retina and possibly relying on the Mu¨ller cells. The role of this novel cycle in mammalian rod-dominant species is still controversial. However, recent physiological experiments with amphibian photoreceptors demonstrate the function of a retina visual cycle under physiological conditions in a rod-dominant retina. Importantly, the combined action of the pigment epithelium and the retina visual cycles is required for the rapid and complete dark adaptation of cones.

Light Adaptation in Cones In contrast to rods, which saturate in moderate light and are not responsive during the day, cones have the ability to

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adapt their sensitivity and remain functional over a very wide range of light intensity. Studies with amphibian and fish photoreceptors indicate that, similar to the case of rods, cone adaptation is mediated by intracellular calcium, modulated by the activation of the phototransduction cascade. In the dark, the continuous current entering the outer segment through the cGMP-gated channels is carried in part by calcium, which is returned to the extracellular space via a Na+/(Ca2+, K+) exchanger. Following photoactivation and the closure of cGMP channels, calcium continues to be exported out of the cell through the Na+/(Ca2+, K+) exchanger until a new equilibrium is reached. As a result, activation by light causes a decline in the concentration of calcium in the outer segment of the cell. This triggers the calcium-mediated negative feedback on phototransduction, which in rods is required for eventually terminating the signal and for adapting the cell in response to light. Interestingly, calcium constitutes a larger fraction of the total ionic flux in and out of the outer segment of cones compared to rods. Thus, in cones of amphibians and fish, the fraction of photocurrent carried by calcium is about 35% compared to 20% in rods. As would be expected from the need to maintain a steady calcium concentration in darkness, the matching rates of extrusion of calcium via the Na+/(Ca2+, K+) exchanger are also higher in cones compared to rods. The combination of faster turnover of calcium in cones and their smaller volume compared to rods allows calcium in cones to decline several times faster upon light stimulation. In addition, their range of calcium concentrations from darkness to bright light is threefold wider than that in rods. These quantitative differences create the potential for more powerful modulation of phototransduction by calcium in cones compared to rods consistent with the ability of cones to adapt better and faster to various light conditions than rods. The mechanisms by which calcium modulates the cone phototransduction cascade are not well understood. However, comparison between cone and rod phototransduction reveals several interesting points. One mechanism by which calcium modulates phototransduction in rods involves inactivation of the visual pigment via phosphorylation by rhodopsin kinase. This reaction is modulated by the calcium-binding protein recoverin (also known as S-modulin). Recoverin is a member of the EF-hand superfamily and exerts its effect by inhibiting phosphorylation of rhodopsin by rhodopsin kinase at high calcium levels. In rods, inhibition of rhodopsin kinase by recoverin regulates phototransduction in darkness, in high calcium conditions, but has little effect during light adaptation, in low calcium conditions. The role of recoverin in modulating cone phototransduction in darkness and during light adaptation is not known. However, rods and cones share the same isoforms of recoverin and rhodopsin kinase. In addition, calcium modulates the sites

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and extent of pigment phosphorylation in cones but not in rods. Finally, unlike in rods, in cones, the calciumdependent inactivation of cone visual pigment could be the rate-limiting step for the shutoff of the cone photoresponse. Another mechanism by which calcium modulates phototransduction in rods involves the synthesis of cGMP by GC. As discussed above, this reaction is modulated by GCAP1 and GCAP2. GCAPs modulate GC in rods up to 20-fold as they inhibit it at high intracellular calcium levels and activate it at low calcium levels. While the simultaneous deletion of GCAP1 and GCAP2 delays the recovery of cone light responses, the extent to which GCAPs modulate cone phototransduction in darkness and during light adaptation is not known. Finally, calcium is also believed to directly modulate the cGMP-gated channels in cones. The Ca2+-dependent modulation of cGMP current is minimal in amphibian and undetectable in mammalian rods. In contrast, cone cGMP channels are directly modulated by Ca2+ both in fish and in mammalian retina. The molecular mechanism of cone channel modulation remains to be discovered. While calmodulin binds to and modulates heterologously expressed cGMP-gated channels, its role in the intact cone photoreceptor has been questioned.

Epilog These are exciting times for studying cone phototransduction. Until recently, technical issues such as the low abundance of cone photoreceptors in rod-dominant retinas and the fragility of mammalian cone photoreceptors have held back the biochemical and physiological studies of cones. As a result, despite the crucial role of cones for our daytime vision, mammalian cone phototransduction has been poorly understood. Recent development of several genetically modified mice has turned the tables. One example is the Nrl knockout mouse. Nrl is a transcription factor required for rod photoreceptor differentiation and its deletion produces a retina populated exclusively by cone-like photoreceptors. This makes possible the purification and biochemical characterization of mammalian cone phototransduction proteins. The Nrl knockout retina has also been used recently for physiological studies of cone photoreceptors. Other examples of useful genetically modified mice include those lacking the rod visual pigment (rhodopsin knockout) and the rod Gta subunit (transducin a knockout). The lack of functional rods in both of these retinas makes possible the physiological identification and study of cone photoreceptors. This approach was most recently used to investigate the role of Arr1 and Arr4 in the inactivation of mouse cone pigments. The combination of new genetic models and improved physiological tools provides

promise for studies of mammalian cone photoreceptors using the full range of tools that have been so successful in characterizing the function of mammalian rods. This should allow not only quantitative characterization of the cone phototransduction cascade but also understanding the mechanisms for cone dark and light adaptation which make cones invaluable as our daytime photoreceptors. See also: Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors; Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Phototransduction: The Visual Cycle.

Further Reading Donner, K. (1992). Noise and the absolute thresholds of cone and rod vision. Vision Research 32: 853–866. Ebrey, T. and Koutalos, Y. (2001). Vertebrate photoreceptors. Progress in Retinal and Eye Research 20: 49–94. Fu, Y. and Yau, K. W. (2007). Phototransduction in mouse rods and cones. Pflugers Archive: European Journal of Physiology 454: 805–819. Hecht, S., Haig, C., and Chase, A. M. (1937). Rod and cone dark adaptation. Journal of General Physiology 20: 831–850. Holcman, D. and Korenbrot, J. I. (2005). The limit of photoreceptor sensitivity: Molecular mechanisms of dark noise in retinal cones. Journal of General Physiology 125: 641–660. Kawamura, S. and Tachibanaki, S. (2008). Rod and cone photoreceptors: Molecular basis of the difference in their physiology. Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology 150: 369–377. Kefalov, V., Fu, Y., Marsh-Armstrong, N., and Yau, K. W. (2003). Role of visual pigment properties in rod and cone phototransduction. Nature 425: 526–531. Kefalov, V. J., Estevez, M. E., Kono, M., et al. (2005). Breaking the covalent bond – A pigment property that contributes to desensitization in cones. Neuron 46: 879–890. Korenbrot, J. I. and Rebrik, T. I. (2002). Tuning outer segment Ca2+ homeostasis to phototransduction in rods and cones. Advances in Experimental Medicine and Biology 514: 179–203. Mata, N. L., Radu, R. A., Clemmons, R. C., and Travis, G. H. (2002). Isomerization and oxidation of vitamin A in cone-dominant retinas: A novel pathway for visual-pigment regeneration in daylight. Neuron 36: 69–80. Nikonov, S. S., Brown, B. M., Davis, J. A., et al. (2008). Mouse cones require an arrestin for normal activation of phototransduction. Neuron 59: 462–474. Rebrik, T. I. and Korenbrot, J. I. (2004). In intact mammalian photoreceptors, Ca2+-dependent modulation of cGMP-gated ion channels is detectable in cones but not in rods. Journal of General Physiology 123: 63–75. Rieke, F. and Baylor, D. A. (2000). Origin and functional impact of dark noise in retinal cones. Neuron 26: 181–186. Tachibanaki, S., Arinobu, D., Shimauchi-Matsukawa, Y., Tsushima, S., and Kawamura, S. (2005). Highly effective phosphorylation by G protein-coupled receptor kinase 7 of light-activated visual pigment in cones. Proceedings of the National Academy of Sciences of the United Sates of America 102: 9329–9334. Wald, G., Brown, P. K., and Smith, P. H. (1955). Iodopsin. Journal of General Physiology 38: 623–681. Yau, K. W. (1994). Phototransduction mechanism in retinal rods and cones. The Friedenwald Lecture. Investigative Ophthalmology and Visual Science 35: 9–32.

Phototransduction: Phototransduction in Rods Y Fu, Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, UT, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Dark current – Also called circulating current. Current generated by constant influx of Naþ/Ca2þ into the rod outer segment through cGMP-gated channels, which is balanced by an outward current flowing across the inner segment membrane that mainly carried by potassium channels. Dark light – Signals produced by thermal activation of rhodopsin in the dark, which adapt the visual system like real background light. Phototransduction – The conversion of a light signal to an electrical signal in a photoreceptor cell. Quantum efficiency – The ratio between the number of photoactivated molecules and the number of molecules that absorbed a photon. Single-photon response – Electrical signal triggered by a single photon in a rod cell. Suction-electrode recording – The recording of light-sensitive current of a single rod (or cone) by drawing its outer segment (or inner segment) into a suction electrode.

Introduction Image-forming vision in vertebrates is mediated by two types of photoreceptors: the rods and the cones. Rods are specialized for dim-light (scotopic) vision while cones mediate vision in bright light (photopic). Great progress has been made in understanding rod phototransduction since the introduction of the suction-electrode recording technique in the late 1970s. The light-sensitive current of individual amphibian and mammalian (including primate) photoreceptors can be recorded with this method. Bovine retina, on the other hand, has been a favorite preparation for studying phototransduction by biochemists because of the abundance of tissue available. The mouse, however, has become an increasingly popular animal model for study in the past decade through the advent of gene-targeting techniques. When combined with electrophysiology, mouse genetics provides unmatched power in elucidating the in vivo functions of key phototransduction proteins, most of which have been knocked out, overexpressed, or mutated in rods, yielding a rich body of information on the mechanisms underlying the amplification, recovery, and adaptation of

rod photoresponses. The details of the activation phase of rod phototransduction are now established. A quantitative description, the Lamb–Pugh model, is achieved that reproduces the activation kinetics of the rod response under physiological conditions. In this article, the focus is on the activation phase of rod phototransduction with particular emphasis on the molecular mechanisms underlying its high signal amplification feature.

Vertebrate Rods Are Highly Efficient Photon Detectors Psychophysical experiments performed by Hecht, Schlaer, and Pirenne in 1942 suggested that human retinal rods can detect single photons. Thirty-seven years later, suctionelectrode recordings from isolated toad rods by Baylor, Lamb, and Yau confirmed this remarkable ability of vertebrate rods (Figure 1). The amazing ability of vertebrate rods to detect single photons can be attributed to at least three factors: high quantum efficiency of photoactivation, low intrinsic noise, and a powerful signal amplification cascade. Two other factors greatly increase the photon capture ability of vertebrate rods, numerical dominance of rods over cones, and a highly specialized outer segment structure. The dense stack of disks of the rod outer segment ensures that virtually every photon traveling axially will be captured. In a sense, vertebrate rods can be viewed as sophisticated three-dimensional photon capture devices.

Phototransduction in Rods: A G-ProteinSignaling Pathway Rod phototransduction is one of the best-characterized G-protein-signaling pathways. The receptor is rhodopsin (R), the G protein is transducin (G), and the effector is cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE or PDE6). Upon photon absorption, the rhodopsin molecule becomes enzymatically active (R*) and catalyzes the activation of the G-protein transducin to G*. Transducin, in turn, activates the effector PDE to PDE*. PDE* hydrolyzes the diffusible messenger cGMP. The resulting decrease in the cytoplasmic-free cGMP concentration leads to the closure of the cGMP-gated channels on the plasma membrane. Channel closure leads to localized reduction on the influx of cations into the outer segment, which results in membrane hyperpolarization, that is, the intracellular voltage becoming more negative (Figure 2).

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Figure 1 Suction-electrode recording on the membrane current of a single toad rod. (a) The outer segment of a rod projecting from a piece of retina was sucked in position in a suction electrode. Proximal end of cell remains attached to retina. Boundary between inner and outer segments is visible. (b) Response of rod outer segment to a series of 40 consecutive dim flashes, 20 ms flash delivering 0.029 photons mm2 at 500 nm, flash timing monitored below. The rod showed no response to some flashes, or a small response of 1 pA to others, and occasionally a larger response. This suggests that the flash response is quantized, as might be expected when on average very few photons are absorbed. With further analysis, the authors demonstrated that each quantal electrical event resulted from a single photo-isomerization with mean amplitude of 1 pA – the single-photon response. Modified from Baylor et al. (1979) Journal of Physiology, 288: 589–611 (a) and 288: 613–634 (b), with permission from Blackwell Publishing.

This hyperpolarization decreases or terminates the dark glutamate release at the synaptic terminal. The signal is further processed by other neurons in the retina before being transmitted to higher centers in the brain. Following light activation, a timely recovery of the photoreceptor is essential so that it can respond to subsequently absorbed photons, and signal rapid changes in illumination. This recovery from light requires the efficient inactivation of each of the activated components: R*, G*, and PDE*, as well as the efficient regeneration of rhodopsin (R) and the rapid restoration of the cGMP concentration. The termination rates of the activation steps set the time course of the photoresponse. Although rod phototransduction is the best-characterized sensory transduction pathway, rods differ from other sensory cells in that light leads to hyperpolarization rather than depolarization. Rods respond to light with graded hyperpolarization whose amplitude increases monotonically as a function of flash intensity until saturation. One hallmark of rod phototransduction is the reproducibility of its single-photon response in both amplitude and kinetics. This is quite remarkable considering the fact that events generated by single molecules are stochastic in nature. The study on the underlying mechanisms has long been a hot topic in the vision field. Recent research pointed to two possible mechanisms: (1) Rhodopsin inactivation is averaged over multiple shutoff steps so that the integrated R* activity varies less than otherwise controlled by a single step. (2) Averaging over the deactivation of multiple G-protein molecules.

High Quantum Efficiency of Photoactivation The quantum efficiency of photoactivation measures the probability that the adsorption of a photon initiates photoactivation. This probability is defined as the ratio between the number of photoactivated molecules and the number of molecules that absorbed a photon. Quantum efficiency of visual pigments is wavelength independent at 0.7 in the spectrum of visible light. This suggests that every absorbed photon in the visible range can activate rhodopsin equally well. The quantum efficiency of 0.7 is very similar across all visual pigments. This high efficiency seems to be a common feature of most vertebrate visual pigments.

The Great Thermal Stability of Rhodopsin Unlike chemosensory systems, phototransduction is not triggered by the binding of a chemical ligand to the receptor, rhodopsin. Instead, the chemical, 11-cis-retinal in birds and land-based animals (or 11-cis-3,4-dehydroretinal in aquatic animals), is prebound to rhodopsin. Photon absorption triggers the cis- to trans-isomerization of the retinoid. This isomerization rapidly converts the ligand from a powerful antagonist to a powerful agonist, leading to the formation of a series of spectrally distinct intermediates of rhodopsin in the order of bathorhodopsin, lumirhodopsin, metarhodopsin I (Meta I), and metarhodopsin II (Meta II) within a few milliseconds. Meta II is


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Figure 2 Schematic representation on the activation of vertebrate rod phototransduction. Following photon absorption, the activated rhodopsin (R*) activates the heterotrimeric G protein, catalyzing the exchange of GDP for GTP, producing the active Ga*-GTP. Two Ga*-GTPs bind to the two inhibitory g-subunits of PDE, thereby releasing the inhibition on the catalytic a- and b-subunits, forming PDE*, which in turn catalyzes the hydrolysis of cGMP. The consequent decrease in the cytoplasmic-free cGMP concentration leads to the closure of the cGMP-gated channels on the plasma membrane and blockage of the influx of cations into the outer segment, which results in the reduction of the circulating dark current.

the active form of rhodopsin (R*), which in turn activates the downstream G protein, transducin. Because free opsin can weakly activate the transduction cascade, the antagonist role of 11-cis-retinal in the dark is important to keep the noise low in rods. Even with 11-cis-retinal attached, rhodopsin occasionally undergoes spontaneous (thermal) activation in the dark, producing responses identical to those triggered by photons. This noise is often expressed as dark light because the noise adapts the visual system like real background light. This activity sets the limit on scotopic sensitivity, the visual sensitivity in darkness or dim light. To achieve the single-photon-detection sensitivity, rods not only need to have a high amplification system, but also need to have extremely low noise, or to be very quiet in the dark. This quietness can be partly attributed to the great thermal stability of rhodopsin. In a toad rod, the rate of thermal activation of rhodopsin was measured to be 0.03 event s1 rod1 at 22 C, corresponding to an average wait of 2000 years for the spontaneous activation of a given rhodopsin molecule to occur, based on a total of 2 109 rhodopsin molecules per cell. This great stability makes it possible for rods to pack many rhodopsin molecules to the rod disks to increase its photon-capture ability while keeping the dark noise low. It should be mentioned that the question of dark noise in vision has had a long intellectual history from the point

of view of psychophysics and system neuroscience. As early as 1940s and 1950s, Hecht and Barlow have estimated the amount of dark light in human rods based on psychophysical experiments. More than 30 years later, Baylor and colleagues used suction-electrode recording technique on primate rods to demonstrate that the very low quantal noise from rhodopsin, corresponding to 0.01 event s1 rod1 in darkness, indeed matches the human psychophysical scotopic threshold. The quantitative agreement between the quantal noise measured from single rods and that measured in human psychophysics was considered a breakthrough in the vision field and a wonderful convergence between cell physiology and human psychophysics/system neuroscience – the goal of modern neuroscience after all.

The Activation of Transducin Constitutes the First Amplification Step The second component of the rod phototransduction is the 81-kDa heterotrimeric G-protein, transducin (Gt, or Gat1b1g1), which forms a subfamily of heterotrimeric G proteins. The molecular weight for a-, b-, and g-subunits of rod transducin is approximately 39, 36, and 6 kDa, respectively. Transducin is present at 10% the amount of rhodopsin in the disk membrane. Although transducin

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subunits are soluble, the holo-transducin is firmly anchored to the disk membranes by a farnesyl lipid group that is post-translationally attached to the carboxy-terminal of the g-subunit and an acyl group on the amino-terminal of the a-subunit. Therefore, photo-activated rhodopsin (R*) can only interact and activate transducin through lateral diffusion at the membrane surface. Like many other G proteins, transducin exists in one of the two states: the GDP-bound inactive state and the GTPbound active state. Binding of R* catalyzes the exchange of GTP for GDP on the a-subunit. The active Ga-GTP (G*) dissociates from R* as well as its native partner, Gbg, and interacts with PDE to carry the signal forward. In the meantime, R* is able to activate additional molecules of transducin. Transducin activation by R* represents the first amplification step in the phototransduction cascade. The estimated rate of transducin activation by a single R* varied from 10 to over 3000 s1 at room temperature. A rate of 120 s1 was later reported to be more consistent with biochemical, light-scattering, and electrophysiological measurements. The rate is roughly doubled in mammalian rods due to the higher body temperature. Until recently, it was believed that over a hundred transducins are activated during the lifetime of a single R* in mammalian rods. This number is now revised to be 20 in mouse rods, based on the shorter life time of R* (80 ms) and activation rate of transducin by R* (240 s1). The importance of transducin for conveying the signal from R* to PDE was manifested from animal models deficient in Ga and human patients carrying Ga mutations. It was found that rods of Gat1-null mice (Gnat1–/–) lost all light sensitivity. In human, a mis-sense mutation in Gnat1 (encoding the rod Gat1) is implicated in autosomal-dominant congenital stationary night blindness of Nougaret, caused by constitutive activation of rod phototransduction. The gnat1–/– mouse line has proven to be a valuable tool for blocking rod phototransduction to study cone phototransduction and circadian photoreception. It was also used successfully to delineate two apoptotic pathways in light-induced retinal degeneration. Bright light triggers apoptosis of photoreceptors through a mechanism requiring the activation of rhodopsin but not transducin signaling. In contrast, low-intensity light induces apoptosis that is predominantly dependent on transducin signaling.

The High Catalytic Power of PDE Accounts For the Second Amplification Step PDE is the third component of rod phototransduction. It is a hetero-tetrameric protein consisting of two catalytic subunits, a- and b-, and two identical g-subunits. PDE is anchored to the disk membrane by a hydrophobic

isoprenyl group (compounds that are derived from isoprene, 2-methylbuta-1,3-diene, linearly linked together) posttranslationally attached to the C-termini of the two catalytic subunits. As for transducin activation, PDE activation by G is through lateral diffusion on the rod disk membrane. Each catalytic subunit has two high-affinity noncatalytic binding sites and one catalytic binding site for cGMP. The noncatalytic sites were suggested to modulate the binding affinity between PDEg and PDEab. The amount of PDE is ~1–2% of rhodopsin. Thus, the first three components of phototransduction are present in the ratio of 100R:10G:1PDE. In the dark, the two g-subunits act as inhibitory subunits by binding to the two catalytic subunits and significantly reducing the hydrolysis of cGMP. In the light, Ga-GTP encounters PDEg and sterically displaces the latter, therefore relieving its inhibitory effect on the catalytic subunits and permitting the hydrolysis of cGMP to proceed (Figure 2). Since each G* can only activate one PDEg, two G*’s are required to fully activate a holo PDE. This is likely the scenario in vivo during light activation due to the excess amount of G over PDE and the presence of many molecules of G* activated by rhodopsin. In contrast to the amplification achieved during transducin activation by R*, the activation of PDE by G* constitutes no gain, that is, with an efficiency approaching 1 (one G*, one activated PDE catalytic subunit) or 0.5 in terms of PDE holoenzyme. It is the catalytic power of PDE* that provides the second amplification step. It was reported that PDE* hydrolyzes cGMP at a rate close to the limit set by aqueous diffusion, with a Km of 10 mM and a Kcat of 2200 s1, making it one of the most efficient enzymes in vivo. In addition to the noise produced by spontaneous activation of rhodopsin, spontaneous activation of individual catalytic PDE subunits produces the continuous noise, which accounts 30–80% (depending on the species) of the total dark noise variance in rods. The basal spontaneous PDE activity balances constitutive guanylate cyclase activity in the dark, therefore maintaining a steady-free cGMP level. It also has the function of increasing the rate of cGMP turnover and consequently speeding up the dim flash response. One might have expected that the deletion of PDEg from mouse rods would unleash the full catalytic power of PDEab. However, it was found, in the absence of PDEg that the PDEab dimer actually lacked catalytic activity, and the photoreceptors of the mutant mouse rapidly degenerated. Thus, the inhibitory PDEg subunit appears to be necessary for the integrity of the catalytic PDEab subunits. The degeneration might be caused by an abnormally high cGMP concentration due to the lack of hydrolysis. A related example is the rd mouse, which is the oldest and one of the best-known models for retinal degeneration. The rod cells in the rd mouse begin to degenerate at about postnatal day 8, followed by cones;


by 4 weeks, virtually no rod photoreceptors are left. Degeneration in this mouse model is preceded by the accumulation of cGMP in the retina, correlated with deficient activity of the rod PDE due to a mutation in the PDEb subunit. It is worth noting that the rd mouse was instrumental in suggesting that inner retinal neurons could mediate non-image-forming vision.

cGMP Is the Second Messenger Mediating Rod Phototransduction By 1970, scientists generally believed that a second messenger was required to mediate the rod photoresponse based on several lines of evidence. First, light absorption occurs on the rod disk membrane, whereas the light-sensitive conductance is in the plasma membrane. Since rod disks are separate from the plasma membrane, a second messenger is required to connect the two. Second, the dim-flash response of rods lasts a few seconds, which is too long to be accounted by the open time of known membrane conductance. However, it took more than a decade before the identity of the second messenger was finally determined to be cGMP. The fierce battle was fought on the validity between two competing candidates, Ca2þ and cGMP. According to the Ca2þ hypothesis, which was first proposed by Hagins, the concentration of intracellular free Ca2þ is low in the dark and rises in the light to block light-sensitive current. The main supporting evidence is that reducing the concentration of external Ca2þ dramatically increases the dark current, suggesting that internal Ca2þ inhibits the dark current. On the other hand, the cGMP hypothesis proposed that the concentration of cGMP was high in the dark to maintain a cGMP-dependent conductance. Light led to the hydrolysis of cGMP and the subsequent closing of the conductance. The supporting evidence is that intracellular injection of cGMP increases the amplititude and latency of the photoresponse. Adding to the complexity is the finding that the free cGMP concentration varies inversely with the free Ca2þ concentration in rods, making it difficult to separate the effect of the two. This debate was finally settled with the discovery of cGMP-gated channels in rods by Fesenko and colleagues in 1985. By using the patch-clamp technique, they showed that cGMP increased a cation conductance of inside-out patches of outer-segment plasma membrane without the need of ATP. The direct channel gating by cGMP is surprising because cyclic nucleotides were generally believed to act through cyclic-nucleotide-dependent kinases and protein phosphorylation on target proteins at that time. This dogma partially explained scientists’ reluctance to embrace the cGMP hypothesis because protein phosphorylation was too slow. Another monumental work by Yau and Nakatani was published at the same year that helped the anointment of cGMP as the

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right candidate. An identical cGMP-gated cation conductance was found on a truncated rod outer segment with an intact plasma membrane. Most importantly, this conductance could be suppressed by light, suggesting that the long-sought light-sensitive conductance is the cGMPgated conductance. The publications by Fesenko and Yau marked the end of the Ca2þ hypothesis.

The cGMP-Gated Channel Provides the Final Step of Signal Amplification The cGMP-gated channel belongs to the family of cyclicnucleotide-gated (CNG) channels, which are nonselective cation channels. The channel is located on the plasma membrane with a density of 400–1000 mm2 and is the last component in the activation phase of phototransduction. Rod CNG channels consist of CNGA1 (or a1) and CNGB1 (or b1) subunits. CNGA1 subunit forms functional homomeric channels by themselves when heterologously expressed. Although CNGB1 does not form functional channels by themselves, it confers several properties typical of native channels when coexpressed with the CNGA1 subunit: flickery opening behavior, increased sensitivity to L-cis-diltiazem, (a CNG channel– specific inhibitor) and weaker block by extracellular calcium. For a long time, the rod channel was believed to be a hetero-tetramer consisting of two CNGA1 and two CNGB1 subunits. In 2002, a number of laboratories made the surprising discovery that the rod channel actually has a 3CNGA1:1CNGB1 subunit composition. In humans, mutations in CNGA1 cause retinitis pigmentosa. CNGB1 subunits were found to be crucial for the targeting of the native CNG channel in rods. Thus, only trace amounts of the CNGA1 subunit were found on the rod outer segments in CNGB1-null mice and the majority of rod photoreceptors failed to respond to light. The gating of the rod channel by cGMP is cooperative with a Hill coefficient of 3; therefore, the light-triggered suppression of the dark current is 3 times larger than the decrease in the intracellular cGMP concentration. This is the last step of signal amplification in rod phototransduction. The combined amplification provided by rhodopsin, PDE, and CNG channels is very high (105–106), ensuring the high sensitivity of rods, including the ability of rods to detect single photons. In the dark, the concentration of free cGMP in the rod outer segment was estimated to be several mM, which is lower than the K1/2 (10–40 mM depending on Ca2þ concentration), the concentration of cGMP necessary to half-maximally activate the channel. As a result, only 1% of the CNG channels are open! In other words, 99% of the channels are already closed in the dark and light can only suppress the remaining 1% channels. This explains

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why current induced by cGMP injection is more than 10 times larger than the dark current. The inward current through the cGMP channel is composed of 85% Naþ because Naþ is the predominant external cation and the channel is nonselective to monovalent cations. The remaining current is mainly carried by Ca2þ with a minor contribution from Mg2þ. Extracellular Ca2þ actually partially blocks the channel to reduce its conductance under physiological conditions. The inward current is balanced by an outward current flowing across the inner-segment membrane, which is mainly carried by potassium channels. This circulating current is also called dark current in both rods and cones. Unlike other ligandgated channels, the CNG channel does not desensitize to cGMP, which is important for rods to maintain a steady dark current ranging between 20 and 70 pA in vertebrate rods. The rod photoresponse is essentially a transient suppression of the circulating current. It was estimated that the dark current was carried by 10 000 channels. The participation of large numbers of micro-channels averages out the channels noise, that is, reduces an otherwise substantial stochastic channel noise if the dark current were carried out by a few macro-channels. This feature improves the sensitivity of rods. Two extrusion mechanisms are critical in maintaining ionic balance in rods. An energy-dependent Na–K ATPase at the inner segment pumped Naþ out and Kþ into the cells. A Na/Ca,K exchanger (NCKX) in the outer-segment plasma membrane extrudes one Ca2þ and one Kþ outward in exchange for four Naþ inward producing the net entry of one positive charge. The exchanger and the CNG channel were found to form a stable complex on the plasma membrane, likely as a way to control the stoichiometry between the two, which is critical for regulating Ca2þ concentration in the rod outer segment. During the light response, the influx of Ca2þ is reduced due to the closure of some CNG channels while

the efflux of Ca2þ through the exchanger is maintained. The resulting Ca2þ decline triggers negative feedback to produce light adaptation. See also: Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: The Visual Cycle; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Secondary Photoreceptor Degenerations: AgeRelated Macular Degeneration; Secondary Photoreceptor Degenerations.

Further Reading Arshavsky, V. Y., Lamb, T. D., and Pugh, E. N., Jr. (2002). G proteins and phototransduction. Annual Review of Physiology 64: 153–187. Baylor, D. A., Lamb, T. D., and Yau, K. W. (1979a). The membrane current of single rod outer segments. Journal of Physiology 288: 589–611. Baylor, D. A., Lamb, T. D., and Yau, K. W. (1979b). Responses of retinal rods to single photons. Journal of Physiology 288: 618–634. Burns, M. E. and Arshavsky, V. Y. (2005). Beyond counting photons: Trials and trends in vertebrate visual transduction. Neuron 48: 387–401. Burns, M. E. and Baylor, D. A. (2001). Activation, deactivation, and adaptation in vertebrate photoreceptor cells. Annual Review of Neuroscience 24: 779–805. Fu, Y. and Yau, K. W. (2007). Phototransduction in mouse rods and cones. Pflugers Archiv – European Journal of Physiology 454: 805–819. Luo, D. G., Xue, T., and Yau, K. W. (2008). How vision begins: An odyssey. Proceedings of the National Academy of Sciences of the United States of America 105: 9855–9862. Pugh, E. N., Jr. and Lamb, T. D. (2000). Phototransduction in vertebrate rods and cones: Molecular mechanisms of amplification, recovery and light adaptation. In: Stavenga, D. G., de Grip, W. J., and Pugh, E. N., Jr. (eds.) Handbook of Biological Physics, Vol. 3: Molecular Mechanisms of Visual Transduction, pp. 183–255. Amsterdam: Elsevier.

Phototransduction: Rhodopsin L P Pulagam and K Palczewski, Case Western Reserve University, Cleveland, OH, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary GPCRs – G protein-coupled receptors are membrane receptor proteins with a seventransmembrane helical topology that are capable of activating G proteins. G proteins – Heterotrimeric intracellular proteins so named because they bind to the guanine nucleotides, guanosine diphosphate in an inactive state and guanosine triphosphate in an active state. GRK1 – G protein-coupled receptor kinase 1 (rhodopsin kinase) is a highly specific protein kinase that catalyzes phosphorylation of photoactivated rhodopsin thereby triggering its deactivation. LCA – Leber’s congenital amaurosis is an inherited degenerative disease of the retina that results in a severe loss of vision. Photoisomerization – The structural change between chromophore geometric isomers (cis to trans) caused by photoexcitation. For rhodopsin, photoisomerization of its chromophore leads to activation of this receptor. ROSs – Rod outer segments are the cylindrical outer portions of rod cells, each containing hundreds of membranous disks enveloped by the cellular (plasma) membrane. RP – Retinitis pigmentosa is the name of a heterogeneous group of progressively blinding degenerations of the human retina caused by mutations in genes encoding photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Schiff base – A functional chemical group containing a carbon–nitrogen double bond. The retinylidene moiety is a chemical link between retinal and an amino group, for example, Lys296 of opsin.

Vision is an important biological sensing mechanism that involves conversion of light signals received by the eye into electrical nerve impulses transmitted to the brain by a process called phototransduction, consisting of a cascade of biological processes that occur in photoreceptor cells (rod and cone cells) of the retina. In the absence of light, photoreceptors are depolarized to a membrane resting potential of –40 mV. In the presence of light, the plasma membrane of the photoreceptor cells becomes hyperpolarized to –70 mV, resulting in a reduced amount of

neurotransmitter released to downstream neurons. This article focuses on rhodopsin structure that relates to its function as a G protein-coupled receptor (GPCR).

Rod Cells and Rhodopsin The vertebrate rod cell, a highly differentiated postmitotic neuron, is characteristically long, cylindrical, and primarily consists of an outer segment connected to an inner segment via a cilium (Figures 1(a) and 1(b)). The rod outer segment (ROS) contains a stack of disk membranes enclosed by the plasma (cell) membrane, whereas the rod inner segment (RIS) contains the metabolic machinery for this cell. Rhodopsin is processed in the endoplasmic reticulum and transferred to the Golgi membranes of the RIS for additional processing of its carbohydrate moieties. Then rhodopsin-containing Golgi vesicles fuse with the apical plasma membrane of the inner segment and the rhodopsin molecules are transported through the rod cell cilium to the ROS where they form disk membranes. Mutations in the C-terminal region of rhodopsin inhibit transport of rhodopsin to ROS, indicating that this region is essential for recognition by the transport machinery. A mammalian ROS consists of a stack of 1000–2000 disks enclosed by the plasma membrane. A cryo-electron tomography image of murine ROS (Figure 1(c)) also reveals that the thickness of a single disk membrane is 8 nm. Rhodopsin comprises >90% of all proteins in disk membranes and occupies 50% of the disk membrane volume. It is also present at a lower density in the plasma membrane of rod cells, and its expression is essential for ROS formation, which is absent in knock-out Rho–/– mice. In wild-type mice, there are approximately 8 104 rhodopsin molecules per disk and 3.96 1014 per eye. The power spectra of negatively stained disk membranes from bovine ROS (Figure 1(d)) reveals a diffuse diffraction ring at (45 A˚)–1, indicating paracrystallinity of rhodopsin. Organization of the seven helices of rhodopsin was first ascertained by using a low-resolution imaging method called electron crystallography. Rhodopsin is unequally distributed in disk membranes. Electron tomographs of the ROS reveal both high- and low-density regions (Figure 1(e)). An atomic force microscopic image of native disk membranes showed that the average packing density of rhodopsin monomers is 48 300 8000 mm–2. Recent atomic force microscopic studies disclosing the arrangement of rhodopsin in native mouse disk membranes revealed that rhodopsin and opsin form structural dimers arranged in

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Figure 1 Vertebrate retina and rhodopsin. (A) Diagram of a rod cell. Vertebrate rod cells are postmitotic neurons with highly differentiated rod outer segments (ROSs) connected to rod inner segments (RISs) that generate proteins and energy to sustain phototransduction. ROSs consist of hundreds of stacked disk membranes enveloped by a plasma membrane. The main component of disk membranes is rhodopsin. Biochemical processes involving rhodopsin in the ROS allow rapid transduction of a light signal to graded hyperpolarization of the plasma membrane resulting from a decrease of light-sensitive conductance in ROS cGMP-gated cation channels. (B) Scanning electron micrograph of mouse retina. Rod cells comprise 70% of all 6.4 million retinal cells, whereas cone cells represent 0.33) represent 71% of the disk volume. Gray values were obtained by computing the gray value for each voxel (three-dimensional pixel) in a disk membrane volume of 10 disks. Spacer proteins connecting two disks are colored red and the plasma membrane is colored blue. (F) Topograph obtained by using atomic-force microscopy shows the paracrystalline arrangement of rhodopsin dimers in the native disk membrane of mouse rod photoreceptors. Vertical brightness ranges: 1.6 nm. Scale bar = 50 nm. (G) Transmission electron microscopy of negatively stained disk membranes solubilized by n-dodecyl-b-D-maltoside. Rhodopsin dimers are clearly discerned on the carbon film. Magnified selected particles marked by broken circles are shown on the right. Scale bar = 500 A˚. Frame size of the magnified particles in the gallery is 104 A˚. (C and E) From Nickell et al. (2007). Originally published in The Journal of Cell Biology. (doi:10.1083/jcb.200612010). (F) Adapted from figure 2a in Fotiadis D. (2003). Atomic-force microscopy: Rhodopsin dimers in native disc membranes. Nature 421: 127–128. (D and G) Adapted from figure 3 in Suda K. (2004). The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes. Molecular Membrane Biology 21: 435–446: Taylor and Francis.

paracrystalline arrays of rows (Figure 1(f )). Previous studies also support the importance of dimerization/oligomerization as necessary for the function of many, if not all GPCRs. Negative staining of detergent (n-dodecyl-b-D-maltoside)solubilized disk membranes shows bi-lobed, roughly conical structures with lengths of 65 A˚, and these lobes are separated from each other by 32 A˚ (Figure 1(g)).

Structure of Rhodopsin Rhodopsin is a transmembrane protein consisting of an apoprotein, the 348 amino acid residue-long opsin

(Figure 2), linked to a chromophore, 11-cis-retinal. The chromophore is bound covalently via a protonated Schiff base to the Lys296-containing side chain of the opsin. Bovine rhodopsin also is post-translationally modified. The N-terminal Met is acetylated, and Cys322 and Cys323 of the C-terminus are palmitoylated, which is very common in GPCRs. In addition, a disulfide bond exists between Cys110 (H-III) and Cys187 (E-II). Rhodopsin is glycosylated at Asn2 and Asn15 by the hexasaccharide sequence (Man)3Glc(Nac)3. The molecular mass of bovine opsin with its post-translational changes (palmitoylation, acetylation of the N terminus and glycosylation) is 42 002.


C-terminal T T V S A

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D D G L 330 E P F H-8 S 240 E K N Cytoplasmic side K H-VI V 320 C-I R T K K R Q T T A H-III C 140 T K L R N K H-IV F K V 230 L G C T 70 K V A C C T M Q E H-II P G H-I H V V 310 N K F H-IV 150 F Q L Y E R V V E N M 60 I E T V L R T 250H-VII H N Y 130 Y V G Q M A L I I L A M I V Y L I V G M 80 L F C Y L T 220 N I V L L V M I P V F W S N F A F A 160 I V V I 260 N V P I I L D A T A V Y I A L V W 50 G F L F 300 I P L F L 120 G E M S A M I V T G C A 210 L I M F I L W K A L H L F 90 V A T 170 L G G P F F A A C F A Y F V 270 F P A F M Y M T G F 290 T P V G 40 A A T P A L E I Y F A V L 110 T I L F V M L N C T Y G Y M G S S F I E I F W 100 L S Q F 200 N TS S H T P R S P W N Y G H G C S C Q P G T I Y Q G S D F E G F V F M E P G 280 A Extracellular side E E-III I L 180 E E-I Y 190 D Y Y T P H 30 N G Q Y P A E F P M1 T E-II S E R N-terminal 10 V G V Y 20 V P F N P G F T K S N P

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Figure 2 Two-dimensional representation of rhodopsin. Rhodopsin has seven transmembrane a-helices, C-I, C-II, and C-III depict its cytoplasmic loops, and E-I, E-II, and E-III represent the extracellular loops in this diagram. Stability of the helical segment is increased by the disulfide Cys110–Cys187 bridge (shown as-S-S-), a highly conserved feature among many GPCRs. The chromophore, 11-cisretinal, not shown here, is attached to Lys296 via a protonated Schiff base. Asn2 and Asn15 are sites of glycosylation by conserved glycans, Met1 is acetylated and Cys322 and Cys323 are palmitoylated. The predominant phosphorylation sites are Ser334, Ser338, and Ser343. The whole C-terminal region is highly mobile but, as shown by using a model peptide, it may become more rigid when bound to arrestin. The highly conserved domains among GPCRs, (D/E)R(Y/W) (colored in pink) in helix 3 and NPXXY in helix VII (colored in green), are important for transforming the receptor from an inactive to a G protein-coupled conformation. Nonsense/missense mutations in rhodopsin leading to retinitis pigmentosa are colored in dark gray.

Rhodopsin was crystallized from detergent solutions. The three-dimensional structure of bovine rhodopsin at 2.8 A˚, the first high-resolution structure reported for a GPCR, reveals the internal organization of this receptor molecule (Figure 3). Rhodopsin folds into seven transmembrane helices (H-I–H-VII) that vary in length from 20 to 33 amino acid residues, and one cytoplasmic helix (H-8). The transmembrane residues are irregular and tilted at various angles due to their Gly and Pro residues. Further advances in rhodopsin crystallization were recently reviewed. The N-terminal region of rhodopsin is located intradiscally (extracellular) and the C-terminal region is cytoplasmic (intracellular), each region possessing three interhelical loops. The extracellular region consists of four distorted b-strands, three interhelical loops and the doubly glycosylated N-terminal domain. The extracellular region from residues 173–198 acts as a plug for the chromophore-binding pocket (Figures 4(a) and 4(b)). The cytoplasmic surface contains 14 positively charged

residues, whereas the extracellular side contains only three positively charged residues, an arrangement that agrees with the positive inside rule for multispanning eukaryotic membrane proteins. The intracellular/cytoplasmic region of rhodopsin is essential for its vectorial transport from the site of synthesis to the ROS and it also plays important roles in G protein activation and photoactivated rhodopsin desensitization. A highly conserved (D/E)R(Y/W) motif in the GPCR A family is formed by the tripeptide, Glu134-Arg135-Tyr136, located in the cytoplasmic region of bovine rhodopsin (Figure 4(a)). The carboxylate group of Glu134 forms a salt bridge with Arg135, a highly conserved residue among GPCRs, and Arg135 also interacts with Glu247 and Thr251 in H-VI. The ionization state of Glu134 is sensitive to its environment, such that protonation of this residue causes rhodopsin activation (from meta IIa to meta IIb). This motif plays an important role in conformational changes in the structure of GPCRs that lead to their activation.

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C-terminal 75Å

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Figure 3 Structure of rhodopsin. (a) Rhodopsin has seven transmembrane helices (H-I to H-VII) and one peripheral helix (H-8). The transmembrane segments are a-helical, but these helices are highly distorted and tilted. Helices are displayed as ribbons and colored from blue (H-I) to red (H-8), as in the visible light spectrum. Helix labels are shown in the same color as their respective helices. N-terminal and C-terminal ends are colored gray. A palmitoyl group (orange-colored ball and stick representation) is attached to each of the two Cys residues at the end of helix H-8. Removal of this group has only a minor effect on phototransduction. b1–b4 are distorted b strands. The carbohydrate moieties (cyan-colored stick representation) are at Asn2 and Asn15. The Gly3 to Pro12 region forms the first b-hairpin that runs parallel to the expected plane of the membrane. Arg177 to Asp190 leave helix H-4 to form a second twisted b-hairpin on the extracellular side. Rhodopsin is represented in a space-filled background and the plane of the lipid bilayer is shown. (b) Opposite side of rhodopsin shown in Figure 2(a). (pdb code: 1U19.pdb)

(a)

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Figure 4 Functional regions of rhodopsin. (a) Conserved NPXXY and (D/E)R(Y/W) motifs in rhodopsin. Amino acid residues of these motifs are rendered as sticks, retinal (RET) is shown in the CPK color scheme as red, and helices are displayed as ribbons. (b) Retinal (RET)-binding site. Amino acid residues within about 5 A˚ are displayed to show the side chain environment surrounding the 11-cis-retinylidene group. Residues are represented as balls and sticks and retinal is shown in stick form (red). See text for explanation/discussion. Helices are colored as in Figure 3.

Another highly conserved NPXXY (Asn-Pro-Xaa-XaaTyr) motif located at the end of helix VII and the beginning of H-8, is also close to the cytoplasmic region. Both the D (E)RY and NPXXY regions control the meta II (active) state of rhodopsin, and the NPXXY sequence is likely to be involved in G protein coupling (Figure 4(a)). The greatest distortion in H-VI is imposed by Pro267 (a highly

conserved residue among GPCRs) and H-VII is elongated due the presence of Pro291 and Pro303 (parts of NPXXY) located in close proximity to Lys296, the retinal-binding residue. Highly conserved Glu122 and His211 are located at the Zn2+-binding site. The interaction between the b ionone ring and H-III occurs at Glu122, one of the residues that determine the rate of meta II decay (Figure 4(b)).


Chromophore-Binding Site Visual pigments of both rod and cone cells contain the chromophore, 11-cis-retinal, bound covalently to a Lys side chain (Lys296 in bovine rhodopsin) via a protonated Schiff base. The absorption maximum (lmax) of free solubilized 11-cis-retinal is about 380 nm. When this chromophore binds to opsins, its lmax shifts toward longer wave lengths (a red shift) ranging from 435 nm (frog rods) to 560 nm (human cones). The protonated Schiff base linkage is responsible for about 70 nm of this shift. A further red shift results from the retinal-binding-pocket environment, especially its counter ion, which is Glu113 in vertebrate rhodopsins. In bovine rhodopsin, the lmax of mutant E113Q (Glu to Gln) is dramatically shifted from 498 nm to 380 nm. The absorption maximum also varies according to the interaction sites of the opsin molecule with the chromophore, especially dipolar interactions near the b ionone ring. Therefore, the lmax absorption of visual pigments varies from species to species that differ with respect to their opsin protein sequences. The chromophore in rhodopsin is located in the core of the seven transmembrane helices, closer to the extracellular side of the disk membrane. 11-cis-Retinal helps maintain rhodopsin in an inactive state (Figure 4(b)). The retinalbinding pocket is formed by helices H-III, H-V, H-VI, and H-VII and the antiparallel b sheet of the N-terminal plug, part of extracellular loop II (E-II in Figure 2). Although the retinal-binding site is very hydrophobic (Figure 4(b)), four charged residues, specifically Glu113 (H-III), Glu122 (H-III), Glu181 (b sheet), and Lys 296 (H-VII), are located near the chromophore. Lys296 (H-VII) donates an amino group to form a protonated Schiff base and Glu113 (H-III), which is 3.6 A˚ away from the Schiff base, acts as a counter ion for this linkage. The positive charge on the protonated Schiff base is energetically unstable in the hydrophobic protein core but the Glu113 (H-III) counter ion stabilizes the base by shifting its pKa from neutral to alkaline. Highly conserved among all known vertebrate visual pigments, Glu113 (H-III) plays three important roles: (1) It keeps rhodopsin in its resting state by participating in the salt bridge with the Schiff base. Disruption of this bridge allows the H-VI motion that occurs upon photoactivation. (2) It prevents spontaneous hydrolysis of the Schiff base by stabilizing the protonated Schiff base via increasing its Ka by as much as 107. (3) It causes a major bathochromic (longer wavelength) shift in the maximum wavelength absorption of visual pigments. Longer wavelength absorption is essential because the front of the eye in most animals does not allow ultraviolet (UV) light to reach the retina. Steric hindrance, resulting either from mutating Gly121 (H-III) or substituting larger R groups at the C9 position, causes transducin (Gt) activation in the dark whereas lack of the C9 methyl group impedes photoactivation.

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Opsin reacts within minutes with 11-cis-retinal to form rhodopsin. Similarly, 7-cis and 9-cis retinal also form visual pigments. In contrast, all-trans-retinal and 13-cisretinal cannot regenerate opsin. The crystal structure of rhodopsin reveals that the chromophore-binding pocket is well defined, suggesting that the binding pocket has high specificity for the Schiff base and the b ionone ring. The exact location of these two components restricts the length of the chromophore-binding site. Therefore, 7-cis, 9-cis, and double and triple cis retinal analogs that have similar lengths and structures can regenerate the opsin, whereas chromophore analogs that are either shorter or longer than 11-cis-retinal cannot.

Rhodopsin Cycle – Retinal Isomerization Inactive rhodopsin is activated upon light absorption, which induces a cis–trans isomerization that converts 11-cis-retinal to all-trans-retinal. The activation process can be divided into three phases (Figure 5). 1. Light-induced cis–trans isomerization of the retinylidene. 2. Thermal relaxation of the retinylidene–protein complex. 3. Hydrolysis of the Schiff base linkage, leading to formation of rhodopsin’s active meta II state. Light absorption induces isomerization of 11-cisretinylidene to all-trans-retinylidene, resulting in a transient intermediate called photorhodopsin, formed by the fastest chemical reaction (200 fs) in the rhodopsin cycle. Photorhodopsin is converted first into a thermally stable and high-energy intermediate product called bathorhodopsin. About 60% of the incident photon energy is stored in bathorhodopsin and then used to drive further conformational changes. In this state, the chromophore is in an 11-trans-15-anti conformation, a distorted all-trans conformation that results from steric restriction caused by the polyene chain of the retinal and the protein side chains. The b ionone ring and Schiff base are located in a conformation similar to that of rhodopsin in the dark, but Thr181 and Glu113 are slightly moved. Then the blue-shifted intermediate (BSI) is produced during the thermal relaxation of bathorhodopsin, but BSI can be observed only by time-resolved measurements during subsequent formation of lumirhodopsin. The distorted all-trans-retinal in bathorhodopsin relaxes by dislocation of the b ionone ring in lumirhodopsin. Displacement of this ring reflects the movement of helix III aided by interactions between other helices, that result in a slightly disordered structure during the transition from the dark state to lumirhodopsin. In particular, Thr181 and Glu122 (which are moved slightly in bathorhodopsin) become significantly moved due to the b ionone ring

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Rhodopsin (λmax = 500 nm) O

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Figure 5 Schematic illustration of the rhodopsin cycle. Rhodopsin consists of an apoprotein called opsin and the bound chromophore, 11-cis-retinylidene, a geometric isomer of vitamin A in aldehyde form that imparts a red color to this protein. Upon light activation, rhodopsin transforms into opsin via many intermediate states. In the dark, rhodopsin contains the 11-cis-retinal chromophore attached to Lys296 (rendered as sticks) of helix H-VII in a protonated Schiff base linkage. Upon absorption of a photon, the chromophore is isomerized with 65% probability from a cis C11–C12 double bond to a trans conformation. In addition, with light activation, rhodopsin transforms into the photointermediate, bathorhodopsin (Batho), which thermally relaxes to BSI followed by lumirhodopsin (Lumi), which then changes to Meta I. During the transition of meta I to meta II, the all-trans-retinylidene Schiff base becomes deprotonated. Meta II, the signaling state capable of G protein activation, ultimately decays to free all-trans-retinal and opsin. The released photoisomerized chromophore, all-trans-retinal, is reduced to an alcohol by short chain alcohol dehydrogenases, such as prRDH, retSDR, and RDH12. The all-trans-retinal then diffuses into the retinal pigmented epithelium. There it undergoes enzymatic transformation back to 11-cis-retinal in a metabolic pathway known as the visual cycle. The replenished 11-cis-retinal then combines with opsin to form rhodopsin, thereby completing the rhodopsin cycle. The lmax in nm as well as the duration (fs-s) of the various components are shown.

displacement. Lumirhodopsin then relaxes further into meta I. Although the conformation of the chromophore in meta I closely resembles that in lumirhodopsin and the Schiff base proton is still hydrogen bonded, the overall structure is similar to rhodopsin. Meta I is further converted to meta II in two steps that can be separated by 20 ms in detergent-solubilized samples: (1) Conversion of meta I to meta IIa, accompanied by proton transfer from the Schiff base to the counterion Glu113. (2) Subsequent uptake of a proton from the cytoplasm leading to meta IIb formation. The proton acceptor here is Glu134 (H-III), thus meta IIa and meta IIb are in a pH-dependent equilibrium regulated by proton uptake at Glu134 (part of E/DRY motif). However, only meta IIb can trigger Gt activation. Deprotonation of the Schiff base is characterized by a large UV shift of the absorption maximum from 478 nm in meta I to 380 nm in meta II. A low-resolution crystal structure of a bovine-deprotonated Schiff base meta IIlike rhodopsin has been elucidated. Finally, meta II decays

into opsin and all-trans-retinal. Free opsin exhibits the largest conformational changes as compared to darkstate rhodopsin (Figure 5).

Visual Cycle – Rhodopsin Regeneration The visual cycle consists of a series of reactions by which all-trans-retinal released from opsin isomerizes back into 11-cis-retinal that again binds to the opsin (Figure 5). This cyclic process does not require light. The released all-trans-retinal is transformed first to all-trans-retinol by a retinal dehydrogenase (RDH) in the ROS. The all-trans-retinol is transferred to the retinal pigment epithelium (RPE) where it is esterified by lecithin:retinol acyltransferase (LRAT), and later isomerized to 11-cis-retinol by a retinol isomerase. 11-cis-RDH then converts 11-cisretinol back to 11-cis-retinal, which leaves the RPE to regenerate the opsin in the ROS.


Vertebrate versus Invertebrate Rhodopsins Enzymatic regeneration of rhodopsin does not occur in invertebrate visual systems where rhodopsin and metarhodopsin are photoconvertible. Upon photon absorption, 11-cis-retinal (or its analogs) of rhodopsin is converted into all-trans-retinal of metarhodopsin, and then irradiation of metarhodopsin changes the all-trans-retinal back to 11-cis-retinal by a process called photoregeneration. Notably, invertebrates differ from vertebrates in the photoactivation of rhodopsin. Absorption of a photon by invertebrate rhodopsin leads to a stable meta II, but

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retinal remains in the retinal-binding pocket. This contrasts to vertebrate rhodopsin where retinal leaves the meta II-binding pocket. Invertebrate phototransduction also involves an inositol-1,4,5-triphosphate signaling cascade (cyclic-guanosine monophosphate (GMP) in vertebrates) and a Gq-type G protein (Gt in vertebrates) that is stimulated by photoactivated rhodopsin. Three-dimensional structures of bovine (vertebrate) rhodopsin and squid (invertebrate) rhodopsin show structural similarities in the arrangement of their transmembrane helices (Figure 6). Squid rhodopsin is a 50-kDa protein composed of 488 amino acid residues. A proline-rich 10-kDa C-terminal extension compared

Bovine rhodopsin Squid rhodopsin Cytoplasm

(a)

(b)

Trp265

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Trp274 Tyr277 Tyr111

Ala269

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Gly114Tyr268 Glu181 Gly188

Tyr191

Ser187

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Gly115

Phe120 Phe188

Tyr177

Met207 lle189

(c)

Phe205

Asn87

Glu122 His211 Phe208

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Met204

Squid

Figure 6 Bovine rhodopsin vs. squid rhodopsin. (a) Superimposition of bovine and squid rhodopsin structures. Helices are displayed in cartoon style, retinal (RET) is rendered as CPK balls, and the planar lipid bilayer is shown. (b) Conserved NPXXY and (D/E)R(Y/W) motifs of both bovine (green) and squid (yellow) rhodopsins are compared. Amino acids of these motifs are rendered as sticks, retinal is rendered as CPK balls, and helices are displayed as ribbons. (c) Retinal-binding site of bovine rhodopsin. Amino acid residues within about 5 A˚ are displayed to show the side-chain environment surrounding the 11-cis-retinylidene group. Residues are rendered as balls and sticks and retinal is shown in stick form (red). (d) Retinal-binding site of squid rhodopsin. Amino acid residues within about 5 A˚ are displayed to reveal the side-chain environment surrounding the 11-cis-retinylidene group. Residues are rendered as balls and sticks and retinal is shown in stick form (red). See text for explanation/discussion.

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with bovine rhodopsin is important for intracellular trafficking of this rhodopsin, and its deletion does not affect G protein activation. Like other GPCRs, squid rhodopsin forms a disulfide bridge between Cys108 and Cys186 for proper folding of the E-II loop. The NPXXY and (D/E)R (Y/W) regions of squid rhodopsin are similar to those of bovine rhodopsin. However, Val in the NPXXY motif, and Glu in the (D/E)R(Y/W) motif of bovine rhodopsin are replaced by Met and Asp, respectively, in squid rhodopsin (Figure 6(b)). Their structural similarity implies that the functional difference between vertebrate and invertebrate rhodopsins might be due to specific interactions between retinal and the amino acid sequence of the retinal-binding site. These binding sites are compared in Figures 6(c) and 6(d). In squid rhodopsin, the highly conserved Glu180 is too far away from the retinal-binding pocket and Asn185 is located between Glu180 and the Schiff base. Asn185 is assumed to move after photoisomerization of retinal, to mediate an indirect interaction between Glu180 and the retinal Schiff base. In the dark state, either Asn87 or Tyr111, which is highly conserved among all invertebrates, might act as a hydrogen-binding partner (counter ion) for the Schiff base. These residues are replaced by Gly89 and Glu113 in bovine rhodopsin.

Signaling Cycle After photoactivation, an active meta II state of rhodopsin triggers the activation of transducin (Gt protein). This form of rhodopsin is capable of activating Gt proteins during the relatively prolonged period of its activation. Accordingly, rhodopsin activity is regulated by its phosphorylation, a common feature among many GPCRs. This regulatory process is also important for rod cells to recover their responsiveness during dark adaptation. Desensitization of rhodopsin involves two steps: (1) phosphorylation of meta II, reducing the rate of transducin activation and (2) binding of arrestin to meta II, completely ending transducin activation by rhodopsin (Figure 7). This phosphorylation is carried out by rhodopsin kinase, a specific kinase also known as GRK1. Rhodopsin kinase phosphorylates specific serines in the rhodopsin C-terminal sequence. However, upon light illumination, GRK1 is released from recoverin and phosphorylates rhodopsin at multiple sites. Ser343, Ser338, and Ser334, located in the C terminal domain on the cytoplasmic side of rhodopsin, are the main sites for this phosphorylation. Some threonine residues in this domain are also phosphorylated. Phosphorylation of this cytoplasmic domain reduces the ability of the Gt protein to bind to meta II, but it does not completely stop Gt activation. By binding to phosphorylated-meta II, arrestin prevents the interaction of Gt with meta II, and thus completely terminates Gt activation. At least three phosphorylated sites are required for high-affinity binding

Rhodopsin 11-cis-retinal

Gt

Opsin

Meta II Gt* Signaling cycle

Pi

ATP GRK1

PrP2A

Pi-opsin

Pi-meta II

All-trans-retinal

P PP

ADP

P PP Arrestin P PP Arrestin

All-trans-retinal Arrestin

Pi-meta II/arrestin

Figure 7 Interaction of rhodopsin with partner proteins. Phototransduction starts with the absorption of light by rhodopsin that causes photoisomerization of 11-cis-retinal to all-trans-retinal. Photoisomerization of this chromophore induces conformational changes in rhodopsin leading to formation of meta II, the signaling state of rhodopsin. Meta II binds and activates a large number of photoreceptor-specific G protein molecules, transducins (Gt), by catalyzing the exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on transducin’s a-subunit, Gta. Deactivation of meta II and consequent Gt-mediated signaling starts with binding of the GPCR kinase called GRK1 (or rhodopsin kinase) that catalyzes subsequent phosphorylation of Ser and Thr residues in the C-terminus of rhodopsin. Phosphorylated rhodopsin then is capped by binding to arrestin, which prevents any residual Gt activation by meta II. The complex of phosphorylated rhodopsin–arrestin loses all-trans-retinal and then arrestin, after which the phosphorylated opsin is dephosphorylated by the action of protein phosphatase 2A (PrP2A). All-trans-retinal is transformed, through a series of steps, to 11-cis-retinal, which rebinds to opsin (as shown in Figure 4), thereby continuing rhodopsin signaling. A fraction of meta II loses all-trans-retinal (without phosphorylation) and directly transforms to opsin.

of the rhodopsin–arrestin complex. Arrestin dissociates from rhodopsin as meta II decays and loses all-trans-retinal. Then phosphorylated opsin is dephosphorylated by protein phosphatase 2A (PrP2A). The resulting free opsin is readily regenerated by 11-cis-retinal and continues recycling through the signaling cascade. A fraction of meta II directly dissociates into all-trans-retinal and opsin.

Rhodopsin Interaction with Other Proteins According to X-ray crystallographic models and atomic force microscopic studies, H-IV–H-V of rhodopsin contact each other in a rhodopsin dimer. The sizes of Gt, GRK1, and


arrestin proteins also favor the hypothesis of their interaction with the rhodopsin dimers. Previously reported models of rhodopsin with these proteins are discussed below. These complexes have yet to be resolved by crystallography.

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Gi-protein

Rhodopsin–Gt In its inactive state, Gt is a membrane-associated protein that consists of a, b, and g subunits with one guanosine diphosphate (GDP) noncovalently bound to the a-subunit. Post-translational modification of the a-(myristoylation) and g-(farnesylation) subunits help this protein to associate with the membrane. A model for the rhodopsin–Gt complex has been reported (Figure 8(a)). Spectroscopic, biochemical, and peptide competition experiments reveal that cytoplasmic loops II, III, H-8, and the C-terminal tail of rhodopsin interact with transducin. The interacting sites of transducin are the C-terminal tail, N-terminal helix, the a4–b6–a5 region of the a-subunit, and the farnesylated C-terminal region of the g-subunit.

(a)

Rhodopsin dimer

GRK1

Rhodopsin–GRK1 GRK1 phosphorylates multiple sites on the C-terminal tail that is freely accessible in both active and inactive states of rhodopsin (Figure 8(b)). A single activated rhodopsin (meta II) molecule can induce the phosphorylation of hundreds of other rhodopsins. Cytoplasmic loops II and III of rhodopsin are the most important sites for binding of GRK1, and the N-terminal 30 residues of GRK1 are important for this interaction. Inactivating mutations in GRK1 are found in human patients with Oguchi disease, a stationary form of night blindness characterized by a substantial delay in recovery of dark vision after photobleaching. Rhodopsin–Arrestin Arrestin binds to photoactivated-phosphorylated rhodopsin (Figure 8(c)). Biochemical analysis of the arrestin–rhodopsin complex reveals that several domains of arrestin are essential for this interaction. In particular, the region from residues 163 to 189 is essential for binding to activated-phosphorylated rhodopsin, but not to unphosphorylated rhodopsin. Lysine and arginine residues of arrestin are also very important for specific binding, but only to phosphorylated rhodopsin.

Mutations in Rhodopsin and Retinal Diseases Mutations in the genes encoding many proteins involved in phototransduction and the visual and signaling cycles have been implicated in causing blinding diseases of humans such as Leber’s congenital amaurosis (LCA), Stargardt

(b) Arrestin

(c) Figure 8 Conceptual models of rhodopsin dimers interacting with Gt, GRK1 and arrestin. (a) Rhodopsin dimer bound to one heterotrimeric Gt. Gta is colored red, Gtb is colored green, and Gtg is colored blue. Gt occupies a single rhodopsin dimer, with only one rhodopsin monomer requiring activation. Helices of rhodopsin are colored as in Figure 2. (b) A rhodopsin monomer is modeled such that its third cytoplasmic loop (C-III) lies close to the proposed receptor-docking site for GRK1. This allows the GRK1 active site to have easy access to the C-tail of activated rhodopsin or of a neighboring unactivated rhodopsin in the same membrane plane, thereby allowing high gain phosphorylation of the ROS. (c) This theoretical model reflects the interaction of one arrestin molecule with a rhodopsin dimer. Molecules are represented in a space-filled background and the plane of the lipid bilayer is shown. No structural optimization was performed.

macular degeneration, congenital cone–rod dystrophy, and retinitis pigmentosa (RP). More than 100 rhodopsin mutants resulting in human eye diseases have been identified (Figure 2). Some mutations result in degeneration of


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Light

Dark 0

0 –5 pA

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–10

–10

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–15

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WT –/– Lrat–/–multigavage Lrat

1000 μV

00

800 600 400

WT –/– Lrat–/–multigavage Lrat

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200 10 20

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0 –40 –30 –20 –10 0 (e)

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Figure 9 Rescue of visual responses measured by single-cell recording and ERG responses of single WT and Lrat–/– mouse rod cells. (a) Flash families measured for a Lratþ/þ mouse (WT) rod, (b) a Lrat–/– rod from a mouse that had received a single gavage with 9-cis-RAc (9-cis-retinyl acetate), and (c) a control Lrat–/– mouse rod. Rods were obtained from 8-week-old mice. Each panel superimposes averaged responses to 5–20 repeats of a flash; flash strength was increased by a factor of 2 to produce each successively larger response. (d and e) Comparisons of scotopic single-flash ERG recordings from Lratþ/þ (WT) control mice, 9-cis-R-Ac gavaged Lrat–/– mice and Lrat–/– untreated mice. Lrat–/– mice were gavaged 9 times with 5 mmol of 9-cis-R-Ac during a 1-month period. (f) Light-induced pupillary constriction of Lrat–/– mice before and after treatment with 9-cis-R-Ac. All together, these experiments show that 9-cis-retinyl acetate restored retinal function in this animal model of LCA. Adapted from figures 4 and 6 in Batten M. L. (2005). Pharmacological and rAAV gene therapy rescue of visual functions in a blind mouse model of Leber congenital amaurosis. PLOS Medicine 2(11): e333.

rod cells, while some affect the function of rhodopsin. Mutations at the C-terminal tail impair rhodopsin trafficking from RISs to the ROSs. Mutations, for example Pro23 to His, which lead to rhodopsin misfolding will not allow the protein to reach disk membranes of ROS. Nonetheless, the ROSs degenerate and finally cause blindness. The Lys296 mutant is unable to bind chromophore, thereby compromising rhodopsin function. Mutations, for example Ala292 to Glu, which lead to human congenital night blindness do not involve ROS degeneration but rather compromise human vision under dim light. Mutations of proteins in the visual cycle also cause eye diseases. For example, inactivating mutations in the LRAT gene cause LCA. The Lrat–/– knock-out mouse with LRAT-mediated retinal dystrophy evidences only traces of retinoid compounds in ocular tissues, resulting in impaired vision from birth. The ROS are shortened in Lrat/ mice, and photoreceptors degenerate very slowly. This disease can be treated by dietary intake of active chromophores or their 9-cisprecursors. Oral supplementation of Lrat/ mice with 9-cis-retinyl acetate restored retinal function (Figure 9). See also: Phototransduction: Phototransduction in Rods; Phototransduction: The Visual Cycle; Rod and Cone

Photoreceptor Cells: Inner and Outer Segments; Rod Photoreceptor Cells: Soma and Synapse.

Further Reading Arshavsky, V. Y., Lamb, T. D., and Pugh, E. N., Jr. (2002). G proteins and phototransduction. Annual Review of Physiology 64: 153–187. Filipek, S., Stenkamp, R. E., Teller, D. C., and Palczewski, K. (2003). G protein-coupled receptor rhodopsin: A prospectus. Annual Review of Physiology 65: 851–879. Fotiadis, D., Liang, Y., Filipek, S., et al. (2003). Atomic-force microscopy: Rhodopsin dimers in native disc membranes. Nature 421: 127–128. Hargrave, P. A., McDowell, J. H., Curtis, D. R., et al. (1983). The structure of bovine rhodopsin. Biophysics of Structure and Mechanism 9: 235–244. Menon, S. T., Han, M., and Sakmar, T. P. (2001). Rhodopsin: Structural basis of molecular physiology. Physiological Reviews 81: 1659–1688. Muller, D. J., Wu, N., and Palczewski, K. (2008). Vertebrate membrane proteins: Structure, function, and insights from biophysical approaches. Pharmacological Reviews 60: 43–78. Okada, T., Sugihara, M., Bondar, A. N., et al. (2004). The retinal conformation and its environment in rhodopsin in light of a new 2.2 A˚ crystal structure. Journal of Molecular Biology 342: 571–583. Palczewski, K. (2006). G protein-coupled receptor rhodopsin. Annual Review of Biochemistry 75: 743–767.

Phototransduction: Rhodopsin Palczewski, K., Kumasaka, T., Hori, T., et al. (2000). Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289: 739–745. Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H. W., and Ernst, O. P. (2008). Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454: 183–187. Park, P. S., Lodowski, D. T., and Palczewski, K. (2008). Activation of G-protein-coupled receptors: Beyond two-state models and tertiary conformational changes. Annual Review of Pharmacology and Toxicology 48: 107–141. Rao, V. R. and Oprian, D. D. (1996). Activating mutations of rhodopsin and other G protein-coupled receptors. Annual Review of Biophysics and Biomolecular Structure 25: 287–314.

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Ridge, K. D. and Palczewski, K. (2007). Visual rhodopsin sees the light: Structure and mechanism of G protein signaling. Journal of Biological Chemistry 282: 9297–9301. Salom, D., Lodowski, D. T., Stenkamp, R. E., et al. (2006). Crystal structure of a photoactivated deprotonated intermediate of rhodopsin. Proceedings of the National Academy of Sciences of the United States of America 103: 16123–16128. Travis, G. H., Golczak, M., Moise, A. R., and Palczewski, K. (2007). Diseases caused by defects in the visual cycle: Retinoids as potential therapeutic agents. Annual Review of Pharmacology and Toxicology 47: 469–512.

Phototransduction: The Visual Cycle G H Travis, UCLA School of Medicine, Los Angeles, CA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Lipofuscin – Fluorescent pigment granules found within cells of the RPE. Lipofuscin contains oxidized fatty acids and condensation of products of retinaldehyde with phosphatidylethanolamine, such as A2E. Lipofuscin is thought to arise from the incomplete digestion of phagocytosed outer segments. Components of lipofuscin, including A2E, are cytotoxic and thought to play a role in the etiology of macular degeneration. Opsin visual pigment – Opsin pigments are light-sensitive complexes containing a protein and an 11-cis-retinaldehyde chromophore. Outer segment – An elongated light-sensitive structure attached to the connecting cilium of rod and cone photoreceptors. The outer segment comprises a stack of approximately 1000 membranous disks. These disks are loaded with rhodopsin or cone opsin visual pigments. Retinyl ester – A conjugate of vitamin A (retinol) with a fatty acid. Retinyl esters represent stable, nontoxic, and water-insoluble storage forms of retinol. Retinyl esters are also the substrate for Rpe65-isomerase in RPE cells. Retinaldehyde – An oxidized form of retinol. Retinaldehydes are highly reactive and potentially cytotoxic. The 11-cis isomer of retinaldehyde (11-cisRAL) is the light-sensitive chromophore in rhodopsin and cone-opsin visual pigments. Schiff base – It is also called an imine. Results from the reaction of a primary amine (as in lysine or phosphatidylethanolamine) with a carbonyl group (as in retinaldehyde) to form a carbon–nitrogen double bond with loss of a water molecule. Formation of a Schiff base is reversible.

The vertebrate retina contains two classes of light-sensitive cells, rods and cones. Both cell types contain a membranous structure called the outer segment (OS), which are loaded with rhodopsin or cone-opsin visual pigments. These pigments are members of the G-protein-coupled receptor superfamily. Each rod OS contains approximately 108 rhodopsin pigments. The ligand for these pigments is 11-cis-retinaldehyde (11-cis-RAL), which is covalently coupled to a lysine in the opsin protein through a Schiff-base linkage. Absorption of a photon

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by an opsin pigment induces photoisomerization of the 11-cis-RAL chromophore to all-trans-retinaldehyde (all-trans-RAL). This isomerization converts the pigment to active metarhodopsin II, which stimulates the visualtransduction cascade. After a brief period, metarhodopsin II is inactivated by rhodopsin kinase-mediated phosphorylation and subsequent capping by arrestin. Next, alltrans-RAL dissociates from the inactivated opsin pigment. To restore light sensitivity, the bleached apo-opsin recombines with another 11-cis-RAL, forming a new rhodopsin or cone-opsin pigment. To maintain continuous vision in light, the all-trans-RAL released by bleached pigments must be converted back to 11-cis-RAL. This process is carried out by a multistep enzyme pathway called the visual cycle (Figure 1). The first two catalytic steps of this pathway occur in photoreceptors, while the remaining steps take place in cells of the retinal pigment epithelium (RPE). The RPE is an epithelial monolayer adjacent to the photoreceptors. Apical processes of RPE cells interdigitate with the photoreceptor OS. The regeneration of visual chromophore is one of the several collaborations between photoreceptors and RPEs; and cone opsins may have access to an alternative source of 11-cis-RAL chromophore. This alternative retinoid pathway is present in Mu¨ller glial cells.

Clearance of All-trans-RAL from OS Disks Following photoactivation and subsequent deactivation of the opsin pigment, all-trans-RAL probably exits between transmembrane (TM) helices, TM1 and TM7, into the lipid bilayer. The all-trans-RAL diffuses within the bilayer until it encounters the amine headgroup of a phosphatidylethanolamine, which may condense with the all-trans-RAL to form the Schiff base, N-retinylidene-phosphatidylethanolamine (N-ret-PE). This condensation reaction is reversible. On the cytoplasmic surface of the OS disk-membrane, alltrans-RAL is reduced to all-trans-retinol (all-trans-ROL), driving dissociation of N-ret-PE (see below). However, alltrans-RAL can be temporarily trapped as N-ret-PE on the intradiscal surface. An adenosine triphosphate (ATP)binding cassette transporter called ABCA4 (also ABCR or rim-protein) is present in the disks of rod and cone OS. Mice with a knockout mutation in the abca4 gene show delayed clearance of all-trans-RAL and elevated N-ret-PE in the retina following exposure to light. In vitro studies suggest that ABCA4 is an outwardly directed flippase for


Opsin 11-cis-RAL + HC= NH (rhodopsin) hv

O 11-cis-RP LRAT

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CH3OC(CH3)4CH3

11-cis-REH 11-cis-RAL

(bound to CRALBP)

Apo-opsin

CHO

Facilitated by ABA4 (bound to IRBP)

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NAD+ (NADP+)

CH2OH

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Rpe65 Isomerase

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3

LRAT

All-trans-RDH’s

CH OH

3

CH2OH

2

All-trans-ROL

RPE cell

All-trans-ROL Rod outer segment

Figure 1 Visual cycle. Following absorption of a photon (hn), 11-cis-RAL Schiff base in rhodopsin is isomerized to all-trans-RAL, converting the receptor to active metarhodopsin II. Subsequently, the all-trans-RAL dissociates from apo-opsin. ABCA4 transports all-trans-RAL (as N-ret-PE) across the disk bilayer from the interior to the cytoplasmic leaflet. The all-trans-RAL is reduced to all-trans-ROL by one or more all-trans-RDH’s that use NADPH as a cofactor. The all-trans-ROL is released by the OS to IRBP in the IPM. The all-trans-ROL is carried by IRBP to the apical RPE, where it is taken up and esterified by LRAT or ARAT to yield an all-trans-RE such as all-trans-RP. The all-trans-RP is isomerized and hydrolyzed by Rpe65 to yield 11-cis-ROL. The 11-cis-ROL may be oxidized by one or more 11-cis-RDH to yield 11-cis-RAL chromophore. Alternatively, the 11-cis-ROL may be secondarily esterified by LRAT or ARAT to yield an 11-cis-RE, such as 11-cis-RP, representing a preisomerized storage form of chromophore precursor. When needed, the 11-cis-RP is hydrolyzed by 11-cis-REH to yield 11-cis-ROL. 11-cis-ROL and 11-cis-RAL are bound to CRALBP in RPE cells. The 11-cis-RAL is released by the RPE into the IPM where it binds to IRBP. Finally, the 11-cis-RAL is delivered to the OS where it recombines with apo-opsin to form a new visual pigment.

N-ret-PE, consistent with the biochemical phenotype in abca4 –/– mice. Thus, ABCA4 appears to facilitate the removal of all-trans-RAL from disk membranes for subsequent reduction to all-trans-ROL. Mutations in the human ABCA4 gene cause Stargardt macular degeneration and a subset of recessive cone–rod dystrophy in humans. Stargardt patients and abca4 –/– mice accumulate toxic lipofuscin pigments in RPE cells. Buildup of these fluorescent pigments is important in the pathogenesis of photoreceptor degeneration in Stargardt’s disease.

Reduction of All-trans-RAL to All-trans-ROL This reaction is carried out in photoreceptor OS by a member of the short-chain dehydrogenase/reductase family called photoreceptor retinol dehydrogenase (prRDH) or RDH8. RDH8 uses nicotinamide adenine dinucleotide phosphate oxidase (NADPH) as a co-factor. In rdh8 –/– knockout mice, reduction of all-trans-RAL to all-transROL is slowed but not halted, suggesting that RDH8 function is complemented in photoreceptors by at least one other retinol dehydrogenase. Photoreceptors contain a second retinol dehydrogenase called RDH12 that also catalyzes NADPH-dependent reduction of all-trans-RAL to all-trans-ROL. Mice with a knockout mutation in the rdh12 gene show mildly slowed reduction of all-trans-RAL to all-trans-ROL, and protection from light-induced

photoreceptor degeneration. Unlike RDH8, which is expressed in photoreceptor OS, RDH12 is expressed in photoreceptor inner segments. This distribution is unexpected given that all-trans-RAL is released following light exposure into the OS. RDH12 may play a detoxifying role in the inner segment by reducing all-trans-RAL that escaped reduction by RDH8 in the OS. Mutations in RDH12 cause a severe recessive blinding disease called Leber congenital amaurosis (LCA). No mutations in RDH8 have been associated with a retinal dystrophy in humans. Mice with a knockout mutation in the rdh8 gene show normal kinetics of rhodopsin regeneration and delayed recovery of sensitivity following exposure to bright light. An identical pattern is seen in abca4 –/– mice. ABCR and all-transROL dehydrogenase act sequentially in the visual cycle to remove all-trans-RAL following a photobleach (Figure 1). Delayed dark adaptation in rdh8 –/– and abca4 –/– mice is probably due to noncovalent reassociation of all-transRAL with apo-opsin to form a noisy photoproduct that activates transducin.

Transfer of All-trans-ROL from Photoreceptors to the RPE Interphotoreceptor retinoid-binding protein (IRBP) is secreted by photoreceptors and present at a high concentration in the extracellular space. Besides IRBP, this space is filled with extracellular matrix material and is called the

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interphotoreceptor matrix (IPM). IRBP contains binding sites for both 11-cis- and all-trans-retinoids. IRBP has been shown to accelerate the removal of all-trans-ROL from bleached photoreceptors. The uptake of all-transROL by IRBP may involve a receptor on the OS plasma membrane. Retinoids bound to IRBP are protected from oxidation and isomerization during transit through the IPM. Mice with a knockout mutation in the irbp gene show accumulation of all-trans-ROL in the retina, and reduced all-trans-REs in the RPE following light exposure. These mice also show accumulation of 11-cis-RAL in the RPE and reduced 11-cis-RAL in the retina following light exposure. These results suggest that IRBP functions to extract all-trans-ROL from bleached photoreceptors, and 11-cis-RAL from RPE cells. Mutations in the RBP3 gene for IRBP cause the inherited blinding disease, recessive retinitis pigmentosa in a small subset of cases. Another all-trans-ROL-binding protein, cellular retinol– binding protein type-1 (CRBP1), is present in RPE cells. CRBP1 is a soluble protein that binds all-trans-ROL with 100-fold higher affinity than does IRBP. This difference in affinity drives the uptake of all-trans-ROL from the IPM into RPE cells. Compared with wild-type mice, crbp1 –/– knockout mice contain reduced all-trans-REs in the RPE and higher all-trans-ROL in the retina following light exposure. This biochemical phenotype is similar to the phenotype in irbp–/– mice.

Synthesis of Retinyl Esters The major retinyl-ester synthase in RPE cells is lecithin: retinol acyl transferase (LRAT), which catalyzes the transfer of a fatty-acyl group from the sn1 position in phosphatidylcholine to all-trans-ROL (see Figure 1). The resulting all-trans-retinyl esters (all-trans-REs) are water-insoluble, and represent a stable and nontoxic storage form of vitamin A. Mice with a knockout mutation in the lrat gene contain virtually no all-trans-REs or other visual retinoids in their ocular tissues. Accordingly, lrat–/– mice are totally blind. Mutations in the human LRAT gene are yet another cause of recessive LCA. Another retinyl-ester synthase activity, called acyl-CoA: retinol acyltransferase (ARAT), is present in RPE cells. Unlike LRAT, ARAT uses palmitoyl coenzyme A (palm CoA) as an acyl donor. Two enzymes have been shown to posses ARAT activity. Diacylglycerol acyltransferase type-1 (DGAT1), which catalyzes palm CoA-dependent synthesis of triglycerides from diacylglycerol, also catalyzes palm CoA-dependent synthesis of all-trans-REs from alltrans-ROL. Multifunctional O-acyltransferase (MFAT) also possesses ARAT catalytic activity. The very low level of all-trans-REs in the RPE of lrat–/– mice despite the presence of ARAT activity is due to the 10-fold higher KM for all-trans-ROL substrate of ARAT versus LRAT. ARAT

preferentially uses free all-trans-ROL as a substrate in contrast to LRAT, which uses holo-CRBP1.

Retinoid Isomerization Conversion of a planar all-trans-retinoid to the strained 11cis configuration is energetically unfavorable. Rpe65-isomerase uses all-trans-REs as substrate and catalyzes two reactions: hydrolysis of the carboxylate ester, and trans to cis isomerization of the C11–C12 double bond in the retinoid. Accordingly, the energy released by ester hydrolysis (–5.0 kcal mole–1) is used to drive isomerization (þ4.1 kcal mole–1). Rpe65 is homologous to b-carotene oxygenase in mammals and apocarotene oxygenase (ACO) in cyanobacteria. The X-ray diffraction analysis showed that ACO has a seven-bladed b-propeller structure, with a Fe2+-4-His arrangement at its axis. The four His residues that define the Fe2+-binding site are conserved in all members of the ACO family including Rpe65. Rpe65 was shown to bind Fe2+, which is required for its catalytic activity. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. Mice with a knockout mutation in the rpe65 gene contain high levels of all-trans-REs in the RPE and no detectable 11-cis-RAL. Accordingly, rpe65 –/– photoreceptors contain only apoopsin, and the mice have no detectable visual function. Despite blocked synthesis of visual chromophore, photoreceptor morphology is nearly normal in rpe65 –/– mice. Visual function has been restored in rpe65 –/– mice and dogs by administering exogenous visual chromophore. Injection of recombinant adeno-associated virus (AAV) containing a wild-type rpe65 gene into the subretinal space (between RPE cells and photoreceptors) of rpe65 –/– mice partially rescued the blindness phenotype. More recently, patients with RPE65-mediated LCA received subretinal injections of a similar RPE65-containing AAV. Encouragingly, these blind patients partially recovered visual function with expression of wild-type Rpe65 in their RPE. The 11-cis-ROL synthesized by Rpe65 binds to cellular retinaldehyde-binding protein (CRALBP) in RPE cells. CRALBP also binds 11-cis-RAL. Mutations in the gene for CRALBP (RLBP1) cause several inherited retinal dystrophies including recessive retinitis pigmentosa. A newly synthesized molecule of 11-cis-ROL has two potential fates. As discussed below, it can be oxidized to 11-cis-RAL for use as visual chromophore. Alternatively, it can be esterified by LRAT to form an 11-cis-RE. 11-cis-REs represent a storage form of preisomerized chromophore precursor. Hydrolysis of 11-cis-REs is catalyzed by 11-cis-retinyl ester hydrolase (11-cis-REH) in the plasma membrane of RPE cells. The protein responsible for 11-cis-REH activity in RPE cells has not yet been identified.


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Synthesis of 11-cis-RAL Chromophore

Regulation of the Visual Cycle

The final step in the visual cycle is oxidation of 11-cis-ROL to 11-cis-RAL. This reaction is catalyzed by 11-cis-ROLdehydrogenase type-5 (RDH5), which uses NAD+ as a cofactor. Mice with a knockout mutation in the rdh5 gene show accumulation of 11-cis-ROL and 11-cis-REs in the RPE, and delayed recovery of rod sensitivity following light exposure. 11-cis-RAL is synthesized inrdh5 –/– mice, albeit at a reduced rate, suggesting that RPE cells express at least one other 11-cis-ROL-dehydrogenase. RDH11 catalyzes NADP+-dependent oxidation of 11-cis-ROL to 11-cis-RAL in the RPE. Surprisingly,rdh5 –/–, rdh11 –/– double-knockout mice also synthesize 11-cis-RAL, although more slowly than in rdh5 –/– or rdh11 –/– single-knockout mice, and much more slowly than in wild-type mice. Thus, extensive functional redundancy exists for the oxidation of 11-cis-ROL in RPE cells, similar to the functional redundancy for reduction of all-trans-RAL in photoreceptors. 11-cis-RAL is strongly bound to CRALBP in RPE cells. CRALBP has been shown to interact with a protein complex on the cytoplasmic surface of the apical plasma membrane. From this position, 11-cisRAL is transferred across the plasma membrane to bind IRBP in the IPM. This process may involve a receptor for IRBP on RPE cells.

In the dark, photoreceptors stop releasing all-trans-ROL. Residual all-trans-ROL is esterified by LRAT. The major retinoids present in a dark-adapted eye are all-trans-REs in the RPE and 11-cis-RALs in photoreceptor visual pigments. How does the visual cycle know to stop converting all-trans-REs into 11-cis-RAL chromophore in the dark? One mechanism is the strong inhibition of Rpe65 by its product, 11-cis-ROL. When rhodopsin is fully regenerated and CRALBP is saturated, further synthesis of 11-cis-ROL by Rpe65 is inhibited. A second mode of visual-cycle regulation involves an opsin protein called RPE-retinal G-protein receptor (RGR) opsin, expressed in RPE cells. Within RPE cells, alltrans-REs are stored in two compartments, internal membranes and oil droplets. Rpe65 associates with internal membrane but not lipid droplets. Hence, RPE internal membranes contain a pool of all-trans-REs available as substrate for isomerization, while lipid droplets contain a storage pool of all-trans-REs. This storage pool is potentially much larger than the isomerase pool in membranes. RGR opsin mediates light-dependent transfer of all-transREs from the storage compartment to the membrane compartment for isomerization. In light, where the requirement for visual chromophore is high, RGR opsin stimulates synthesis of 11-cis-ROL by increasing substrate availability to Rpe65. Consistently, mice with a knockout mutation in the rgr gene synthesize less 11-cis-RAL in the light and accumulate all-trans-REs. Mutations in the human RGR gene cause autosomal dominant retinitis pigmentosa.

Regeneration of Rhodopsin or Cone Opsin The final step in the visual cycle is regeneration of a visual pigment from an apo-opsin and 11-cis-RAL. The mechanism whereby 11-cis-RAL is transferred from IRBP in the IPM to apo-opsin in the OS disk is unknown. It may involve an IRBP receptor on the OS plasma membrane, or simple diffusion of the 11-cis-RAL. No retinoid-binding protein has been identified in OS. The interaction of 11-cis-RAL with an apo-opsin involves a two-step process. First, a weak noncovalent complex is formed with 11-cisRAL binding to a hypothesized entrance site on the opsin. Second, the 11-cis-RAL moves into the hydrophobic pocket and forms a Schiff base. This step is virtually irreversible in the case of rhodopsin. Once formed, rhodopsin is extremely quiet, with a spontaneous thermalactivation rate of one isomerization every 2000 years. In contrast to rhodopsin, recombination of 11-cis-RAL with the apo-cone-opsins is less favorable thermodynamically. Unlike rhodopsin, 11-cis-RAL freely dissociates from coneopsins. For example, a dark-adapted red cone contains approximately 10% apo-cone-opsin due to spontaneous dissociation of chromophore. This effect contributes to the higher noise and much lower sensitivity of cones versus rods. It is also explains the tendency of rods to steal visual chromophore from cones when the availability of 11-cisRAL is limited.

See also: Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin.

Further Reading Batten, M. L., Imanishi, Y., Maeda, T., et al. (2004). Lecithin–retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver. Journal of Biological Chemistry 279: 10422–10432. Beharry, S., Zhong, M., and Molday, R. S. (2004). N-retinylidenephosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR). Journal of Biological Chemistry 279: 53972–53979. Cideciyan, A. V., Aleman, T. S., Boye, S. L., et al. (2008). Human gene therapy for rpe65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proceedings of the National Academy of Sciences of the United States of America 105: 15112–15117. Gollapalli, D. R. and Rando, R. R. (2003). All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle. Biochemistry 42: 5809–5818.

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Jin, M., Li, S., Moghrabi, W. N., Sun, H., and Travis, G. H. (2005). Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium. Cell 122: 449–459. Kaschula, C. H., Jin, M. H., Desmond-Smith, N. S., and Travis, G. H. (2006). Acyl coa:retinol acyltransferase (ARAT) activity is present in bovine retinal pigment epithelium. Experimental Eye Research 82: 111–121. Kefalov, V. J., Estevez, M. E., Kono, M., et al. (2005). Breaking the covalent bond – a pigment property that contributes to desensitization in cones. Neuron 46: 879–890. Lamb, T. D. and Pugh, E. N. (2004). Dark adaptation and the retinoid cycle of vision. Progress in Retinal and Eye Research 23: 307–380. Maeda, A., Maeda, T., Imanishi, Y., et al. (2006). Retinol dehydrogenase (RDH12) protects photoreceptors from light-induced degeneration in mice. Journal of Biological Chemistry 281: 37697–37704. Mata, N. L., Weng, J., and Travis, G. H. (2000). Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration. Proceedings of the National

Academy of Sciences of the United States of America 97: 7154–7159. Radu, R. A., Hu, J., Peng, J., et al. (2008). Retinal pigment epitheliumretinal g protein receptor-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells. Journal of Biological Chemistry 283: 19730–19738. Redmond, T. M., Yu, S., Lee, E., et al. (1998). Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nature Genetics 20: 344–351. Travis, G. H., Golczak, M., Moise, A. R., and Palczewski, K. (2007). Diseases caused by defects in the visual cycle: Retinoids as potential therapeutic agents. Annual Review of Pharmacology and Toxicology 47: 469–512. Weng, J., Mata, N. L., Azarian, S. M., et al. (1999). Insights into the function of rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in ABCR knockout mice. Cell 98: 13–23. Winston, A. and Rando, R. R. (1998). Regulation of isomerohydrolase activity in the visual cycle. Biochemistry 37: 2044–2050.

Physiological Anatomy of the Retinal Vasculature S S Hayreh, University of Iowa, Iowa City, IA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Blood flow autoregulation – The property of a tissue or an organ (i.e., retina) to maintain constant blood flow during changes in perfusion pressure. Blood–retinal barrier – The system of occluding cellular junctions in the retinal pigmented epithelium and retinal vascular endothelium that prevents free movement of fluid and macromolecules from the blood into the retina. Cilioretinal artery – A retinal artery which arises from the choriod or the posterior ciliary artery, and is found as a variant in some individuals. Cotton wool spots – White patches on the retina observed on fundus examination and are caused by local obstruction of the tiny arteries supplying that area. Fluorescein fundus angiography – Fundus photographs taken in rapid sequence following injection of the fluorescent dye, fluorescein. This provides important information about blood flow as the dye reaches the retinal and choroidal vasculature.

The retina has a dual blood supply; the retinal vasculature supplies only the inner retinal layers up to the inner part of the inner nuclear layer (Figure 1), while the choroidal vascular bed supplies the outer 130 mm – up to the outer part of the inner nuclear layer, so that retinal vessels supply only 20% of the retina while the choroid supplies 80%. In this article, the discussion is restricted to the retinal component.

Arterial Supply of the Retina The main arterial supply of the retina is by the central retinal artery (CRA). In some eyes, another artery, called the cilioretinal artery, may supply a highly variable part of the retina. Central Retinal Artery The CRA is usually the first branch of the ophthalmic artery, arising as an independent branch or in common

with one of the posterior ciliary arteries (Figure 2). Its course can be divided into three distinct parts: (1) intraorbital (lying below the optic nerve (ON) – (Figure 2)), (2) intravaginal (lying in the space between the ON and its sheath), and (3) intraneural (lying in the ON) (Figure 3). It enters the ON about 10 mm posterior to the eyeball (Figures 2 and 3). A variable number of branches arise from each of its three parts, which anastomose with the surrounding branches from other arteries, mostly in the pial plexus of the ON (Figure 3). At the optic disk, the CRA usually first divides into two and then each of them further divides into its various branches (Figure 4). The lumen of the intraneural part of the CRA is approximately 200 mm. Cilioretinal Artery The cilioretinal artery is either a direct branch of one of the posterior ciliary arteries or arises from the peripapillary choroid and enters the retina by hooking around the Bruch’s membrane at the disk margin – usually on the temporal side (Figures 3 and 5). Based on ophthalmoscopy, the incidence of the occurrence of the cilioretinal artery reported by different authors varies from 6% to 25%. However, fluorescein fundus angiography provides the most reliable data because the cilioretinal artery fills synchronously with the choroidal filling, which usually starts to fill before the retinal circulation (Figure 6(a)). An artery which, on ophthalmoscopy, may look similar to a cilioretinal artery may in fact be an intraneural branch of the CRA emerging at the optic disk – not a true cilioretinal artery. A fluorescein fundus angiographic study of 2000 eyes showed one or more cilioretinal arteries in 32% of the eyes and in both eyes in 15% of persons. There is great variability in size, number, and distribution of the cilioretinal arteries. The area of the retina supplied by the cilioretinal arteries varies markedly, from a tiny region to a large sector of the retina. Eyes where one-fourth to half of the retina is supplied by a cilioretinal artery have been observed (Figure 6(a)). Rarely, the CRA is missing and the entire retina is supplied by the cilioretinal artery (Figure 6(b)). The outer part of the entire retina is always supplied by the posterior ciliary artery. When a cilioretinal artery is present, in the part of the retina supplied by it, the entire thickness of the retina receives its blood supply from the posterior ciliary artery. The blood flow in the retinal vascular bed depends upon the perfusion pressure, which is equal to the difference

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RA

Vitreous (a) (b)

Eyeball

(c) (d) (e) LPCA

(f)

ON PPS

(g)

MPCA

CAR

(h)

OA

Choroid

Col.br. of OA

Intra Canalicular part of ON

Sclera

Figure 1 Light micrograph of the retina, choroid, and sclera. The retinal layers are identified as: (a) nerve fiber layer; (b) ganglion cell layer; (c) inner plexiform layer; (d) inner nuclear layer; (e) outer plexiform layer; (f) outer nuclear layer; (g) rod and cone layer; and (h) retinal pigment epithelial layer. RA, retinal arteriole.

between the retinal arterial and venous pressures. The CRA and cilioretinal artery belong to two arterial systems with different physiological properties. This raises an important physiological issue in eyes with a cilioretinal artery when that eye develops central retinal vein occlusion in which the retinal venous pressure rises suddenly to a high level. The CRA arises directly from the ophthalmic artery and the retinal vascular bed supplied by it has an efficient blood flow autoregulation (see below), so that when there is a fall in perfusion pressure in the retinal arterial bed, caused by a rise in the retinal venous pressure, the autoregulatory mechanism in the central retinal arterial vascular bed kicks in trying to maintain retinal circulation. By contrast, the cilioretinal artery belongs to the choroidal vascular system, which has no autoregulation, so that when the venous pressure rises, there is no corresponding compensatory autoregulatory mechanism. Moreover, the perfusion pressure in the choroidal vascular bed normally is lower than that in the CRA, and there is no corresponding rise of pressure in the choroidal venous bed. In view of all these factors, the following scenario occurs in an eye with cilioretinal artery developing central retinal vein occlusion: sudden occlusion of the central retinal vein results in a

Figure 2 View from under surface of the human eyeball and optic nerve (ON) showing central artery of the retina (CAR) and its site of penetration into the optic nerve sheath (PPS), medial (MPCA) and lateral (LPCA) posterior ciliary arteries, and ophthalmic artery (OA). Reproduced with permission from Singh (Hayreh), S. and Dass, R. (1960). The central artery of the retina I. Origin and course. British Journal of Ophthalmology 44: 193–212.

marked rise of intraluminal pressure in the entire retinal capillary bed; when that intraluminal pressure rises above the pressure in the cilioretinal artery, the result is a hemodynamic block in the cilioretinal artery, producing cilioretinal artery occlusion (Figure 7). Intraretinal Branches of the CRA Each of the two main branches (superior and inferior) of the CRA at the optic disk usually divides into temporal and nasal branches, which supply the four quadrants of the retina (Figure 4); however, there is marked variation in their vascular pattern. In the retina, the arrangement of the branches and their subdivisions is highly variable, so much so that each eye has a different pattern (Figures 4 and 7). It has been suggested that the pattern could be used for personal identification like a finger print. Usually, there is a dichotomous or right-angle branching pattern. The various branches, by multiple divisions, finally end in terminal or precapillary arterioles, which are usually not visible on ophthalmoscopy. Terminal arterioles play an important role in the regulation of retinal blood flow by constriction or dilatation.


C R S

Retinal vein Retinal artery

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Col. Br.

PCA D

A Pia

ON

OD PR LC

SAS

Cilioretinal artery

PCA

CRA

CRV

Figure 3 Schematic representation of blood supply of the optic nerve. A, Arachnoid; C, choroid; CRA, central retinal artery; Col. Br., collateral branches; CRV, central retinal vein; D, dura; LC, lamina cribrosa; OD, optic disc; ON, optic nerve; PCA, posterior ciliary artery; PR, prelaminar region; R, retina; S sclera; SAS, subarachnoid space. Modified with permission from Hayreh, S. S. (1974). Anatomy and physiology of the optic nerve head. Transactions of the American Academy of Ophthalmology and Otolaryngology 78: OP240–OP254.

There has been a controversy about the true nature of the arteries in the retina. According to some accounts these are small arteries. Others, however, consider them as arterioles after the first branching in the retina because they possess the following anatomic properties, typically seen in arterioles: (1) the widest part of the lumen of the retinal arterioles is near the optic disk and there its diameter is about 100 mm, which is typically the diameter of an arteriole; and (2) unlike arteries, they possess neither an internal elastic lamina nor a continuous muscular coat. This differentiation from the arteries is important in understanding their pathological involvement in some diseases, such as giant cell arteritis. In the retina, there are no interarterial or arteriovenous anastomoses, so that the retinal vascular bed is an end-arterial system. These intraretinal arterial branches mainly lie in the nerve fiber and ganglion cell layer, usually under the internal limiting membrane (Figure 1); however, at the arteriovenous crossing they may extend down to the inner nuclear layer.

Figure 4 Normal human fundus – left eye.

Retinal Capillary Bed Each terminal arteriole gives out a plexus of 10–20 interconnected capillaries (Figure 8). Capillaries lie between the feeding arterioles and venules (Figure 8). Around the retinal arteries, there is a capillary-free zone (Figure 8). The retinal capillaries are arranged in two layers (Figure 9): (1) a superficial layer in the ganglion cell and nerve fiber layers, and (2) a deeper layer in the inner nuclear layer which is denser and more complex than the superficial layer. However, in the posterior retina, there may be three layers in the peripapillary region and there is only one layer in the perifoveal region. Furthermore, in the peripheral retina the deep layer disappears and only the superficial layer is left, with a wider network. At the extreme periphery of the retina, there is an avascular zone about 1.5 mm width.

Figure 5 Ophthalmoscopic appearance of right eye with central retinal artery occlusion (pale retina) and a normal cilioretinal artery in the area of normal retina (arrow).

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Figure 7 Fundus photograph of right eye with nonischemic central retinal vein occlusion associated with cilioretinal artery occlusion (arrow).

Figure 6 Fluorescein fundus angiograms of (a) right and (b) left eyes of a person. (a) The right eye has one large cilioretinal artery (one asterisk) supplying the superior one-third of the retina that starts to fill before the central retinal artery (two asterisks) which supplies the rest of the retina. (b) The left eye has two large branching cilioretinal arteries (white) – the one above branches in the upper half and the other below branches in the lower half. This eye lacks a central retinal artery.

In addition to the retinal capillary bed described above, there is a distinct retinal capillary bed called the radial peripapillary capillaries, which were first described in 1940 (Figures 9 and 10). They have the following special characteristics compared to other retinal capillaries: 1. They are long, straight capillaries, measuring several hundred microns to several millimeters. 2. They form the most superficial layer (Figure 9) lying among the superficial nerve fibers, along the superior and inferior temporal arcades of retinal vessels and the peripapillary region (Figure 10). 3. They rarely anastomose with one another.

Figure 8 Fluorescein fundus angiogram showing retinal vessels and capillary network. A, retinal arteriole; V, retinal vein.

4. They arise from the peripapillary retinal arterioles lying deeper in the retina, and drain into retinal venules or veins on the optic disk (Figure 3). Because of these characteristics, the radial peripapillary capillaries assume importance in the development of several lesions. For example, cotton wool spots are often located in the distribution of the radial peripapillary capillaries, which indicates that the latter may play a role in the pathogenesis of cotton wool spots. In addition, in chronic optic disk edema these capillaries become dilated and develop microaneurysms and hemorrhages.


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Figure 9 Schematic representation of two layers of the retinal capillaries and radial peripapillary capillaries (RPC). Reproduced with permission from Henkind, P. (1969). Microcirculation of peripapillary retina. Transactions of the American Academy of Ophthalmology and Otolaryngology 73: 890–897.

Figure 11 A cast of retinal capillaries in the macular region of a monkey showing the foveal avascular zone in the center. Courtesy of Professor Koichi Shimizu.

tight-cell junctions, which constitute a blood–retinal barrier (see below). In addition to the endothelial cells, there are also pericytes which form a discontinuous layer within the basement membrane of the capillaries. They have a contractile property, by virtue of which they may play a role in regulating blood flow in the capillaries and autoregulation of blood flow (see below). Pericytes are lost preferentially in diabetes so that they may have a role in diabetic retinopathy; it is suggested that diabetic pericyte loss is the result of their migration. Migration of pericytes is also involved in the regulation of angiogenesis.

Retinal Venous Drainage Figure 10 Schematic representation of radial peripapillary capillaries. Site of foveola (X). Reproduced with permission from Henkind, P. (1967). Radial peripapillary capillaries of the retina: I. Anatomy: Human and comparative. British Journal of Ophthalmology 51: 115–123.

In the macular region, the capillaries are supplied by arterioles arising from the superior and inferior temporal arteries (Figure 4). Their thickness decreases toward the center of the macula where they are arranged in a single layer. The capillaries are absent in the foveal region, with a capillary-free zone of about 400–500 mm in diameter (Figure 11). The wall of the retinal capillaries consists of endothelial cells, pericytes, and basement membrane. Their diameter varies from 3.5 to 6 mm. The endothelial cells have

The postcapillary venules drain the blood from the capillaries but, occasionally, capillaries may join a major vein directly. The terminal arterioles and postcapillary venules are situated in an alternating pattern, with the capillary bed in between the two (Figure 8). The postcapillary venules drain into bigger venules and finally into the branch retinal veins. The lumen of the major branch retinal veins, just before they join to form the central retinal vein, is about 200 mm. In the central part of the retina, the branch retinal veins and arteries usually run in close association and at places cross one another (Figures 4, 5, and 7). On the other hand, in the peripheral retina, the veins do not follow the course of the arteries. Various retinal arteries and veins in the retina cross each other at arteriovenous crossings (Figures 4, 5, and 7). In a study of 189 normal eyes, at the sites of arteriovenous crossing, the artery crossed over the vein in 68% and was the reverse

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in the remainder. However, in eyes with branch retinal vein occlusion, the artery crossed over the vein at the site of occlusion in 98% of the cases, indicating that pattern of arteriovenous crossing plays a role in the development of branch retinal vein occlusion. At the site of arteriovenous crossing, the artery and vein share a common fibrous coat and are separated by only a thin endothelial lining and basement membrane. The superior branch veins usually join to form a superior trunk and the inferior branch veins, an inferior trunk. These superior and inferior trunks join on the optic disk to form the central retinal vein (Figure 3). However, in 20% of eyes, the superior and inferior trunks do not join together at the disk but enter the optic disk as two separate trunks; this represents a congenital anomaly (Figure 12). During the third month of intrauterine life, there are always two trunks of the central retinal vein in the ON, one on either side of the CRA (Figure 13), and one of the two trunks usually disappears before birth; however, in 20% of eyes, a dual-trunked central retinal vein persists into adult life. In such eyes, only one of the two trunks may develop occlusion in the ON, resulting in development of the clinical entity called hemi-central retinal vein occlusion (Figure 12). The central retinal vein travels in the ON temporal to the artery, where the central retinal vein and artery lie in the center of the ON, surrounded by a fibrous tissue envelope (Figure 14). During its intraneural course, the vein receives many tributaries (Figure 3). The central retinal vein exits the ON and its sheath (Figure 15), and finally drains into either the superior ophthalmic vein or directly into the cavernous sinus.

Figure 12 Fundus photograph of an eye with inferior hemicentral retinal vein occlusion, involving the lower trunk of the central retinal vein. Two trunks (arrows) of the central retinal vein enter the optic disk separately above and below.

Nerve Supply The intraorbital and intraneural portions of the CRA have an adrenergic nerve supply from a sympathetic nerve called the nerve of Tiedemann (Figure 16); however, the retinal branches of the CRA have no adrenergic nerve supply. Therefore, there is no autonomic innervation of the retinal vascular bed.

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Figure 13 Schematic representation of two trunks of the central retinal vein in the anterior part of the optic nerve. A, Arachnoid; C, choroid; CRA, central retinal artery; Col. Br., collateral branches; CRV, central retinal vein; D, dura; LC, lamina cribrosa; OD, optic disc; ON, optic nerve; PCA, posterior ciliary artery; PR, prelaminar region; R, retina; S, sclera; SAS, subarachnoid space.

Figure 14 Histological sections (Masson’s trichrome staining) showing the central retinal vessels and surrounding fibrous tissue envelope, as seen in a transverse section of the central part of the retrolaminar region of the optic nerve, in a normal rhesus monkey (above) and in a rhesus monkey with experimental arterial hypertension, atherosclerosis and glaucoma (below). CRA ¼Central retinal artery, CRV ¼ central retinal vein; FTE ¼ fibrous tissue envelope.


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Figure 15 View from under surface of optic nerve in a rhesus monkey showing the intraorbital part of the central retinal vessels and their site of penetration into the sheath of the optic nerve. CRA, central retinal artery; CRV, central retinal vein; ON, optic nerve; SOV, superior ophthalmic vein. Reproduced with permission from Hayreh, S. S. (1965). Occlusion of the central retinal vessels. British Journal of Ophthalmology 49: 626–645.

The retina has two types of blood–retinal barriers. Inner blood–retinal barrier. This lies in the retinal vessels. It is produced by the tight cell junctions between the endothelial cells of the vessels (due to the presence of extensive zonulae occludentes). The tight interendothelial cell junctions block movement of macromolecules from the lumen toward the interstitial space. Pericytes, Mu¨ller cells, and astrocytes also contribute to the proper functioning of this barrier. Outer blood–retinal barrier. Tight cell junctions between the retinal pigment epithelial cells (Figure 1) also produce a blood–retinal barrier, preventing the leakage of fluid from the choroid into the retina. This barrier breaks down when the retinal pigment epithelial cells are destroyed or subjected to ischemia, as in hypertensive choroidopathy. The blood–retinal barrier plays an important role in the regulation of the microenvironment in the retina. An intact blood–retinal barrier is essential for maintaining retinal structure and function. Breakdown of this barrier results in increased vascular permeability of the capillaries, which causes retinal edema, as seen in a variety of retinopathies. Breakdown of the inner blood–retinal barrier may be caused by acute distension of the vessel walls, ischemia, chemical influences, defects in the endothelial cells, or failure of the active transport system. The retinal tissue itself has no barrier in its stroma, therefore fluid may diffuse from one part to the adjacent areas.

Autoregulation of Retinal Blood Flow

Figure 16 Light micrograph of a longitudinal section of optic nerve of a rhesus monkey, showing central retinal artery (CRA) in the center of the nerve and nerve of Tiedemann (arrow) running parallel with the wall of the central retinal artery. (Gros-Schultze’s stain). Reproduced with permission from Hayreh, S. S., Vrabec, Fr. (1966). The structure of the head of the optic nerve in rhesus monkey. American Journal of Ophthalmology 62: 136–150.

The object of blood flow autoregulation in a tissue is to maintain relatively constant blood flow during changes in perfusion pressure. This is an important mechanism to regulate blood flow. The retinal circulation has efficient autoregulation. The exact mechanism and site of autoregulation are still unclear except that it most probably operates by altering the vascular resistance. It is generally considered as a feature of the terminal arterioles; so with the rise or fall of perfusion pressure beyond normal levels, the terminal arterioles constrict or dilate, respectively, to regulate the vascular resistance and thereby the blood flow. Recent studies have suggested that pericytes in the retinal capillaries play a role in autoregulation as well because of their contractile property. The metabolic needs of the tissue also regulate the autoregulation. Autoregulation works within a critical range of perfusion pressure, and it breaks down with any rise or fall of the perfusion pressure beyond the critical autoregulatory range. The vascular endothelium plays an active role in the vasomotor function of both macro- and microvasculatures,

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including maintenance of vascular tone and regulation of blood flow. Recent studies suggest that vascular-endothelial-derived vasoactive agents (e.g., endothelin-1, thromboxane A2, and prostaglandin H2 – vasoconstrictors; and nitric oxide – a vasodilator) profoundly modulate local vascular tone and, thereby, may also play a role in autoregulation. Mechanical stretching and increases in arteriolar transmural pressure induce the endothelial cells to release contracting factors affecting the tone of arteriolar smooth muscle cells and pericytes. Therefore, damage to vascular endothelium (as in arteriosclerosis, atherosclerosis, hypercholesterolemia, aging, diabetes mellitus, ischemia, and possibly from other causes) may be associated with abnormalities in the production of endothelial vasoactive agents, and consequent autoregulation abnormalities. See also: Blood–Retinal Barrier; Breakdown of the Blood–Retinal Barrier; Breakdown of the RPE Blood– Retinal Barrier; Central Retinal Vein Occlusion; Pathological Retinal Angiogenesis.

Further Reading Anderson, D. R. (1996). Glaucoma, capillaries and pericytes 1. Blood flow regulation. Ophthalmologica 210: 257–262. Cunha-Vaz, J. G. (1976). The blood–retinal barriers. Documenta Ophthalmologica 41: 287–327. Duke-Elder, S. and Wybar, K. C. (1961). Anatomy of the visual system. In: Duke-Elder, S. (ed.) System of Ophthalmology vol. 2, pp. 363–382. London: Kimpton.

Haefliger, I. O., Meyer, P., Flammer, J., and Lu¨scher, T. F. (1994). The vascular endothelium as a regulator of the ocular circulation: A new concept in ophthalmology? Survey of Ophthalmology 39: 123–132. Hayreh, S. S. (1963). The cilio-retinal arteries. British Journal of Ophthalmology 47: 71–89. Hayreh, S. S. and Hayreh, M. S. (1980). Hemi-central retinal vein occlusion. Pathogenesis, clinical features, and natural history. Archives of Ophthalmology 98: 1600–1609. Hayreh, S. S., Fraterrigo, L., and Jonas, J. (2008). Central retinal vein occlusion associated with cilioretinal artery occlusion. Retina 28: 581–594. Henkind, P. (1967). Radial peripapillary capillaries of the retina: I. Anatomy: Human and comparative. British Journal of Ophthalmology 51: 115–123. Henkind, P. (1969). Microcirculation of peripapillary retina. Transactions of the American Academy of Ophthalmology and Otolaryngology 73: 890–897. Justice, J. Jr. and Lehmann, R. P. (1976). Cilioretinal arteries. A study based on review of stereo fundus photographs and fluorescein angiographic findings. Archives of Ophthalmology 94: 1355–1358. Kaur, C., Foulds, W. S., and Ling, E. A. (2008). Blood–retinal barrier in hypoxic ischaemic conditions: Basic concepts, clinical features and management. Progress in Retinal and Eye Research 27(6): 622–647. Pournaras, C. J., Rungger-Bra¨ndle, E., Riva, C. E., Hardarso, S. H., and Stefansson, E. (2008). Regulation of retinal blood flow in health and disease. Progress in Retinal and Eye Research 27: 284–330. Singh (Hayreh), S. and Dass, R. (1960). The central artery of the retina I. Origin and course. British Journal of Ophthalmology 44: 193–212. Singh (Hayreh), S. and Dass, R. (1960). The central artery of the retina II. Distribution and anastomoses. British Journal of Ophthalmology 44: 280–299. Weinberg, D., Dodwell, D. G., and Fern, S. A. (1990). Anatomy of arteriovenous crossings in branch retinal vein occlusion. American Journal of Ophthalmology 109: 298–302. Wise, G. N., Dollery, C. T., and Henkind, P. (1971). The Retinal Circulation, pp. 20–54. New York: Harper and Row.

The Physiology of Photoreceptor Synapses and Other Ribbon Synapses W B Thoreson, University of Nebraska Medical Center, Omaha, NE, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Ca2þ microdomains – Local submembrane regions of elevated intracellular Ca2þ caused by the influx of Ca2þ through nearby Ca2þ channels. Calcium-induced calcium release (CICR) – Release into the cytoplasm of Ca2þ ions stored in the endoplasmic reticulum that is triggered by the Ca2þ-dependent activation of ryanodine receptors. ERG b-wave – The electroretinogram (ERG) is a massed electrical response of the retina to light that can be recorded by electrodes on the surface of the cornea. ON bipolar cells provide the source of the b-wave of the ERG. A selective reduction in the b-wave indicates a reduction in signaling between photoreceptors and ON bipolar cells. L-type calcium currents – Currents from highvoltage-activated CaV1.1 (alpha 1S), CaV1.2 (alpha 1C), CaV1.3 (alpha 1D), or CaV1.4 (alpha 1F) calcium channels which show sustained activation and can be selectively blocked by dihydropyridine antagonists. OFF bipolar cells – Second-order retinal bipolar cells which exhibit a hyperpolarizing response to light mediated by non-NMDA ionotropic glutamate receptors. ON bipolar cells – Second-order retinal bipolar cell subtypes which exhibit a depolarizing response to light mediated by mGluR6 metabotropic glutamate receptors. Readily releasable pool – A pool of synaptic vesicles primed for rapid release by elevation of intracellular calcium. SNARE proteins – SNAP and NSF attachment receptors are a family of proteins participating in vesicle fusion. They can be subdivided into vesicle SNAREs (v-SNAREs), which attach to vesicles, and target SNAREs (t-SNAREs), which associate with the plasma membrane. Synaptic ribbon – An electron dense presynaptic structure that tethers synaptic vesicles in nerve terminals of sensory neurons including photoreceptors, retinal bipolar cells, hair cells, pinealocytes, and electroreceptors. Total internal reflectance (TIRF) microscopy – By taking advantage of the subwavelength evanescent

field of light created by reflections at the interface between a cell and coverslip, TIRF microscopy can be used to visualize subwavelength structures such as synaptic vesicles.

Anatomy of the Ribbon Synapse Structures of the electron dense ribbons found at the synapses of retinal photoreceptors and bipolar cells differ depending on cell type. In cross section, photoreceptor ribbons appear as 35 nm thick bars, but in three dimensions they form flat, ribbon-like structures. Mammalian rods have one to two ribbons that can be up to 2 mm in length and extend up to 1 mm into the cytoplasm. Each wraps around the synaptic ridge to form crescent or horseshoe shapes. The ribbons in mammalian cone are smaller, typically less than 1 mm long and extend only a few hundred nanometers into the cytoplasm. They are shaped like surfboards, and typically a dozen or more ribbons are in each cone terminal. Sitting just below the photoreceptor ribbon is a trough-like arciform density. Ribbons in bipolar cells are planar, like those in rods, but they are smaller than both rod and cone ribbons. The focus of this review is on ribbon synapses in retina; however, ribbon synapses are not unique to retina. They are present in other sensory neurons, including pinealocytes, electroreceptors in the lateral line organ of fishes, and hair cells of the cochlea and vestibular apparatus. Again, the ribbons in the synapses of these cells have different structures depending on cell type. For example, hair cell ribbons are small spheres. The ribbon synapses of photoreceptor terminals contain many more synaptic vesicles than conventional synapses. Conventional synapses have 10–100 vesicles near each presynaptic density compared to the synaptic terminal of a lizard cone, which contains 170 000 vesicles or 7000 vesicles per ribbon. Furthermore, 85% of the vesicles at ribbon synapses are freely mobile and readily participate in release compared to 20% of vesicles at conventional synapses. The expanded mobility of vesicles at ribbon synapses may be due to the absence of synapsins which have been proposed to tether synaptic vesicles at conventional synapses. The small subset of vesicles in photoreceptor terminals that are tethered to synaptic ribbons are attached by fine filaments (Figure 1).

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Figure 1 Ribbon-style active zones (arrows) in a salamander rod photoreceptor. A tangential section through the ribbon at right shows the hexagonal packing of vesicles on the ribbon (arrowheads). Scale ¼ 200 nm. Adapted from Thoreson, W. B., Rabl, K., Townes-Anderson, E., and Heidelberger, R. (2004) A highly Ca2þ-sensitive pool of vesicles contributes to linearity at the rod photoreceptor ribbon synapse. Neuron 42: 595–605, with permission from Elsevier.

Vesicle Pools and Vesicular Release at Synaptic Ribbons Newly tethered vesicles can be found throughout the ribbon, indicating that vesicles either freely enter the ribbon at any position or redistribute rapidly about the ribbon face after attachment. However, once attached to the ribbon, vesicles exit only from the base. Vesicles tethered on the bottom first to third rows of the ribbon contact the plasma membrane along the synaptic ridge. These vesicles constitute a pool that can be released rapidly in response to increased Ca2þ levels. In bipolar cells, the fastest component of vesicular release equals the number of vesicles tethered along the bottom row of the ribbon, and maintained depolarization stimulates release of the total number of vesicles lining the entire ribbon. Similarly, the total releasable pool in rod photoreceptors equals the number of vesicles tethered to the entire ribbon (700 vesicles in amphibian rods), and there is an ultrafast component similar to the number of vesicles tethered at the ribbon base (30 vesicles). Because of their smaller size, cone ribbons have a smaller releasable pool than rod ribbons. In contrast with bipolar and photoreceptor cells, the ultrafast release component in hair cells exceeds the number of vesicles lining the bottom row by nearly 10-fold, and the total releasable pool exceeds the number of vesicles lining the ribbon six- to eightfold. The large ultrafast or rapidly releasable pool could be explained by compound fusion between adjacent vesicles as discussed later. The large size of the total pool suggests that vesicles released from the ribbon are replenished rapidly from the surrounding cytoplasm. Vesicular transmitter release clearly occurs at the ribbons in ribbon synapses, but the ribbons may not be the only site of transmitter release. Photoreceptors also

contact bipolar cell dendrites at flat or basal junctions identified by pre- and postsynaptic membrane densities. In mammalian retina, the basal junctions of cones only contact OFF-type bipolar cells and the dendrites of many OFF-type bipolar cells are not directly apposed to synaptic ribbons. In salamander retinas, the architecture of photoreceptor input to bipolar cells is different, and OFF bipolar cells receive 80% of their contacts from ribbon synapses and only 20% from basal junctions. On the other hand, salamander ON-type bipolar cells receive 20% of their contacts from ribbons synapses and 80% from basal junctions. Basal junctions lack the vesicle clusters which typify conventional synapses; however, the absence in mammals of direct ribbon contacts onto many OFF bipolar cells led to the suggestion that they necessarily receive synaptic input from basal junctions. This idea was tested in experiments that compared synaptic events recorded simultaneously from two OFF bipolar cells – one that contacted photoreceptors only at basal junctions and a neighbor whose dendrites approach the ribbon. These studies showed that glutamate released at a synaptic ribbon can diffuse rapidly to bipolar cell processes at basal junctions. This finding shows that glutamate released at the ribbon can reach basal junctions, although it does not exclude the possibility of additional nonribbon release events. The question of whether nonribbon release events occur in ribbon synapses has been addressed further by using total internal reflectance (TIRF) microscopy to visualize single-vesicle fusion events. Studies of release from bipolar cells indicate that up to 1/3 of fusion events occur at ectopic sites away from the ribbon including much of the release during sustained depolarization. Studies on hair cells from mice with disrupted ribbon anchoring also suggest a role for nonribbon sites in


sustained release by showing that sustained release is unchanged although fast release is diminished. However, in photoreceptors, as discussed later, there is evidence suggesting that sustained release occurs predominately at the ribbon.

Role of the Ribbon in Release The functions of the ribbon in release are not fully understood. It has been widely suggested that the ribbon may operate like a conveyor belt, acting as a molecular motor to accelerate delivery of vesicles to their release sites. Consistent with this possibility is the presence of a kinesin motor protein, KIF3A, at the ribbon. However, release of vesicles attached to the ribbons does not require ATP, although ATP is needed for subsequent replenishment and priming of vesicles. Ribbons are also not necessary for sustaining high-frequency release since it can be observed at conventional central nervous system (CNS) synapses without ribbons. In fact, during sustained release from photoreceptor ribbons, the delivery of vesicles to the base of the ribbon is actually slower than the rate predicted from delivery by simple diffusion. Thus, rather than accelerating release, synaptic ribbons in cones appear to slow the rates of sustained release by constraining the rate of vesicle delivery to the base. By making release more regular, such a mechanism could improve the ability to detect small intensity changes that produce small changes in release rate. Another hypothesis of ribbon function is that it may operate as a vesicle trap. In this scenario, vesicles moving about the terminal by Brownian motion occasionally collide with the ribbon face where they are captured like flies to fly paper and then delivered to the base for release. Ribbons may assist in vesicle priming. The match between releasable pool size and the number of vesicles tethered to rod or bipolar cell ribbons indicates that tethered vesicles are primed for release. Furthermore, members of the RIM family of proteins involved in vesicle priming localize to the ribbon. RIM proteins are synaptic proteins required for normal neurotransmitter release. Another proposed role for the ribbon is to facilitate compound fusion during depolarization by promoting fusion between neighboring vesicles on adjacent rows of the ribbon. Compound fusion could explain the finding in hair cells that many more vesicles fuse rapidly after stimulation than are anchored at the base of the ribbon. The possibility of compound fusion in hair cells is supported by statistical properties of fluctuations in postsynaptic currents and presynaptic exocytotic capacitance changes. There is also ultrastructural and electrophysiological evidence for compound or coordinated fusion of multiple vesicles at bipolar cell synapses.

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Ribbons may perform more than one of these functions. They may capture vesicles, prime them for release, regulate their delivery to release sites, and facilitate compound fusion. To sort out the unique functions of ribbons and ribbon synapse, the biochemistry and electrophysiological properties of ribbon synapse are being studied in detail.

Synaptic Proteins Ribbon synapses contain many of the same proteins as conventional synapses. For example, conventional and ribbon synapses both employ SNARE proteins in vesicle fusion: synaptobrevin (VAMP1 and 2), SNAP-25, and syntaxin. Ribbon synapses also possess Munc 18-1, Munc 13-1, RIM, and rab3A proteins which assist with assembly of the SNARE complex and vesicle priming. RIM1 is distributed across the ribbon, whereas RIM2 localizes to the ribbon base, suggesting that they may have different roles. In addition to many similarities, there are also differences among the proteins at ribbon and conventional synapses. In place of syntaxin-1 found at many conventional synapses, ribbon synapses utilize syntaxin-3b. In place of complexins 1 and 2 that interact with the SNARE complex at many synapses, ribbon synapses possess complexins 3 and 4. Ribbon synapses lack synapsins. The functional consequences of these differences remain to be explained. As in conventional synapses, a rise in intracellular free Ca2þ is required for neurotransmitter release, but the identity of the calcium sensor(s) at ribbon synapses is unsettled. Synaptotagmin I is the principal calcium sensor at most conventional synapses. Antibodies to synaptotagmin I/II label photoreceptor and bipolar cell ribbon synapses in mouse and bovine retina but do not label these synapses in goldfish and salamander retina, which are instead labeled by antibodies to synaptotagmin III. In hair cells, it has been proposed that otoferlin, not synaptotagmin, is the principal calcium sensor for exocytosis. The most conspicuous difference between ribbon and conventional synapses is the presence of the ribbonspecific protein, ribeye. Ribbons are constructed from interdomain interactions between adjoining ribeye molecules. Each bipolar cell ribbon is formed from 4000 ribeye molecules and the 10-fold greater surface area of rod ribbons suggests that they are built from 40 000 ribeye molecules. Ribeye is an alternative transcript of the gene for transcriptional repressor C-terminal-binding protein 2 (CtBP-2) with a unique ribbon-specific A domain and an enzymatic B domain. Interactions between ribeye molecules are regulated by NAD and NADH levels, suggesting a mechanism by which changes in the metabolic state of the terminal could contribute to observed circadian changes in ribbon structure.

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As mentioned above, the kinesin KIF3A is located at the ribbon, but since ATP-driven molecular motors are not needed for vesicle release from the ribbon, the role of KIF3A at the ribbon is unclear. However, kinesin motor proteins interact with plant homologs of CtBP, indicating that the interaction of kinesins with the major structural protein in ribbon synapses is highly conserved. It has also been proposed that KIF3A may assist with circadian changes in ribbon structure. Interactions among synaptic proteins help maintain the structure of the ribbon. Interactions between ribeye and the cytomatrix scaffold protein bassoon anchor ribbons to the active zone. Bassoon also tethers ribbons in hair cells and pinealocytes. Bipolar cells lack bassoon but possess a related protein, piccolo. In photoreceptors, piccolo is located further up the ribbon than bassoon. Interactions between cytoskeletal proteins, membranespanning dystroglycan proteins, and extracellular matrix pikachurin proteins are important for maintaining contacts between photoreceptor ribbon synapses and their postsynaptic targets. Disrupting these interactions leads to reductions in the electroretinogram (ERG) b-wave (indicating a reduction in ON bipolar cell responses) in patients with muscular dystrophy. Interactions between ribeye and Unc119, a protein that is highly expressed in photoreceptors, may help localize Ca2þ channels to the base of the ribbon. Unc 119 can bind to both ribeye and the calcium-binding protein CaBP4. CaBP4 binds in turn to Ca2þ channels in photoreceptor terminals. Mutations in Unc 119 lead to cone/rod degeneration.

ribeye-binding peptides and antibodies to L-type calcium channels co-localize with antibodies to bassoon and ribeye. Properties of single-calcium channels recorded from amphibian rod photoreceptors are similar to those of L-type calcium channels in other tissues. Single-CaV1.4 channels expressed in HEK293 cells showed a tiny singlechannel conductance and extremely low open probability. However, it is unlikely that these properties are retained in vivo since they imply an unrealistically large number of channels per ribbon (>15 000). Photoreceptor calcium currents (ICa) exhibit a sigmoidal voltage dependence (Figure 2). When measured under the same experimental conditions, the voltage dependence of ICa (calcium current) in rods and cones of different species are remarkably similar. Typically, photoreceptor ICa activates above –60 mV and is fully activated around –20 mV. ICa attains about a third of its

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Glutamate release from photoreceptors requires an influx of Ca2þ through Ca2þ channels. However, unlike conventional synapses that use N- and P-type calcium channels, photoreceptors and other ribbon synapses rely on dihydropyridine-sensitive, L-type calcium channels. Ltype calcium channels are classified by their a1 pore forming subunits into CaV1.1-CaV1.4 subtypes. Mutations in CaV1.4 (also known as a1F) cause incomplete congenital stationary night blindness and antibodies to CaV1.4 label mammalian rod terminals, suggesting that CaV1.4 is the principal subtype in rods. Rods and longwavelength sensitive cones also appear to possess CaV1.3 channels but CaV1.3 antibodies do not label short-wavelength-sensitive cones, suggesting that they possess a different channel subtype. Calcium channels cluster beneath the ribbon. Freeze fracture electron micrographs from mammalian cones show clusters of 500 polyhedral transmembrane particles, each with a central dimple, beneath the arciform density of each ribbon. Sites of calcium influx co-localize with

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Figure 2 Influence of light-evoked changes in membrane potential on cone calcium currents. (a) Response of a salamander cone to a bright flash of light. (b) Calcium current averaged from eight salamander cones evoked by a ramp voltage protocol (0.5 mV ms1). By convention, the influx of positively charged Ca2þ ions into cones is shown as negative or inward current. The dark potential of the cone in (a) is denoted by the dashed line and the reduction in ICa caused by the hyperpolarizing light response is shown by the arrow.


peak amplitude at the dark resting potential (ca. –40 mV). The hyperpolarizing light response of a rod or cone photoreceptor diminishes ICa and thereby diminishes Ca2þ-dependent release. The sigmoidal voltage dependence of ICa contributes to response compression at higher intensities. As illustrated in Figure 2, the first 10 mV of membrane hyperpolarization during a cone light response causes a much greater decrease in ICa than the next 10 mV. This diminished responsiveness at more hyperpolarized potentials is sufficiently pronounced at rod synapses that it has been described as response clipping. There is evidence that Ca2þ influx through cGMP-gated cation channels can stimulate synaptic release from cones but the role that these channels play in release under normal physiological conditions is not clear. Photoreceptor ICa shows limited and slowly developing inactivation involving both voltage- and calciumdependent mechanisms. Limited inactivation is important for sustaining synaptic release in darkness when photoreceptors are continuously depolarized. Although the amplitude of ICa declines slowly in darkness, changes in ICa produced by brief changes in illumination mirror the sigmoidal voltage dependence of ICa. Along with pore-forming a1 subunits, calcium channels possess accessory b and a2/d subunits. Knockout of b2 subunits almost completely abolishes both the ERG b-wave and staining for CaV1.4 in the outer plexiform layer indicating that b2 subunits are the predominant subtype at photoreceptor synapses. Mutations in a2/d type 4 subunits lead to disordered ribbons, reduced scotopic b-waves, absent photopic b-waves, and a human cone dystrophy, suggesting that this accessory subunit is associated with calcium channels in photoreceptors, particularly cones. The calcium-binding protein, CaBP4, is closely associated with photoreceptor calcium channels. When heterologously expressed in the presence of CaBP4, the voltage dependence of CaV1.3 and CaV1.4 ICa is shifted to more negative potentials, similar to the voltage dependence of ICa in photoreceptors. By shifting activation to more positive potentials, mutations in CaBP4 reduce the amplitude of ICa in the normal physiological range and thereby reduce synaptic output from photoreceptors. The reduction in rod output accompanying CaBP4 mutations causes congenital stationary night blindness.

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One way to remove Ca2þ from the cytoplasm is to pump it into the endoplasmic reticulum using sarcoand endoplasmic reticulum ATPases (SERCA). SERCA 2A predominates in photoreceptor terminals. The release of calcium from these sequestered stores by calciuminduced calcium release (CICR) can amplify calcium entry through L-type calcium channels. CICR in retina is mediated by a retina-specific variant of the type 2 ryanodine receptor. In rods, CICR amplifies synaptic release and increases the likelihood of the simultaneous fusion of multiple vesicles. CICR also contributes to synaptic release from ribbon synapses in vestibular hair cells. Calcium imaging studies show evidence for CICR in cone cell bodies but it does not appear to contribute to synaptic release. Immunohistochemical studies suggest IP3 receptors, which mediate the release of intracellular Ca2þ in many cell types, may be present in cone terminals. However, there is no physiological evidence for IP3-mediated release of calcium in photoreceptors. Depletion of calcium from intracellular stores triggers the opening of calcium-permeable channels in the plasma membrane to facilitate store refilling. Store-operated calcium entry can influence synaptic release by regulating basal calcium levels in photoreceptor terminals. Calcium can also be removed from the cytoplasm by pumping it out of the cell. Calcium is removed from outer segments by a Na/Ca exchanger whereas extrusion from inner segments and synaptic terminals rely more on plasma membrane calcium ATPases (PMCA). PMCA2 antibodies label photoreceptor terminals and PMCA2 knockout mice show significant reductions in rod-driven responses, suggesting that this subtype is particularly important in regulating calcium levels in rod terminals. Calcium buffering by cytoplasmic proteins provides a much more rapid way to reduce free calcium levels than extrusion. The principle calcium buffers are calbindin, calretinin, and parvalbumin although many signaling proteins (e.g., calmodulin, synaptotagmin, and CaBP4) also bind calcium. There is considerable species variability, but cones typically possess the fast, low mobility buffer calbindin whereas the higher mobility buffers calretinin and parvalbumin are less common in photoreceptors. Supplementing these mechanisms, large calcium increases in rods and cones can be buffered by mitochondrial uptake.

Role of Intracellular Ca2þ in Release

Physiology of Release at Photoreceptor Synapses

Free calcium levels in the photoreceptor synapse are tightly regulated by a variety of mechanisms to maintain synaptic output and prevent calcium overload during the continual influx of Ca2þ in darkness.

Photoreceptors hyperpolarize to light and decreases in light intensity cause photoreceptors to depolarize. Depolarization increases the open probability of Ca2þ channels clustered beneath the ribbon. The opening of Ca2þ

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channels increases [Ca2þ]i, stimulating fusion of vesicles at the base of the ribbon. The calcium sensors in bipolar and hair cells have a low affinity for calcium requiring >10 mM calcium to stimulate exocytosis. Release from bipolar cells and most other CNS neurons exhibits high cooperativity consistent with the binding of as many as five Ca2þ ions required for release. The release mechanism employed by photoreceptors differs from these synapses by showing a much higher affinity for Ca2þ whereby submicromolar calcium levels can stimulate release. This high affinity is consistent with the possible involvement of synaptotagmin III, which has a higher affinity for calcium than synaptotagmin I or II. In addition to higher Ca2þ affinity, release from photoreceptors shows lower cooperativity for Ca2þ binding (N 3). Ca2þ channels are close to the ribbon and thus opening of a channel will expose nearby release sites to high [Ca2þ]. Opening only a few channels is sufficient to stimulate fusion of a vesicle. Increasing the number of active Ca2þ channels increases the number of active Ca2þ microdomains and this in turn increases the number of active release sites. Because the number of active release sites increases linearly with ICa amplitude, there is a linear relationship between ICa and release at hair cell synapses. This mechanism may also contribute to linearity between ICa and release at photoreceptor synapses although linearity at this synapse is also promoted by use of a sensor with a low cooperativity for calcium binding (N 3). Synaptic release at ribbon synapses involves two components: a transient burst of release stimulated by abrupt membrane depolarization and the slower, sustained release that accompanies maintained depolarization. In bipolar cells, as with many other neurons, fast release requires very high levels of Ca2þ whereas slow sustained release is triggered by low Ca2þ levels. The separate control of fast release by low Ca2þ affinity sensors and slow release by high Ca2þ affinity sensors is consistent with the idea that fast and slow release in bipolar and hair cells may occur at ribbon and nonribbon sites, respectively. Unlike bipolar cells, fast and slow release from photoreceptors exhibit the same high affinity for Ca2þ, suggesting that both components of release occur at the same site. Thus, sustained release from photoreceptors appears to be predominately due to continued release of vesicles from the ribbon, albeit at lower rates than those attained during fast transient release. Because of the high affinity for Ca2þ exhibited by the release apparatus in photoreceptors, micromolar levels of Ca2þ present at the base of the ribbon in darkness are sufficient to stimulate fusion of vesicles at the base of the ribbon almost immediately after docking. As a consequence, the base of the cone ribbon is largely devoid of vesicles in darkness. This means that in darkness, calcium channel openings often occur beneath empty release sites. The rate of sustained release in darkness is

therefore not directly controlled by the stochastic opening of individual Ca2þ channels, but by the rate at which vesicles are delivered and readied for release at the base of the ribbon. Release rates decline when photoreceptors hyperpolarize, allowing vesicles to be replenished at release sites along the base of the ribbon. With a sufficiently long and bright flash of light, the entire readily releasable pool of vesicles can be replenished. When the cone depolarizes at light offset, the rapid release of this replenished pool of vesicles can evoke a large off response in second-order neurons. Photoreceptors release vesicles continuously at a rate of 10–20 vesicles per ribbon per second in darkness. Cones can respond to light intensities spanning a 10 000fold range, but this sustained release rate can encode only 10–20 distinguishable levels of steady light if synaptic release exhibits Poisson release statistics. If sustained release is controlled by the rate of vesicle delivery down the ribbon rather than the stochastic openings of individual calcium channels, this will make the rate of sustained release more regular. Regularization allows discrimination of a greater number of light levels than predicted for a Poisson release process. The high rates of release from cones that can be attained at light offset allow for the encoding of up to 100 distinguishable light decrements. This may account for psychophysical results showing a greater sensitivity to decrements than increments of light. Rods exhibit slower release kinetics than cones, roughly matched to the slow kinetics of rod light responses. Rod and cone synapses have similar ribbons, ICa with similar properties, and similarly rapid, high affinity calcium sensors, suggesting that differences in Ca2þ handling and buffering may be responsible for rod/cone differences in release kinetics. The continuous release of vesicles from photoreceptor synapses in darkness is balanced by compensatory endocytosis of vesicles. Photoreceptors rely largely on clathrin-mediated endocytosis whereas bipolar cells and hair cells rely more on bulk retrieval of large endosomes. Visualization of single vesicles at bipolar cell terminals by TIRF microscopy show that the vast majority of vesicles undergo full collapse during fusion indicating that kissand-run retrieval of fully formed vesicles is minimal at this synapse.

Disease-Related Mutations in Synaptic Proteins at the Photoreceptor Synapse Given that all visual information must pass through the photoreceptor synapse, it is not surprising that mutations in synaptic proteins of photoreceptors can produce visual deficits. For example, rod–cone dystrophies can be caused by mutations in Rab3 interacting protein (RIM1),


UNC-119, or Ca2þ channel a2/d subunits. Congenital stationary night blindness can be caused by mutations in the rod CaV1.4 Ca2þ channel or CaBP4. Misregulation of glutamate release by photoreceptors and bipolar cells may also contribute to excitotoxic damage in neurodegenerative diseases of the retina. See also: Cone Photoreceptor Cells: Soma and Synapse; Rod Photoreceptor Cells: Soma and Synapse.

Further Reading Choi, S. Y., Borghuis, B. G., Rea, R., et al. (2005). Encoding light intensity by the cone photoreceptor synapse. Neuron 48: 555–562. Daiger, S. P., Sullivan, L. S., and Browne, S. J. (2009). RetNet – Retinal Information Network. http://www.sph.uth.tmc.edu/retnet (accessed July 2009). DeVries, S. H., Li, W., and Saszik, S. (2006). Parallel processing in two transmitter microenvironments at the cone photoreceptor synapse. Neuron 50: 735–748. Dowling, J. E. (1987). The Retina: An Approachable Part of the Brain. Cambridge, MA: Harvard University Press.

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Heidelberger, R., Thoreson, W. B., and Witkovsky, P. (2005). Synaptic transmission at retinal ribbon synapses. Progress in Retinal and Eye Research 24: 682–720. Jackman, S., Choi, S.-Y., Thoreson, W. B., et al. (2009). Role of the synaptic ribbon in transmitting the cone light response. Nature Neuroscience 12: 303–310. Kolb, H., Fernandez, E., and Nelson, R. (2009). Webvision: The Organization of the Retina and Visual System. http://webvision.med. utah.edu (accessed July 2009). Krizaj, D. and Copenhagen, D. R. (2002). Calcium regulation in photoreceptors. Frontiers in Bioscience 7: 2023–2044. LoGiudice, L. and Matthews, G. (2007). Endocytosis at ribbon synapses. Traffic 8: 1123–1128. Prescott, E. D. and Zenisek, D. (2005). Recent progress towards understanding the synaptic ribbon. Current Opinions in Neurobiology 15: 431–436. Rodieck, R. (1998). The First Steps in Seeing. Sunderland, MA: Sinauer. Sterling, P. and Matthews, G. (2005). Structure and function of ribbon synapses. Trends in Neuroscience 28: 20–29. Thoreson, W. B., Rabl, K., Townes-Anderson, E., and Heidelberger, R. (2004). A highly Ca2þ-sensitive pool of vesicles contributes to linearity at the rod photoreceptor ribbon synapse. Neuron 42: 595–605. tom Dieck, S. and Branstatter, J. H. (2006). Ribbon synapses of the retina. Cell and Tissue Research 326: 339–346.

Polarized-Light Vision in Land and Aquatic Animals T W Cronin, University of Maryland Baltimore County, Baltimore, MD, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Chromophore – As used here, a small molecule that when bound to a protein causes the complex to absorb light at visible or near-visible wavelengths. The chromophore for visual pigments, in which the protein component is opsin, is either 11-cis retinal or a very similar molecule. Circular polarization – As used here, a type of polarization of light in which the electric vector, or e-vector, rotates one full circle for each wavelength traveled by the light, thus describing a circle as seen from the wave front or a helix as seen from the side. Circular polarization can be either right-handed or left-handed, depending on the direction of rotation. Dichroism – The property of a substance to absorb polarized light of one e-vector orientation more strongly than of other orientations, thus transmitting linearly polarized light. Dielectric – Refers to chemical compounds or substances that do not conduct electricity. Water and most biological molecules are dielectric. e-Vector – The electrical vector of an electromagnetic wave. For polarized light, the e-vector orientation is usually taken to be the plane of polarization. Linear polarization – Sometimes called plane polarization. Refers to light in which the e-vectors of the constituent photons are all oriented on the same axis, or in the same plane. Microvillus – A membranous protrusion from a cell surface shaped like a tiny tube, typically only a few cell membrane thicknesses in radius. Polarized light – The light in which the e-vector lies in a plane (for linearly polarized light) or rotates through a full circle once for each wavelength (circularly polarized light). Rayleigh scattering – A type of scattering of electromagnetic energy caused by interactions of the energy with particles much smaller than the wavelength of the energy. Rayleigh scattering produces the blue color of the sky and also produces a celestial polarization pattern by scattering of sunlight. Specular reflection – Reflection as from a mirror, where the reflected ray leaves the surface at the same angle that the incident ray arrived. Specular reflection is typical of shiny surfaces; examples in nature include shiny leaves, insect cuticle, wet skin, or the surface of smooth water.

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Light is made up of streams of photons, the elementary particles that carry electromagnetic energy. Each of these photons can be thought of as a miniature electromagnetic wave, which has a single wavelength related to the energy it carries (the distance the photon travels from one energy maximum to the next, inversely proportional to the photon’s frequency) and a single plane within which the electrical energy vibrates – the polarization angle, properly called the e-vector (for electrical vector) angle. Note that since the energy is electromagnetic, there are both electrical vectors and magnetic vectors present, normal to each other. For consistency, throughout this article reference is made only to the e-vector. Therefore, a beam of light, containing countless photons, is characterized by its intensity (the number of photons delivered per unit time), its spectrum (the distribution of wavelengths of all the photons in the beam), and its polarization (the distribution of the planes of vibration, or e-vector angles, of all the photons in the beam). The most common form of polarization, linear (or plane) polarization, has two descriptors: the overall e-vector angle, which is the mean angle of all planes of vibration of the constituent photons, and the degree of polarization, which is the fraction of energy of all photons vibrating within the plane of the e-vector angle. Of course, in a typical beam consisting of photons of mixed wavelengths, these polarization parameters generally vary with wavelength, creating a polarization spectrum. In this article, only linearly polarized light is discussed unless otherwise noted. Like many vertebrates, humans are not generally aware of light’s polarization properties, but the visual systems of most animals perceive light’s polarization and use this ability to regulate their behavior. To help us understand what visualizing polarization would be like, the polarization properties of light can be analogized to its color properties. The spectrum of light produces the sensation of color, with a perceived hue (the predominant wavelength of constituent photons) and purity or saturation (the overall distribution of wavelengths around that of the hue itself). Hue is therefore analogous to the e-vector (the predominant angle of polarization of constituent photons) and saturation to the degree of polarization (the distribution of angles around this). In fact, polarization fields are often portrayed as images using false colors where angle is coded into hue and degree of polarization into saturation. Such a display can also include the coding of overall brightness as the intensity at each point, to provide a complete description of the polarized-light field.

Polarized-Light Vision in Land and Aquatic Animals

Polarized Light in Nature There are no natural sources of polarized light of known biological significance. Nevertheless, linearly polarized

Transmission

Reflection

Scattering

Figure 1 The three most common ways by which linearly polarized light is created either in nature or in the laboratory. At the top part of the figure, the polarization is produced by transmission through some dichroic material, with its preferential plane of transmission symbolized by the vertically parallel lines. Light emerging from a perfect dichroic material becomes fully linearly polarized. Dichroic polarizers are relatively rare in nature, and account for only a minor fraction of the polarized light observed in natural light fields. The middle part of the figure illustrates polarization by reflection from a smooth, dielectric surface. At a particular angle, known as Brewster’s angle, the reflected light is fully polarized parallel to the surface. Most biological surfaces are dielectric, as is the surface of water, so much light reflected from shiny natural surfaces is highly polarized. The bottom section of the figure illustrates polarization induced by scattering. When the scattering angle is orthogonal to the axis of the ray being scattered, the scattered light is fully polarized at an e-vector angle perpendicular to the plane containing the original ray and the scattered ray.

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light is abundant in natural scenery. Light can become polarized in many ways, but the most important processes in nature are through differential absorption, differential reflection, or differential scattering (Figure 1). Some natural or artificial transparent materials preferentially transmit one e-vector plane while absorbing others, usually because of aligned molecules within the material. This property, known as dichroism, is not particularly common in biological systems, but there are important exceptions, including the inherent dichroism of visual pigment molecules described later. Reflection of light from dielectric surfaces produces polarization parallel to the surface (Figure 1). Therefore, bodies of water and many surfaces in natural scenery reflect horizontally polarized light. Rayleigh scattering from molecules and suspended particles in air produces a well-known pattern of polarization in the sky. Scattering-induced polarization varies with the scattering angle, being greatest (often near 100% polarization) for scattering perpendicular to the axis of the incoming ray (Figure 1). As a result, skylight polarization reaches its maximum in a band that stretches across the sky at 90 to the sun. The axis of the e-vector of the scattered ray is perpendicular to the plane defined by the incoming ray and the scattered ray, such that the band of maximum sky polarization has its e-vectors oriented tangentially to the great circle 90 from the sun’s position. At dawn or dusk, this band stretches vertically across the celestial hemisphere (Figure 2). Since Rayleigh scattering is most effective at short wavelengths, skylight polarization is strongest in the ultraviolet. Scattering from water molecules and very small particles suspended in natural waters also produces polarization (Figure 3), although it rarely reaches the very high degrees of polarization seen in the sky. Light scattering in water is optically different from the processes operating in air, and polarization in

Figure 2 Polarization in the sky at twilight produced by Rayleigh scattering, imaged through a fisheye lens fitted with a linear polarizer with the transmission axis oriented to the right and left. Thus, vertically polarized light is not transmitted to the camera and shows as a dark band in the sky. In these photographs, taken at the same location and not enhanced or retouched in any way, North is to the top and West to the right. (a) Polarization in a clear sky at dusk. Note the clearly visible band of strong polarization passing from North to South through the zenith. (b) Polarization in a partly cloudy sky at dawn. The polarization is still clearly visible, but the presence of clouds depolarizes the skylight.

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Figure 3 Polarization of light underwater produced by scattering from water molecules and suspended particles. (a) An unaltered image of an underwater scene at a depth of about 7 m, showing coral reef and rubble. (b) The same scene shown as a polarization image, with the degree of polarization encoded by brightness. The maximum degree of polarization in this scene is about 50%. Note that the parts of the scene that are fairly near the camera and that appear darkest in the normal photograph are most polarized. This occurs because the water between these dark regions and the camera scatters mostly horizontally polarized light.

water typically reaches its maximum value at blue-green wavelengths. Thus, in the sky and underwater, scattering of incoming light produces partial polarization that varies with solar position and direction of view, and reflection of light from the air–water interface or from shiny surfaces (e.g., leaves, wet surfaces, animal skin, scales, or cuticle) produces strong polarization in geometrically favorable circumstances. If a terrestrial animal has polarized-light vision, the sky presents a reliable pattern useful for navigation, but in contrast, the chaotic and unpredictable pattern of polarized-light reflection produces false, pointillistic images that can mask or taint the true colors and locations of objects. Consequently, as described later, photoreceptors in animals that would normally be sensitive to the polarization of light are sometimes structurally modified to destroy polarization sensitivity. The situation is almost always simpler in water than in air, particularly at depths greater than a few meters. Due to refraction at the air/water interface, illumination from the sun or moon is confined to within 46 of overhead position. The resulting polarization field, while variable to some extent, has horizontally oriented e-vectors much of the time, and the degree of polarization is almost always lower than in air. The pointillistic reflection of polarized light from objects is virtually gone underwater, as the refractive index gradient between water and most natural objects is much lower than in air, such that there is little specular reflection of light (required to produce polarization from dielectric surfaces). The predictable surround, typically low degree of polarization, and minimal polarizedlight reflective noise would seem to make polarization vision in water of little utility, yet many aquatic animals have excellent polarization sensitivity. Currently, it is not

always clear what biological advantages are provided by such visual abilities.

Polarization Sensitivity and Polarization Vision Polarization Responses of Photoreceptor Cells Light is absorbed in visual photoreceptors of all animals by molecules of visual pigment, which consist of a chromophore (derived from vitamin A or a close chemical analog) linked to a protein, termed opsin. Just as each visual pigment molecule has its characteristic absorption spectrum, which ultimately determines the spectral sensitivity of the photoreceptor within which it resides, it also has an inherent polarization sensitivity. This exists because the chromophore itself is dichroic, absorbing preferentially when the e-vector of an incident photon is parallel to the long axis of the molecule. Since the chromophores of visual pigment lie nearly parallel to the membranes of photoreceptors, when light arrives perpendicular to these membranes the incident photons are likely to be polarized parallel to the absorption axes of some of the visual pigment chromophores. The way in which these chromophores are aligned within the photoreceptor cell as a whole determine whether or not the receptor responds differentially to polarized light, and thus whether it has inherent polarization sensitivity (Figure 4). In the rod and cone photoreceptors of vertebrate retinas, photoreceptive membranes are arranged in a series of parallel layers, either in flattened disks (rods) or lamellae formed from folded membrane sheets (cones). Since light generally strikes these layers normal to their surfaces, it encounters visual pigments that are arrayed at


Surface view (as light arrives)

Side view Rod disk

Side view (as light arrives)

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parallel to the axis of the microvillus. The microvilli typically extend out perpendicular to the axis of the receptor cell as a whole, such that light arrives perpendicular to each microvillus. In this orientation, each microvillus preferentially absorbs light polarized parallel to its axis, such that if microvilli are arranged parallel throughout the receptor as a whole, the cell will be polarizationsensitive. Note that this property requires no other cellular specializations, and as a result, almost all microvillar photoreceptor cells have some level of polarization sensitivity.

End view Microvillus

Figure 4 An illustration to show absorption of polarized light by vertebrate rod photoreceptors (left; cone photoreceptors would have similar properties) and by microvillar photoreceptors like those of arthropods or cephalopod mollusks (right). In life, light arrives normal to the flat surfaces of rod disks and encounters randomly oriented chromophores of visual pigment lying within the disk membrane (symbolized by double-headed arrows to indicate the preferred axis of polarization for best absorption). Since the orientation is fully random, there is no preferential absorption of any given e-vector angle. If light were to arrive from the side of the disk, it would encounter chromophores that are restrained to angles near that of the membranes themselves, favoring the absorption of horizontally polarized light. In microvillar photoreceptors (right), light arrives orthogonal to the long axis of each microvillus, and encounters visual pigment chromophores that are oriented roughly parallel to the axis of the microvillus. Thus, the microvillus as a whole preferentially absorbs light polarized parallel to its axis. If light were to impinge on the microvillus from the end, it would encounter chromophores arrayed at all possible angles around the circumference of the microvillus, and no preferred absorption orientation would exist.

all possible orientations (Figure 4, top-left). Consequently, rods and cones rarely have an overall polarization response, even though the individual molecules of visual pigment are dichroic. The situation would be very different if light impinged on rods or cones from the side (i.e., normal to the long axes of their outer segments). It would then meet chromophores lying in the planes of the membrane layers, and all chromophores would preferentially absorb light polarized nearly parallel to the membrane. Note that while the individual chromophores have random arrangements in the membrane’s plane, and thus absorb light from this direction with varying effectiveness (suggested by the variable lengths of the double-headed arrows), they always absorb light polarized in the membrane’s plane most effectively. The photosensitive membranes of photoreceptor cells of arthropods (crustaceans, insects, etc.) and cephalopods (octopus, squid, and cuttlefish) are constructed from bundles of microvilli. In each microvillus, for reasons that are not yet fully understood, the molecules of visual pigments are arranged such that their chromophores are roughly

Polarization sensitivity Having receptor cells that respond differentially to polarized light is only the first requirement for polarization sensitivity at higher levels of neural analysis. For the nervous system to be able to analyze light’s polarization, sets of photoreceptors with different preferred polarization orientations must be compared, typically through opponent processing. This type of analysis is like that of color vision, where sets of photoreceptors with differential spectral sensitivity are compared for color processing. Recall that polarization of light has three attributes: intensity, degree of polarization, and polarization angle. Thus, for full awareness of light’s polarization at a given point in the visual field, independent inputs from three receptor sets must be analyzed. Interestingly, few animals do this; in almost all cases, only two receptor sets with orthogonal microvilli are compared. This is reasonably effective in practice, because natural polarization tends to be predictable, such that if the receptor sets are appropriately oriented, the polarization is well analyzed. Two-channel polarization analysis can be extended to full polarization sensitivity if the receptors are rotated relative to the stimulus, although this has rarely been observed in practice. The animal groups for which the mechanisms of polarization sensitivity are best understood are the insects, the crustaceans, and the cephalopod mollusks (octopus and squid). All of these have microvillar photoreceptors, and in most species the receptors are arranged orthogonally. Crustaceans and insects have compound eyes that are particularly well designed for analyzing polarized light. Each unit of the eye contains a group of photoreceptor cells, such that each unit of the compound eye can potentially serve as an independent polarization detector. For two-axis polarization sensitivity, subsets of receptors in each group have orthogonal microvilli. In insects, these subsets often extend into a central, fused photoreceptor from four sides, with microvilli entering from opposite sides abutting near the center. Viewed from the tip of the receptor group, the overall arrangement ends up having two cell sets with horizontal microvilli and two with vertical microvilli (Figure 5). Crustaceans have a similar system, but here the orthogonal sets of microvilli exist in successive layers, making the entire composite

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Cell #1

Cell #2

Cell #4

Cell #3

Figure 5 A schematic diagram of the structure of typical polarization-sensitive photoreceptors such as would be found in the compound eyes of insects. The receptor is viewed in a section as seen on the axis of the photoreceptor group. In life, these receptors would form a bundle of cells arranged in a circle and together forming a tall cylinder with the microvilli arranged in a smaller cylinder running down its middle. Each cell forms a section of the overall receptor (a single cell in this diagram can represent either one receptor cell or two cells lying side-by-side with parallel microvilli). Note that cells on opposite sides of the receptor extend parallel microvilli toward the junction in the center, and thus have parallel polarization sensitivity. Since two sets of receptors exist, with either horizontally oriented or vertically oriented microvilli, the receptor group as a whole can provide information for two-axis polarization analysis. Receptors of crustaceans are similar to this, but each layer of the joint photoreceptor contains microvilli from only one subset of cells, with either horizontally arrayed or vertically arrayed orientations. Successive layers of the receptor contain microvilli from the other subset of cells, and thus the receptor cylinder has stacks of mutually orthogonal microvillar layers which can contribute to two-axis polarization analysis. Photoreceptors of cephalopods are somewhat different from these, since they are arrayed continuously side-by-side throughout a retina, but each junction of four cells forms a set of microvilli organized like those in the center of the diagrammatic insect photoreceptor illustrated here. Cephalopod receptor cells also form parallel microvilli on the opposite side of each cell. Consequently, each cell contributes to two junctions of microvilli, one on each side of the cell. Again, with two primary axes of orientation of microvilli, separate cells can contribute to two-axis polarization sensitivity.

photoreceptor like a pile of a large number of circular segments, each having microvilli orthogonal to the segments immediately above and below. Finally, the cephalopods (which do not have compound eyes, but instead have a single lens eye structured much like a vertebrate camera eye) arrange their microvillar receptors such that each cell has microvilli on two opposing sides (like a twosided toothbrush). The mosaic of cells forms junctions similar to what is pictured in the center of Figure 5, except that each of the four cells in this figure would form another junction on the opposite side with yet other cells. In all these cases, the cells with parallel microvilli viewing one point in space join to form one polarization channel, and those with microvilli orthogonal to these join for the opponent channel.

Vertebrate polarization sensitivity is more difficult to explain, and it is fair to say that we are still not able to account for it satisfactorily. Nevertheless, there is no doubt at all that some vertebrates sense light’s polarization. Recall that end-on stimulation of rods and cones is unlikely to produce any differential sensitivity to the plane of polarization, because chromophores are randomly oriented for such light (Figure 4). If vertebrate photoreceptor cell outer segments were slanted relative to the axes of impinging rays of light, this would confer some polarization sensitivity. It appears that in at least some fishes, the outer segments of some classes of cones lie on their sides, tilting their lamellae vertically in the retina. If all cones of a given class lay parallel, or were organized into orthogonal classes, this could permit the retina as a whole to achieve an overall polarization sense. There is recent evidence that some rod or cone classes are measurably dichroic to end-on illumination. The origin of this dichroism is unclear, but it could be caused by parallel tilting of the rod disks or cone lamellae. Polarization vision If an animal has polarization sensitivity, it can obviously respond in some way to a polarization stimulus. As described later, these responses are frequently hardwired and inflexible, and the polarization sense that drives them does not correspond to what is normally conceived of as vision, which implies a perception of space, form, and individual objects. The term polarization vision refers to a polarization sense analogous to color vision, whereby animals visualize polarization attributes of features within the overall field of view and use polarization variations to enhance the visibility, contrast, or features of particular objects. In principle, an animal that is capable of polarization vision perceives the visual world as a pattern varying in polarization features among receptive fields. While polarization sensitivity is most useful for orientation or for organizing simple responses, polarization vision offers the potential to direct complex behavior such as predation, camouflage generation or breaking, and signal detection. There is only weak evidence of this ability in some vertebrates, but many species of both arthropods and cephalopods probably use true polarization vision in ways that are discussed later. Disentangling polarization and color sensitivity Many – perhaps most – animals that are sensitive to polarized light also have color vision. This presents both perceptual and sensory-processing challenges, as it is generally not desirable to mix these visual modalities. For example, if a receptor cell that contributes to a perceptual color channel has some residual polarization sensitivity, color appearance will be altered by stimuli that contain polarized light. This is most often a problem for


animals with microvillar photoreceptors due to their inherent polarization bias. The cephalopods have firmly dealt with this issue by discarding color vision entirely – the great majority of octopuses, squids, and cuttlefishes have only a single spectral receptor class in their retinas, restricting vision entirely to the intensity and polarizational domains. Many crustaceans have reached a similar solution, devoting nearly all of their receptors to polarizedlight reception. Some crustacean species, however, separate color and polarization processing, using a single spectral class for polarization analysis while reserving a set of other polarization-insensitive classes for color vision. This solution is used, for example, by stomatopod crustaceans, also known as mantis shrimps. Insects also commonly separate color-sensitive from polarizationsensitive receptors, isolating their polarization receptors to just one part of the visual field and often using only ultraviolet receptors for polarization analysis. Some insect species destroy polarization sensitivity in photoreceptors by twisting the entire receptor group around its long axis. In a few cases, surprisingly, insects unify polarization and color perception in the same receptor cells, interpreting some stimuli by combining these two modalities into a single signal. Some butterfly species, for instance, examine potential ovoposition sites in this way. In vertebrates, however, as with other aspects of polarized-light photoreception, it is unknown how (or even if) polarized-light processing is kept separate from color processing. This could be a difficult problem, as it is thought that some vertebrates use different spectral types of cones to sense different polarization planes, a technique that immediately must mix color and polarization information at the first level of light detection.

The Contributions of Polarized-Light Perception to Behavior Sensing polarized light seems strange to us, but for most animals it is as fundamental to their visual perception as color vision is to humans. Indeed, as will be described shortly, in many animals polarized-light perception plays similar roles to those assumed by color vision, and it can even work together with color vision to improve visual interpretation of stimuli. However, there are many situations where polarized light is used for special purposes unique to this modality. Among these are water surface detection and skylight navigation. Water surfaces reflect horizontally polarized light, as illustrated in Figure 1. This is why sunglasses with polarizing lenses make it easier for fisherman to see fish – the lenses are oriented to block horizontally polarized light, reducing the glare from the water’s surface and clarifying the visibility of objects in the water itself. Many flying insects, including adult water beetles and mayflies, use the

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reflected polarization in the opposite way – their eyes are adapted to respond strongly to large expanses of horizontal polarization below the horizon, which in nature invariably correspond to water surfaces. The insects respond by diving into the water, or by alighting on its surface to ovoposit. This simple response is extremely reliable in nature, but can lead to disastrous consequences for the insects today, when many shiny, horizontal surfaces are man-made. Parking lots, oil ponds, and even the painted surfaces of cars and other manufactured objects induce the same response, which in these cases is frequently lethal. Scattering of sunlight in the clear sky produces a highly reliable pattern of polarization (Figure 2), recognized by the visual systems of many insects (including bees, ants, and crickets) as well as by other arthropods including some spiders and crustaceans. The pattern is used for navigation, as it is a perfect indicator of the current position of the sun, persisting even when the sun is not visible behind an obscuring object or landscape feature, or when the sun is hidden by clouds. Thus, navigation is possible even on quite cloudy (but not wholly overcast) days. Most insects that navigate using skylight polarization devote a small region of the compound eye, called the dorsal rim, to perceiving the pattern, and most require only a small patch of clear sky to orient. Navigation using the location of the sun or skylight polarization patterns is not simple, as the solar position drifts through the sky with changing dynamics throughout the seasons, and insects must be able to compensate for these changes each day as they manage their foraging excursions. Some insects, including dung-foraging scarab beetles, use skylight polarization created by moonlight to navigate during their nocturnal rambles. In an interesting vertebrate example, migrating birds are thought to use skylight patterns of polarization at twilight to calibrate their magnetic compasses. The tasks described so far are analogous to map senses, simple reflexes, or other perceptual abilities that are not strictly visual in the sense that we humans understand it. In other words, these types of abilities do not examine features or objects in the outside world except in very general ways. However, there are animals that actually see patterns of polarization in a fashion that is quite analogous to the way that we perceive the external world – they use polarization vision to recognize objects, to enhance contrast of prey, or to see signals of conspecific animals. Some animals, in fact, can be trained to discriminate objects that we see as identical but that differ in the patterns of polarization that they reflect or transmit. Octopus and mantis shrimps learn such tasks. Near the water’s surface, the skylight polarization pattern penetrates and is therefore available as an orientation cue. Deeper than this, underwater polarization is only rarely usable for navigation (although it can be used to orient vertical migration) because it is frequently weak,

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often oriented near the horizontal plane, and quite variable depending on the background (Figure 3). Nevertheless, the background polarization can be used to enhance the contrast of the visual world and make midwater objects more visible. Squids take advantage of this, making them more effective predators on fishes and transparent planktonic prey against a polarized background than against a depolarized one. Presumably, they use their polarizedlight sense to detect differences in polarization between the prey and the background, making objects of similar overall brightness appear distinguishable. If squids and cuttlefishes can distinguish objects based on polarization, it should not be surprising that they also have body patterns that reflect polarized light, undetectable by many marine animals, and apparently employ these patterns to communicate with each other. The polarization patterns are actively controlled by the animal producing them and can appear and disappear in fractions of seconds. When displayed, the polarization reflections remain highly visible to a conspecific individual even as the signaler changes its posture or moves its arms about. Two other groups of animals, one marine and one terrestrial, are currently known to recognize and respond to polarization signals. Mantis shrimps produce an abundance of signals based on patterns of polarized light reflected from their carapace (Figure 6), and use these signals during mating and aggressive displays. Like the cephalopods, of course, they are marine invertebrates and conduct their diplays underwater. One group of insects, however, uses polarized-light signals in the open air. Many species of tropical butterflies find mates in the diffuse light under the rainforest canopy. Here, the background polarization is relatively weak, and the strong polarization pattern of the sky is rarely visible. Thus, the polarization produced by reflection from scales on butterfly wings can act as an unusually strong, visible signal.

Sensitivity to Circularly Polarized Light This discussion of polarized-light sensitivity would not be complete without mention of a recently discovered visual modality, sensitivity to circularly polarized light. Circular polarization differs from linear polarization, the type discussed exclusively until now, in that the e-vector does not remain within a single plane, but instead rotates around the axis of the beam of light. Circularly polarized light is not common in nature, and its presence cannot be detected with standard polarization-sensing systems. Despite this, one group of animals, the mantis shrimps, perceives circularly polarized light and produces circularly polarized signals by reflection. This ability is particularly unexpected because there is no known source of circular polarization underwater other than signals from other mantis shrimps, so it is difficult to explain how and why the ability originally arose. It is possible that circular polarization sensitivity in these animals first appeared as an accidental epiphenomenon related to the unusual way in which their linear polarization system is assembled, and that this led to the elaboration of signals based on circularly polarized light. See the suggested reading for a more detailed account of this unusual finding.

Summary The ability to perceive and respond to linearly polarized light is widespread among animals, occurring in many vertebrates and invertebrates. Some of these species use polarization for general tasks that do not require precise imaging, such as finding water or navigating using patterns of scattered polarized light in the sky. Others truly see polarized objects and use this imaging ability to detect prey and recognize signals from conspecifics. Our poor understanding of the biology of polarized-light sensitivity

Figure 6 Polarization signals reflected from the shed carapace (or molt) of the stomatopod crustacean, or mantis shrimp, Odontodactylus cultrifer. These patterns of polarization are visualized through a linearly polarizing filter rotated to two orientations at 90 to each other. Signals like these are used during aggressive or mating displays of mantis shrimps. Photograph by T. H. Chiou.


in vertebrates, and the recent discovery of circular polarization sensitivity in mantis shrimps, suggest that there are other aspects to polarized-light sensitivity and to polarization vision that still remain to be revealed. See also: Microvillar and Ciliary Photoreceptors in Molluskan Eyes; The Photoresponse in Squid; Phototransduction: Rhodopsin; Rod and Cone Photoreceptor Cells: Inner and Outer Segments; The Colorful Visual World of Butterflies; Evolution of Opsins.

Further Reading Chiou, T. -H., Kleinlogel, S., Cronin, T. W., et al. (2008). Circular polarisation vision in a stomatopod crustacean. Current Biology 18: 429–434.

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Cronin, T. W., Shashar, N., Caldwell, R. L., et al. (2003). Polarization vision and its role in biological signaling. Integrative and Comparative Biology 43: 549–558. Dacke, M., Doan, T. A., and O’Carroll, D. C. (2001). Polarized light detection in spiders. Journal of Experimental Biology 204: 2481–2490. Hawryshyn, C. W. (1992). Polarization vision in fish. American Scientist 80: 164–175. Horva´th, G. and Varju´, D. (2004). Polarized Light in Animal Vision: Polarization Patterns in Nature. Berlin: Springer. Muheim, R., Phillips, J. B., and Akesson, S. (2006). Polarized light cues underlie compass calibration in migratory songbirds. Science 313: 837–839. Shashar, N., Rutledge, P., and Cronin, T. W. (1996). Polarization vision in cuttlefish: A concealed communication channel? Journal of Experimental Biology 199: 2077–2084. Sweeney, A., Jiggins, C., and Johnsen, S. (2003). Polarized light as a butterfly mating signal. Nature 423: 31–32. Waterman, T. H. (1981). Polarization sensitivity. In: Autrum, H. (ed.) Handbook of Sensory Physiology VII/6B, pp. 281–469. Berlin: Springer. Wehner, R. (2001). Polarization vision – a uniform sensory capacity? Journal of Experimental Biology 204: 2589–2596.

Post-Golgi Trafficking and Ciliary Targeting of Rhodopsin D Deretic, University of New Mexico, Albuquerque, NM, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Cell polarity – The asymmetry in cell shape, protein distributions, and cell functions. Golgi complex – A biosynthetic organelle comprised of a stack of membranous cisternae that are involved in protein modifications, such as processing of N-linked sugars as well as sorting and transport of membrane proteins. Newly synthesized membrane and secretory proteins enter the stack through the cis-Golgi, progress through the medial cisternae and exit at the trans-Golgi. Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) – An essential second messenger as well as lipid regulator of membrane trafficking. Along with its precursor phosphatidylinositol-4-phosphate (PI(4)P), it regulates Arfs and is regulated by them, providing a positive-feedback loop that regulates membrane trafficking. Post-Golgi transport carriers (TCs) – The postGolgi vesicles that carry cargo to the plasma membrane. It is now clear that these carriers are large pleiomorphic structures, rather than small vesicles, as previously believed, thus the term vesicles has been replaced with transport carriers. Small GTPases – The members of the lowmolecular-weight (20–25 kDa) Ras super family of guanosine-triphosphate (GTP)-binding proteins comprised of at least four large families, including the Arfs and the Rabs. Small GTPases function by providing directionality to membrane traffic through the molecular switch whose ON and OFF states are triggered by binding and hydrolysis of GTP. The nucleotide-bound state determines the affinity of interactions with regulatory proteins and the downstream effectors of small GTPases. SNARE proteins – The soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins are major components of the intracellular machinery responsible for targeted membrane delivery. SNAREs were identified as membrane receptors for the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) in the cell-free system that reconstituted intra-Golgi trafficking. SNARE proteins form complexes, which are generally composed of a four helical bundle that bridges opposing membranes and brings them into close proximity to initiate fusion.

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The trans-Golgi network (TGN) – A tubular network in the close proximity to the trans-Golgi cisternae that represents the central sorting station of the cell, where proteins and lipids destined for different subcellular domains are segregated from each other and sorted into post-Golgi TCs.

Introduction Retinal rod photoreceptors are exquisitely complex polarized cells that carry photon detection and visual transduction that are essential as the first step in vision. Following the final cell division of their precursors, photoreceptors attain a level of polarity that is nearly unmatched in other cells of the body. Maintaining this organization throughout the lifetime of the organism is a prerequisite for vision. Proper targeting and retention of the macromolecular complexes involved in the visual transduction cascade are accomplished by the highly coordinated action of protein and lipid regulators that together constitute the membrane trafficking machinery. Specific components of this machinery involved in the directed delivery of rhodopsin and its associated proteins and lipids are only beginning to emerge.

Photoreceptor Polarity Rod photoreceptors are modified neurons with specialized light-sensing organelle, the rod outer segment (ROS). The ROS is filled with membranous disks housing the phototransduction machinery that converts photon absorption by rhodopsin into changes in neurotransmitter release, thus transmitting photosensory information to the visual cortex. The light-sensing machinery is comprised of peripheral and integral membrane proteins of the ROS. It is continuously replenished through ROS disk membrane renewal, followed by its removal through daily shedding and phagocytosis by retinal pigment epithelial (RPE) cells. The ROS disk membrane proteins are embedded in a low-viscosity lipid bilayer milieu comprised of unsaturated long-chain phospholipids highly enriched in omega-3 docosahexaenoic acid (DHA, 22:6(n-3)), which is essential for sensory membrane function and for cell


survival. The exceptionally high content of polyunsaturated DHA phospholipids renders ROS membranes highly susceptible to light and oxidative damage. ROSs initially form from primary cilia, and a short 9+0 (nonmotile) connecting cilium remains in the adult as the only path of communication between the ROS and the photoreceptor rod inner segment (RIS). The RIS houses mitochondria and biosynthetic membranes involved in oxidative metabolism and membrane protein and lipid biosynthesis, respectively. The photoreceptor-connecting cilium corresponds to the transition zone of primary cilia, which is considered a gateway for the admission of specific proteins to this privileged intracellular compartment. The primary cilium is the site of assembly of large molecular complexes involved in intraflagellar transport (IFT). IFT protein 20 (IFT20) subunit links mammalian IFT complex with the microtubule motor, kinesin II. The base of the cilium is a region of particularly high lipid ordering, separating the ciliary membrane from the surrounding plasma membrane due to high cholesterol content and glycosphingolipid products of the phosphatidylinositol 4-phosphate (PI(4)P)- and Arf-dependent effector four-phosphate-adaptor protein 2 (FAPP2). Cholesterol rings have also been reported to surround the photoreceptor connecting cilium. Lipid ordering might be important for the docking of the basal body to the plasma membrane or the extension of the ciliary axoneme, both essential processes in ciliogenesis. The RIS is separated from the nuclear and synaptic domains by adherens junctions (AJs) that comprise a continuous adhesion belt, the outer limiting membrane (OLM). Interestingly, this junctional region lacks tight junctions that normally confine plasma membrane proteins to their respective domains. Nonetheless, rod membrane proteins are strictly confined to their specialized domains and the maintenance of polarity is essential for the cell’s function and survival. The photoreceptor synaptic terminal contains specialized ribbon synapses that are responsible for the tonic release of neurotransmitters, which is interrupted by photon capture. Photoreceptor cytoskeletal networks and molecular motors play a major role in the cell polarity. Microfilaments provide structural support by encircling the RIS beneath the plasma membrane and are anchored at the AJs. Other polar dynamic actin networks and filaments are also dispersed through the cell, most notably in the distal portion of the cilium, at the sites of ROS disk formation where they regulate the growth of nascent disks. Actinbased motility through the cilium is thought to be mediated by myosin VIIa, the product of the Usher syndrome (USH) 1B (Usher1B) gene. An array of microtubules radiates into the RIS from the microtubule-organizing center nucleated by a pair of centrioles located below the cilium. Microtubules generate polar networks that generally determine the position of membrane organelles

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and allow intracellular motility. Heterotrimeric molecular plus end-directed motor kinesin-II and homodimeric KIF17 mediate microtubule-dependent trafficking into the ROS. The absence of KIF3A subunit of kinesin-II causes membrane accumulation in the RIS and cell death. Cytoplasmic dyneins 1 and 2 mediate retrograde trafficking to the centrosome from the RIS and the ROS, respectively.

Photoreceptor Biosynthetic Membrane Trafficking: Endoplasmic Reticulum, Golgi, and Post-Golgi Transport Carriers ROS integral membrane proteins are synthesized on the rough endoplasmic reticulum (ER), pass through the Golgi apparatus, and are then incorporated into post-Golgi vesicles or, in current terminology, rhodopsin transport carriers (RTCs) that bud from the trans-Golgi network (TGN), the central membrane sorting station of the cell, and are transported to a docking site near the base of the cilium. Along with rhodopsin, DHA phospholipids are co-transported on RTCs, which fuse with the specialized domain separating the ciliary membrane from the surrounding RIS plasma membrane, thereby regulating the replenishment of light-sensitive ROS membranes. Rhodopsin represents 90% of the newly synthesized protein in rod photoreceptors, and its biosynthetic pathway is best understood. In the 1980s, the laboratories of David S. Papermaster and Joseph C. Besharse combined electron microscope (EM) immunocytochemistry, autoradiography, and freeze-fracture analysis and demonstrated that newly synthesized rhodopsin is transported vectorially to the base of the cilium on membranous carriers. Membrane biosynthesis was also studied by pulse–chase experiments that established the kinetics of movement of newly synthesized rhodopsin through membrane compartments separated by subcellular fractionation. This methodology was subsequently refined by Deretic and Papermaster to incorporate high-resolution linear sucrose gradients, which separated the low-density post-Golgi carriers from other subcellular organelles, including the Golgi, TGN, plasma membrane, and synaptic vesicles. Successful isolation of post-Golgi RTCs provided not only insight into their molecular composition, but was also the basis for the development of the retinal cell-free assay that reconstitutes RTC budding in vitro. The development of this assay in our laboratory led to the discovery that rhodopsin contains a sorting signal within its five C-terminal amino acids that regulates its incorporation into RTCs as they bud from the TGN. Abundant evidence points to the role of the amino acid sequence valine-xproline-x (VxPx motif) in rhodopsin’s C-terminal domain as a sorting signal into RTCs and delivery to the cilium and the ROS. In our retinal cell-free system a monoclonal antibody, whose antigenic site is within the five C-terminal

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amino acids of rhodopsin, and synthetic peptides corresponding to the C-terminus of rhodopsin inhibit RTC budding from the TGN. Studies in a number of laboratories employing transgenic animals expressing rhodopsin lacking the sorting signal, or rhodopsin–C-terminal fusion proteins carrying autosomal dominant retinitis pigmentosa (adRP) mutations, confirmed that the absence of the correct sorting information results in the targeting to the RIS plasma membrane and the synapse in vivo. Because of the exceptionally high membrane turnover, photoreceptors are vulnerable to mutations that affect membrane trafficking. Among the rhodopsin mutations with the most severe phenotypes are those that alter the rhodopsin C-terminal VxPx targeting motif. Rhodopsin C-terminal mutations cause rapid photoreceptor cell death, retinal degeneration, and blindness in adRP. Within the VxPx targeting motif, V345 and P347 are the primary sites of C-terminal adRP mutations involving single amino acid substitutions. To dissect the sorting machinery that regulates RTC budding, targeting, and fusion, it was essential to identify the resident proteins of this organelle. These studies indicated a succession of binding on RTCs of members of the small guanosine triphosphate (GTP)-binding protein families (G proteins or small GTPases) that are the known regulators of membrane trafficking.

Small GTPases of the Rab and Arf Families and Their Regulators in Rhodopsin Trafficking Rabs Directed delivery of membrane cargo is mediated through vesicular transport regulated by the small GTPases of the Rab and Arf families, which play a central role in organizing intracellular membrane trafficking. All GTPases function by providing a universal molecular switch whose ON and OFF states are triggered by binding and hydrolysis of GTP. Small GTPases are in a dynamic equilibrium between the cytosol and the membranes maintained by the interactions with a number of regulatory proteins, including nucleotide exchange factors (GEFs), GTPaseactivating proteins (GAPs), and their downstream effectors, and the affinity of these interactions is determined by the nucleotide-bound state. The Rab family of small GTPases includes over 60 members that are generally designated numerically (i.e., Rab6, Rab8, and Rab11). Upon GTP binding, activated Rabs recruit a multitude of effectors that organize membrane domains involved in the tethering of membranes to other membranes and to cytoskeletal elements, thus conferring directionality to membrane traffic. Macromolecular complexes organized by Rabs provide a unique identity to membrane microdomains within cellular organelles. Consequently, a change in Rabs,

termed Rab conversion, changes the membrane identity and accompanies cargo progression through intracellular compartments. Rab6 regulates retrograde transport between the Golgi and the ER, but in cells with hypertrophied synthesis of simple membranes it also associates with post-Golgi carriers. In photoreceptor cells, Rab6 is associated with the Golgi, TGN, and RTCs. It was demonstrated in Drosophila that Rab6 regulates rhodopsin trafficking, and that the expression of the GTPase-deficient mutant of Rab6 leads to retinal degeneration. Rab11 has been localized to both the Golgi and the recycling endosomes, which are involved in return of plasma membrane receptors to the cell surface through endocytosis. However, the Golgi-associated function of Rab11 is less well understood. The specific functions of Rab11 in apparently divergent cellular processes are probably based on its ability to interact with different effector molecules that belong to the family of Rab11interacting proteins (FIPs), which are localized in different trafficking pathways. Rab11 is associated with photoreceptor TGN and RTCs where it interacts with FIP3. At the TGN, Rab11 and FIP3 are incorporated into a ciliary targeting complex regulated by the small GTPase Arf4, described below. Sustained presence of Rab11 on RTCs suggests that Rab11 might also interact with the conserved octameric Sec6/8 complex, also known as the exocyst in yeast. The Sec6/8 complex tethers the Rab11/FIP3positive membranes and is involved in tethering RTCs to the RIS plasma membrane. Exocyst complex localizes to the cilia in polarized epithelial cells, and we also find it at the base of the photoreceptor cilium. In Drosophila, interaction of the Sec6/8 complex with Rab11 plays a role in the tethering of membranes carrying rhodopsin. Rab11 may also cooperate with another RTC-associated Rab, Rab8. A handover from Rab11 to Rab8, or Rab conversion, may occur at the base of the cilium to couple the final stages of traffic along the ciliary pathway. Rab8 regulates polarized trafficking in epithelial cells and neurons through its activity on cytoskeleton remodeling necessary for membrane outgrowth and the formation of cellular protrusions. It has recently emerged as a major player in ciliogenesis. In retinal photoreceptors, Rab8 regulates RTCs fusion and ROS biogenesis. It acts at the base of the cilium in conjunction with another small GTPase (Rac1), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), actin and the phosphoinositide- and actinbinding protein moesin. In addition, Sec6/8 complex is also likely to function as one of Rab8 effectors in ciliogenesis. Transgenic Xenopus with photoreceptor-specific expression of GFP-Rab8Q67L dominant-active mutants show normal photoreceptor cell morphology, but cause slow retinal degeneration, whereas GFP-Rab8T22N GTPase-deficient dominant-negative mutants show a defect in membrane tethering and accumulate RTCs in


the vicinity of the cilium, leading to rod cell death and rapid retinal degeneration. Emerging evidence points to Rab8 as a central regulator of the biogenesis of primary cilia, suggesting that its regulation of rhodopsin trafficking may be a part of a broad and more general role for Rab8 in the regulation of ciliogenesis. Mutations that affect regulatory proteins of small GTPases and their interacting proteins have been found to cause X-linked retinitis pigmentosa (RP), autosomal recessive Leber congenital amaurosis (LCA), and choroideremia. Rab escort protein 1 (Rep1), encoded by the choroideremia gene is a subunit of geranylgeranyl transferase, the enzyme that isoprenylates Rab proteins. Rab8interacting proteins, Optineurin and Huntingtin, are also linked to the neurodegenerative diseases primary openangle glaucoma (POAG) and Huntington’s disease, respectively. Rab8, Optineurin, and Huntingtin are collectively involved in the linkage of membrane organelles to the cytoskeleton, suggesting that the breakdown of this linkage may be a common theme in retinal degeneration and in other neurodegenerative diseases. Arfs The Arf family of small GTPases includes three different groups of proteins: the Arfs, Arf-like proteins (Arls), and SARs. Arfs were originally discovered as ADPribosylation factors, but in 2006 the new nomenclature for the human Arf family of GTP-binding proteins, formerly known as ARF, ARL, and SAR proteins, has been established. Arfs are no longer called ADP-ribosylation factors (ARFs), since ADP-ribosylation appears unrelated to their physiological function. Arf family members regulate membrane trafficking, lipid metabolism, organelle morphology, and cytoskeleton dynamics. These functions were elucidated for the abundant class I Golgi Arfs, Arf1 and Arf3, and the plasma membrane-associated Arf6. The loss of a single class I or class II Arf has little effect on membrane trafficking, but the deletion of pairs of Arfs causes distinct defects. This suggests a pair-wise engagement of Arfs and a certain redundancy in their function. Arf function depends on GTP hydrolysis mediated by Arf GAPs, which are essential for coupling the proofreading of cargo incorporation to the budding of membrane carriers and are often incorporated into protein coats. The selection and packaging of sensory receptors and membrane cargo targeted to the primary cilia and ciliaderived sensory organelles are critical to replenish the ciliary membrane, yet it remains poorly understood. Our recent studies have demonstrated that rhodopsin C-terminal VxPx targeting signal binds Arf4 to regulate incorporation of rhodopsin into RTCs at the TGN. The function of the class II Golgi-associated Arfs, Arf4 and Arf5, is least understood, yet the direct and specific binding of the VxPxtargeting motif to Arf4 suggests a distinct role for this

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particular Arf in the generation of RTCs. The targeting VxPx motif binds Arf4 and recruits it to the TGN, leading to assembly of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and an Arf-GAP/ effector ASAP1. The localization of the ciliary targeting complex in photoreceptors is illustrated in Figure 1. ASAP1 catalyzes phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent GTP hydrolysis on Arf4. Transgenic frogs expressing an Arf4 mutant impaired in ASAP1mediated GTP hydrolysis, display dysfunctional rhodopsin trafficking and cytoskeletal and morphological defects, resulting in retinal degeneration. FIP3, which binds Arf4, also forms a ternary complex with Rab11 and ASAP1 and stimulates Arf GAP activity of ASAP1. Emerging evidence points to the role of ASAP1 and FIP3 as a functional module that provides temporally and spatially restricted hydrolysis of GTP bound to Arf4 at the TGN. Since ASAP1 and FIP3 act as homodimers, they may oligomerize to form a protein coat that regulates ciliary targeting, a specialized form of the TGN-to-plasma membrane trafficking. Rhodopsin provides the spatial control for the ciliary targeting module by recruiting Arf4 to the carrier budding sites at the TGN through its VxPx targeting signal. Surprisingly, the VxPx motif is not unique to rhodopsin, but is present in other membrane proteins targeted to primary cilia such as polycystins 1 and 2, and the cyclic nucleotide-gated channel CNGB1b subunit. The VxPx from polycystin-2 also binds Arf4, suggesting that the targeting complex recruited through Arf4 is a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

SNAREs and their Regulators in Rhodopsin Trafficking In addition to GTPases and their effectors, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are major components of the intracellular machinery responsible for targeted membrane delivery. SNAREs are considered to be directly involved in membrane fusion. After the tethering step, SNAREs are activated on the opposing donor and target membranes to form a complex that bridges the two membranes and brings them into close proximity to initiate fusion. Although SNARE pairing alone is not sufficient to determine the specificity of organelle fusion, cognate SNAREs are correctly paired in biological membranes, based on proofreading and polarized distribution leading to their relative enrichment at the appropriate fusion sites. Rabs also function by concentrating and activating SNAREs, accessory proteins and lipids, at the sites of membrane fusion and are thus required for carrier docking and fusion with the target organelle.

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Figure 1 The Arf GAPASAP1 co-localizes with Rab11 on nascent RTCs at the trans-Golgi network (TGN). (a) A confocal optical section (0.7 mm) of frog retina labeled with anti-rhodopsin C-terminal mAb 11D5 (red) and anti-ASAP1 (green). Anti-rhodopsin antibody labels the rod outer segment (ROS) and the Golgi (G) in the rod inner segment (RIS), where ASAP1-positive puncta (yellow, arrows) line up with regular periodicity. ASAP1 is also detected in calycal processes (CP) that evaginate from the RIS and surround the base of the ROS. Nuclei (N) are stained with TO-PRO-3 (blue). (b) A confocal optical section labeled with anti-rhodopsin C-terminal mAb 11D5 (red) and anti-Rab11 (green). Rab11-positive puncta (yellow, arrows) aligned with rhodopsin-laden Golgi. Rab11 is also present on RTCs (arrowheads). Nuclei (N) are stained with TO-PRO-3 (blue). (c) ASAP1 (blue) and Rab11 (red) colocalize in the bud-like profiles at the tips of the trans-Golgi (Rab6, green) (boxed area magnified in (d). (d) Magnified trans-Golgi area from panel C, with ASAP1- and Rab11positive buds (arrows), which likely represent the TGN. (d0 and d00 ). Rab11 (red) and ASAP1 (blue) are shown separately. Scale bar ¼ 3 mm in (a)–(c), 0.7 mm in (d), 1 mm in (d0 ) and (d00 ). Modified from Mazelova, J., Astuto-Gribble, L., Inoue, H., Tam, B. M., Schonteich, E., Prekeris, R., Moritz, O. L., Randazzo, P. A., and Deretic, D. (2009). Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4. EMBO J 28: 183–192.

SNARE complexes are generally composed of a fourhelical bundle bridging opposing membranes and bringing them into close proximity to initiate fusion. Fusion with the plasma membrane requires formation of a complex between syntaxins (Qa SNAREs) and VAMPs (R-SNAREs), each contributing one helix to the fourhelix SNARE bundle, and Qbc SNAREs, either neuronal SNAP-25 or non-neuronal SNAP-23, which provide two helices to the central layer of the core complex. SNAREs are targeted to appropriate membrane domains based on specific sequences. The polarized distribution of Qa SNAREs is likely to contribute additional specificity of membrane targeting by promoting fusion with only certain target membranes. Recent evidence suggests that the local lipid environment, particularly phospholipids enriched in omega-3 and omega-6 fatty acids, also contributes to regulate SNARE function. The membrane fusion event through which RTCs deliver rhodopsin to the cilium is mediated by a SNARE complex. Syntaxin 3 and SNAP-25 are the Q-SNAREs for the fusion of incoming RTCs with the RIS plasma membrane and, therefore, regulators of ROS biogenesis in photoreceptors. The distribution of these SNAREs in photoreceptors is illustrated in Figure 2. Remarkably, omega-3 DHA enhances syntaxin 3 incorporation into SNARE complexes at RTC fusion sites and promotes ciliary membrane expansion and ROS biogenesis. Microtubules direct the restricted distribution of syntaxin 3, consistent with the membrane cytoskeleton playing an essential role in

concentrating RTC fusion regulators around the cilium. Syntaxin 3 is the major partner for SNAP-25 in photoreceptor cells; however, SNAP-25 pairing with syntaxin 1A, or 1B, may regulate a distinct trafficking pathway in the RIS. Interestingly, Syntaxin 3 is also found at the base of mouse ROS, suggesting an additional role for this SNARE in rodent rods.

ROS is a Modified Primary Cilium Almost all cells possess primary cilia that house an array of signal transduction modules. Long underappreciated, the cilium has recently received a great deal of attention due to the ciliary involvement in a wide range of human diseases, including retinal degeneration, polycystic kidney disease (PKD), Bardet–Biedl syndrome (BBS), and neural tube defects. Many cilium disease proteins were detected in the mouse photoreceptor ciliary proteome. Ciliary involvement in a wide range of retinal diseases has come into sharp focus in the past several years, with the molecular mechanism underlying these diseases being rapidly elucidated. Several human syndromes, including Senior–Loken syndrome, Jeune syndrome, and BBS, are also characterized by both cystic kidneys and retinal degeneration, which are often found in combination with skeletal defects or other abnormalities such as obesity, polydactyly, hypogenitalism, and developmental delay that might also be caused by defects in cilia. The organization of the small GTPases,


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Figure 2 SNAP-25 is a photoreceptor RIS plasma membrane (PM) and synaptic SNARE, whereas syntaxin 3 is concentrated in the RIS PM. (a) A confocal optical section (0.7 mm) of frog retina labeled with anti-SNAP-25 N-terminal mAb (green) and anti-syntaxin 3 (STX3, red). Nuclei are stained with TO-PRO-3 (blue). Anti-SNAP-25 (green) outlines the photoreceptor PM. The calycal processes (CP), which are in continuum with the RIS PM, also contain SNAP-25. The ROS, which are visible by DIC, are completely devoid of this SNARE. AJ, adherens junctions that form the outer limiting membrane (OLM, dotted line). The retinal layers are: ONL-outer nuclear, OPL-outer plexiform, INL-inner nuclear. SNAP-25 co-localizes with synaptophysin (SYP, red) in the OPL, which encompasses the synapses of rods and cones with the rod bipolar, cone bipolar, and horizontal cells. Asterisks indicate the protruding RIS of green rods, a minor subpopulation that accounts for 5% of total rods. (b) A confocal optical section labeled with anti-syntaxin 3 (STX3, red), which is highly concentrated in the RIS PM (arrows), but is absent from the ROS, and from the RPE. Syntaxin 3 is also abundant in the OPL. Nuclei are stained with TO-PRO-3 (blue). (c) SNAP-25 and syntaxin 3 co-localize (yellow) in the RIS at the RTC fusion sites in the vicinity of cilia (arrows). Syntaxin 3 is also abundant in the inner (lower) half of the OPL (large bracket), where bipolar and horizontal cells are localized, but not in the outer (upper) half where photoreceptor synapses are localized. (d–f). SNAP-25 (green in (d)) co-localizes with synaptophysin (red in (e)) in the photoreceptor synapses (yellow in (f)). However, SNAP-25 is abundant in the bipolar and horizontal cell processes in the OPL (green in (f)), where synaptophysin is not detected. Scale bar ¼ 8 mm in (a), 10 mm in (b), 7 mm in (c)–(f). Modified from Mazelova, J., Ransom, N., Astuto-Gribble, L., Wilson, M. C., and Deretic, D. (2009). Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid (DHA), controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments. Journal of Cell Science 122: 2003–2013.

SNAREs and their regulators involved in the ciliary targeting of rhodopsin is schematically illustrated in Figure 3. Twelve BBS genes have been identified. A complex composed of seven BBS proteins, the BBSome, localizes to the base of the cilium and is required for ciliogenesis. BBS3, which is not a part of BBSome encodes the Arf family GTPase Arl6. Strikingly, Rabin8, the GDP/GTP exchange factor that activates Rab8, localizes to the basal body and contacts the BBSome. In cultured epithelial cells, activated Rab8 enters the primary cilium and promotes extension of the ciliary membrane. This explains the accumulation of RTCs below the cilium in photoreceptors expressing mutant Rab8. Strikingly, activated Rab8, in its GTP-bound form interacts with another centrosomal/ciliary protein CEP290/BBS14/NPHP6, which is not a part of the BBSome. Thus, BBS may be caused by defects in Rab8-mediated vesicular transport to the cilium. An extraordinary array of retinopathy-associated ciliary proteins includes X-linked RP1, which is localized to the proximal cilium and involved in disk morphogenesis, and RP2, which was recently identified as a GAP for the

Arf family GTPase Arl3 that is involved in kidney and photoreceptor development. A major player in ciliary morphogenesis is retinitis pigmentosa GTPase regulator (RPGR), which is homologous to RCC1, the nucleotide exchange factor for the small GTPase Ran. Mutations in the retina-specific ORF15 isoform of RPGR (RPGR (ORF15)) were found in X-linked RP3, which is associated with 10–20% of RP. RPGR appears to be a part of a ciliary and basal body protein network that, when disrupted, can result in Leber congenital amaurosis, Senior–Loken syndrome, nephronophthisis, or Joubert syndrome. RPGR (ORF15) co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin. RPGRIP1, which is affected in patients with LCA, anchors RPGR to the photoreceptor connecting cilium and participates in disk morphogenesis. RPGR also interacts with calmodulin and nephrocystin-5, a ciliary IQ domain protein, which is mutated in Senior–Loken syndrome, and with the centrosomal/ciliary protein CEP290/BBS14/NPHP6, which is truncated in earlyonset retinal degeneration in the rd16 mouse. In addition,

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Ciliary targeting motif: VxPx Vx P x C

ADRP mutations Rhodopsin

N

Cilium Rhodopsin transport carriers (RTCs)

ROS

Ellipsoid RIS Myoid Adherens Junctions

Cilium Basal body RTCs Mitochondria TGN

Basal body

RIS TGN

Golgi Nucleus

GTpases:

Synapse

Arf4 Rab6 Rab11 Rab8 Effectors: ASAP1 FIP3

(a)

(b)

SNAREs: SNAP-25 Syntaxin 3 VAMP

Figure 3 Post-Golgi trafficking and ciliary targeting of rhodopsin (a) Diagram of the rod photoreceptor cell. Cilium protrudes from the cell body (RIS) and elaborates the ROS filled with membranous disks containing photopigment rhodopsin and associated phototransduction machinery. Following synthesis in the RER, newly synthesized rhodopsin traverses the Golgi and the TGN, localized in the myoid region of the RIS, where it is incorporated into transport carriers (RTCs). RTCs travel from the TGN, though the mitochondria-laden ellipsoid region of the RIS, to the base of the cilium where they fuse with the RIS PM. Adherens junctions separate the RIS from the synapse. Little is known about membrane targeting to the RIS PM and the synapse. (b) Polarized trafficking of post-Golgi RTCs is dependent on the rhodopsin C-terminal VxPx ciliary targeting motif. ADRP mutations in the VxPx motif are indicated. Selected proteins involved in the recognition of the VxPx motif, sorting of rhodopsin into the RTCs and their targeting to the cilium are shown in the enlarged area of the RIS. Rhodopsin C-terminal binds to, and recruits Arf4 to the TGN membrane, leading to assembly of a ciliary targeting complex. This complex is comprised of two small GTPases Arf4 and Rab11, the Rab11/Arf effector FIP3, and an Arf-GAP/effector ASAP1. The small GTPases Rab6 regulates trafficking through the Golgi, whereas Rab8 regulates RTC fusion. Syntaxin 3 and SNAP-25 are a part of the SNARE complex that catalyzes RTC fusion at the base of the cilium.

mutations in the gene encoding the basal body protein RPGRIP1L (RPGRIP-like), a nephrocystin-4 interactor, cause Joubert syndrome. Another ciliary and basal body protein network linked to myosin VIIa is disrupted in human USH, the most frequent cause of combined deafness–blindness. USH is genetically heterogeneous with three clinical types, USH1–3. The scaffold protein harmonin (USH1C) integrates USH1 and USH2 molecules into protein networks. The Usher protein network is organized by the scaffold proteins SANS (USH1G), which provides a linkage to the microtubule transport machinery, and whirlin (USH2D), which anchors USH2Ab and very large G-proteincoupled receptor 1b (VLGR1b). Remarkably, the USH protein network is also a part of the periciliary ridge complex (PRC), a specialized membrane domain for docking and fusion of RTCs in Xenopus photoreceptors. Finally, the Usher protein network is linked to the Crumbs polarity complex in the retina and mutations in Crumbs cause retinitis pigmentosa (RP12).

Conclusions and Summary Numerous diseases arise from defects in proteins that participate in membrane protein trafficking, vectorial transport, and assembly of outer segment membranes. Thus, maintenance of photoreceptor cell polarity is of utmost importance for their health and survival, and ultimately for vision. See also: Genetic Dissection of Invertebrate Phototransduction; The Photoreceptor Outer Segment as a Sensory Cilium; The Physiology of Photoreceptor Synapses and Other Ribbon Synapses; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Retinal Degeneration through the Eye of the Fly; Rod and Cone Photoreceptor Cells: Inner and Outer Segments; Rod and Cone Photoreceptor Cells: Outer Segment Membrane Renewal; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration; Xenopus laevis as a Model for Understanding Retinal Diseases.


Further Reading Cai, H., Reinisch, K., and Ferro-Novick, S. (2007). Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Developmental Cell 12: 671–682. Deretic, D., Schmerl, S., Hargrave, P. A., Arendt, A., and McDowell, J. H. (1998). Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA. Proceedings of the National Academy of Sciences of the United States of America 95: 10620–10625. Deretic, D., Williams, A. H., Ransom, N., et al. (2005). Rhodopsin C-terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ARF4. Proceedings of the National Academy of Sciences of the United States of America 102: 3301–3306. Gillingham, A. K. and Munro, S. (2007). The small G proteins of the Arf family and their regulators. Annual Review of Cell and Developmental Biology 23: 579–611. Green, E. S., Menz, M. D., LaVail, M. M., and Flannery, J. G. (2000). Characterization of rhodopsin mis-sorting and constitutive activation in a transgenic rat model of retinitis pigmentosa. Investigative Ophthalmology and Visual Science 41: 1546–1553. Leroux, M. R. (2007). Taking vesicular transport to the cilium. Cell 129: 1041–1043. Malsam, J., Kreye, S., and Sollner, T. H. (2008). Membrane fusion: SNAREs and regulation. Cellular and Molecular Life Sciences 65: 2814–2832. Mazelova, J., Astuto-Gribble, L., Inoue, H., et al. (2009). Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4. EMBO Journal 28: 183–192. Mazelova, J., Ransom, N., Astuto-Gribble, L., Wilson, M. C., and Deretic, D. (2009). Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid (DHA), controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments. Journal of Cell Science 122: 2003–2013.

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Moritz, O. L., Tam, B. M., Hurd, L. L., et al. (2001). Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods. Molecular Biology of the Cell 12: 2341–2351. Nachury, M. V., Loktev, A. V., Zhang, Q., et al. (2007). A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129: 1201–1213. Papermaster, D. S., Schneider, B. G., and Besharse, J. C. (1985). Vesicular transport of newly synthesized opsin from the Golgi apparatus toward the rod outer segment. Ultrastructural immunocytochemical and autoradiographic evidence in Xenopus retinas. Investigative Ophthalmology and Visual Science 26: 1386–1404. Shi, G., Concepcion, F. A., and Chen, J. (2004). Targeting od visual pigments to rod outer segment in rhodopsin knockout mice. In: Williams, D. S. (ed.) Photoreceptor Cell Biology and Inherited Retinal Degenerations, pp. 93–109. Singapore: World Scientific Publishing. Tam, B. M., Moritz, O. L., Hurd, L. B., and Papermaster, D. S. (2000). Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis. Journal of Cell Biology 151: 1369–1380. Wandinger-Ness, A. and Deretic, D. (2008). Rab8a. UCSD-Nature Molecule Pages. Nature Publishing Group. doi:10.1038/mp. a001997.001901.

Relevant Websites http://www.retina-international.com – Retina International. http://www.signaling-gateway.org – The UCSD-Nature Signaling Gateway. http://www.sph.uth.tmc.edu/RetNet/ – Retinal Information Network. http://webvision.med.utah.edu – WEBVISION: The organization of the retina and visual system.

Primary Photoreceptor Degenerations: Retinitis Pigmentosa M E Pennesi, P J Francis, and R G Weleber, Oregon Health and Sciences University, Portland, OR, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Allied disorders – Retinitis pigmentosa (RP) is often grouped with a class of more stable, inherited retinal disorders collectively referred to as RP and allied disorders. Some of these allied disorders cause similar clinic findings as RP, e.g., nyctalopia (night blindness), but usually do not show progression and deterioration with time. An example is congenital stationary night blindness (CSNB), which can present with nyctalopia and decreased rod and cone function on the electroretinogram (ERG). Unlike RP, most patients with CSNB have stable visual function. X-linked CSNB is caused by mutations in nyctalopin (NYX) and L-type voltage dependent calcium channel (CACNA1F). Although the majority of mutations of rhodopsin causes typical RP, rare mutations, such as G90D in rhodopsin, produce night blindness with such mild progression late in life that they have been called stationary night blindness. Another allied disorder is achromatopsia, which is caused by mutations in cyclic nucleotide-gated channel subunits (CNGA2, CNGB3) or guanine nucleotide alpha-binding protein 2 (GNAT2). Achromatopsia is associated with severely decreased central and color vision, photophobia, and nystagmus. These symptoms are similar to those that can be seen with some cone–rod dystrophies. Indeed, later in life some modest foveal atrophy can occur and cases of progressive cone–rod dystrophy have been associated with mutations of some of the achromatopsia genes. However, unlike cone–rod dystrophies, which invariably progress, achromatopsia is, in the vast majority of cases, stationary. Cone dystrophy – Cone photoreceptors are affected and rod photoreceptors are minimally affected or spared in cone dystrophy. Many cases of early cone dystrophies with time will develop significant rod abnormalities. Cone–rod dystrophy – Cone-rod dystrophy, as a group, involves both photoreceptors with cones affected more than rods. Certain forms of RP present with greater cone than rod involvement on ERG and these patients have been termed to have cone–rod RP. However, in cone–rod dystrophies as a group the primary defect lies in cones and secondaryrod loss

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occurs with time. Most investigators consider primary cone–rod dystrophy separate from RP. Extrinsic factor – An agent external to the organism that contributes to or is causative of a disease state. This can include drugs, foods, normal nutrients (excess or deficiency), toxins, inhaled chemicals, infectious agents, and exposures to radiation such as light, sound, and high-energy particles. Intrinsic factor – An agent that is inherent to the organism that contributes to or is causative of a disease state. Mixed intrinsic and extrinsic etiology for a secondary photoreceptor degeneration – This occurs when a person has a genetic variant that creates a toxic metabolite in the presence of an extrinsic molecule that would normally not be encountered. Mixed model of primary and secondary photoreceptor degeneration – This is considered when a genetic alteration within the photoreceptors is insufficient to cause photoreceptor degeneration by itself, but predisposes to degeneration in the presence of an extrinsic or intrinsic agent. A second mode of combined primary and secondary photoreceptor degeneration is when one group of photoreceptors, such as the rod photoreceptors, undergoes a primary degenerative process that is due to a mutation in a gene that is expressed in those photoreceptors and precipitates apoptosis, which leads to a secondary degenerative process, in this example cones, due to alterations in the cellular environment induced by death of neighboring cells. Primary retinal degeneration – This occurs when cells in the retina, usually photoreceptors, die secondary to a process that originates within the retina itself. An example of a primary retinal degeneration is RP, which is caused by mutations in genes that encode proteins important for retinal function. A disease can be classified as a primary retinal degeneration if the genetic defect is such that correction of expression of the normal gene product in the photoreceptors is required to correct the abnormality and arrest the degeneration. Primary retinal degeneration with secondary photoreceptor degeneration – This occurs when photoreceptor degeneration is the result of mutation(s) of a gene that exists in other retinal cells, for example,

Primary Photoreceptor Degenerations: Retinitis Pigmentosa

retinal pigment epithelial (RPE) cells. Correction of the genetic defect would require modification of the effects of those other retinal cells (e.g, RPE cells). Retinal atrophy – A broad term encompassing not only processes that occur with retinal degenerations, but also abnormal retinal tissue or cellular loss due to developmental defects and malnutrition. Retinal degeneration – A process whereby cells in the retina undergo cell death by apoptosis. Most retinal degenerations affect both rod and cone photoreceptors, but some disorders reflect damage that occurs principally in other cell types, e.g., the RPE in Stargardt’s disease and other ABCA4-related retinopathies. Secondary degeneration of the RPE is also common. Transsynaptic degeneration of higher-order cells, bipolar and ganglion cells, can also occur. The general term retinal degeneration should be distinguished from the more specific term, photoreceptor degeneration. Retinal dystrophy – A broad term that not only encompasses retinal degenerations, but also includes abnormal retinal function due to developmental defects and malnutrition. Retinitis pigmentosa (RP) – A heterogeneous group of diseases that result in degeneration of the rod and cone photoreceptors and secondarily the RPE. This degeneration usually leads to a loss of night vision due to the early degeneration of rods, constricted visual fields, decreased responses on ERG, and ultimately a decrease in visual acuity once macular cones begin to degenerate. Typical fundus findings include midperipheral atrophy of the pigment epithelium, bone spicule pigments, retinal vessel attenuation, and waxy pallor of the optic nerve. The term RP usually refers to only rod-cone dystrophies; however, cone-rod dystrophies and cone dystrophies are sometimes grouped under this term. Rod–cone dystrophy – A retinal dystrophy in which the rod photoreceptors are affected more than the cones. Most forms of RP manifest as rod–cone dystrophies. Secondary photoreceptor degeneration of the extrinsic type – A secondary photoreceptor degeneration of the extrinsic type exists if, despite the underlying molecular defect, one could avoid the photoreceptor degeneration by preventing an individual’s exposure to an extrinsic agent or condition (e.g., toxin, drug, infectious agent, light, and trauma). Secondary photoreceptor degeneration of the intrinsic type – If one can prevent photoreceptor degeneration by correcting or reversing a systemic or

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ocular metabolic or immune process, then it is a secondary photoreceptor degeneration of the intrinsic type.

Background Retinitis pigmentosa (RP) is caused by a large number of genetic defects that result in a characteristic pattern of degeneration of the rod and cone photoreceptors and the retinal pigment epithelium (RPE). This degeneration usually leads to a loss of night vision due to the early degeneration of rods, constricted visual fields, decreased responses on electroretinogram (ERG), and ultimately a decrease in visual acuity once macular cones begin to degenerate. The typical fundus exam in RP reveals midperipheral atrophy of the pigment epithelium, bone spicule pigmentation, retinal vessel attenuation, and waxy pallor of the optic nerve (Figure 1). RP was first named by the Dutch ophthalmologist, Frans Cornelius Donders, in the mid-nineteenth century, although earlier clinical descriptions of the disease exist. The term retinitis pigmentosa is somewhat of a misnomer because inflammation is not thought to be the primary pathological mechanism. Rather, mutations in over 100 genes have been shown to cause RP and its allied disorders, and there still remain a significant number of genes yet to be identified. To keep track of the ever-growing list of genes implicated in this disease, a comprehensive online database, Retnet, has been established by Dr. Stephen Daiger. One of the most fascinating aspects of RP is that mutations in genes that encode functionally distinct

Figure 1 Classic fundus appearance in retinitis pigmentosa demonstrating bone spicule pigmentation, vascular atrophy, retinal pigment epithelium atrophy, and waxy pallor of the optic nerve. From Weleber, R. and Evan, K. G. (2006). Retinitis pigmentosa and allied disorders. In: Ryan, S. J. (ed.) The Retina, 4th edn., vol. 1, chap. 17, pp. 395–498. Philadelphia, PA: Elsevier.

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proteins result in a common degenerative pathway. Some of the many examples include genes involved in the structural integrity of the photoreceptors and cilia, the retinoid cycle, the phototransduction cascade, the extracellular matrix, cellular metabolism, intracellular trafficking, and RNA processing.

Prevalence The worldwide prevalence for all forms of RP has been reported to be approximately 1:4000. While most studies have focused on the prevalence in European/Caucasian populations, the occurrence of RP has been reported throughout the world.

Inheritance All forms of Mendelian inheritance have been reported but autosomal dominant, recessive, and X-linked traits are most frequently seen. Rarely RP is inherited as a digenic disorder or through the maternal line as a mitochondrial disease. Autosomal Recessive RP patients with a family history of similarly affected relatives are called multiplex, whereas those with no family history are classified as simplex. Simplex individuals are usually assumed to represent autosomal recessive inheritance, although some of these cases may be de novo dominant mutations or unrecognized X-linked inheritance. When simplex cases are included, autosomal recessive cases of RP have been reported to account for approximately 50–60% of all cases, with the exact percentage varying from country to country. Some of the most commonly affected genes are usherin (USH2A), a gene that is involved in both Usher syndrome and autosomal recessive RP, and the phosphodiesterase beta subunit (PDE6B), a gene involved in phototransduction. X-Linked RP X-linked RP results from mutations of genes on the X chromosome and represents approximately 5–15% of patients with RP. To date, six genes that cause retinal degeneration have been linked to the X chromosome. Two genes, retinitis pigmentosa GTPase regulator (RPGR) and retinitis pigmentosa 2 (RP2), are known, and several genes remain to be identified. Males with X-linked RP typically have more severe retinal degeneration compared to autosomal recessive and dominant forms of the disease. The actual rate of degeneration is likely similar to the other forms, but the age of onset appears to be earlier.

Female carriers are thought to have a mosaic retina in which some of the cells express the normal allele, while others express the mutant allele. The fundus findings in female carriers of X-linked RP can vary from very subtle changes, such as mottling of the RPE, to more severe disease with some patients showing the classic bone spicule pigmentation. Even in the cases of female carriers with a normal appearing fundus, changes are usually apparent on the ERG.

Autosomal Dominant Patients with autosomal dominant RP often have a family history of the disease, although there are cases of incomplete penetrance and de novo mutations. Autosomal dominant mutations account for approximately 30–40% of patients with RP. In general, patients with autosomal dominant RP tend to be less affected than patients with X-linked or autosomal recessive RP. Some of the most commonly mutated genes include rhodopsin (RHO) and retinitis pigmentosa 1 (RP1).

Nonsyndromic versus Syndromic Retinal Degeneration Most cases of RP are nonsyndromic and the pathology is limited to the eye. However, RP can also be associated with dysfunctions in other organ systems, with many of these cases comprising defined syndromes. The most common syndromic association with RP is Usher syndrome, which is an autosomal recessive disorder and is divided into three subtypes based on clinical findings. Patients with type I Usher syndrome present with severe, but nonprogressive congenital hearing loss, balance problems, and RP. In type II Usher syndrome, patients have less severe hearing loss, RP, and normal balance. Patients with type III Usher syndrome, start with symptoms similar to type II but later progress to type I. The retinal findings in Usher syndrome are indistinguishable from those characteristic of nonsyndromic autosomal recessive RP. Eleven genes have been found to cause Usher syndrome. Considering the presumed shared evolutionary ancestry of photoreceptors and cochlear hair cells, it is likely that some of these genes share similar functions. Bardet–Biedl syndrome (BBS) is an autosomal recessive disorder in which RP is a universal finding. Other commonly associated features include postaxial polydactyly, truncal obesity, abnormalities of cognition, and renal disease. Mutations in 12 genes have been implicated in BBS. Many of these genes encode proteins that are important for the formation or function of the cilia (Figure 2). Some other syndromes that can present with RP include: abetalipoproteinemia (Bassen–ornzweig disease), Alstro¨m


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(a) (c)

(b) Figure 2 (a) Fundus photos of patient with Bardet–Biedl syndrome (BBS), demonstrating the classical changes of RP that include bone spicule pigmentation, vascular attenuation, and waxy pallor of the optic nerve. (b) Scars on the foot of a patient with BBS from removal of an extra digit. (c) Similar scars on the hand.

syndrome, chronic progressive external ophthalmoplegia (CPEO), Friedreich’s ataxia, incontinentia pigmenti (Bloch–Schulzberg syndrome), Joubert syndrome, Kearns– Sayre syndrome, mucopolysaccharide disorders, neuronal ceroid lipofuscinoisis (Batten disease), Refsum disease (infantile and adult), Senior–Loken syndrome, and spinocerebellar ataxia type 7.

Table 1 Most common genes causing retinitis pigmentosa by inheritance

Classification of RP

X-linked RP

One often confounding feature of RP is the many different ways in which the disease can be classified. RP can be classified by its mode of inheritance, age of onset, fundus appearance, pattern of functional vision loss, or by genetic mutation. As mentioned previously, RP is often characterized by its pattern of inheritance. With the advent of genetic testing, patients are increasingly being tested and classified according to which genes are mutated (see Table 1 for the most common mutations and Table 2 for description of genes). The phenotype and course of the disease can show significant variation with different mutations in the same gene. Likewise, there can also exist significant phenotypic variations between two people who harbor the same mutation. Ultimately, classification by genetic

Most common genes causing retinitis pigmentosa Autosomal recessive RP (including Usher syndrome) Autosomal dominant RP

USH2A, PDE6B, PDE6A, MYO7A, CRB1, RGR, CNGB1, RPE65 Rho (rhodopsin), RP1, PRPF31, PRPF3, RDS/ROM RPGR, RP2

defects will likely prove to be the most useful way to segregate and treat patients with RP. However, many genes remain to be discovered and genetic testing is not yet universally available to test for all mutations in known genes. For these reasons, it is useful to examine the ways in which RP has been categorized in the past. Classification by Age of Onset Severe forms of RP that manifest before the first year of life are referred to as Leber congenital amaurosis (LCA). The forms of RP occurring between 1 and 5 years have been termed juvenile RP or severe early childhood-onset retinal dystrophy (SECORD). LCA is characterized by

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Table 2

Genes, protein, diseases, and function

Gene symbol

Protein

Diseases

Function

ALMS1

Alstro¨m syndrome protein 1

Alstro¨m syndrome

CACNA1F

Calcuim-channel, voltagedependent, alpha 1F subunit

Incomplete CSNB, AIED, and other X-linked CRD (CORDX3, Maori disease, CSNB with retinal and optic atrophy)

CEP290 CHM

Centrosomal protein CEP290 Rab escort protein 1 (REP-1)

Joubert syndrome, LCA Choroideremia

CNGA2

Achromatopsia

CRB1

Cyclic nucleotide-gated channel – subunit alpha 2 Cyclic nucleotide-gated channel – subunit B1 Cyclic nucleotide gated channel – subunit beta 1 Homolog of crumbs

arRP, LCA

GNAT2

Transducin alpha 2

Achromatopsia

MYO7A

Myosin-VIIA

Usher syndrome type IA

NYX

Nyctalopin

CSNB

OAT

Ornithine-@aminotransferase

Gyrate atrophy

PDE6A

Phospodiesterase alpha subunit

arRP

PDE6B

Phospodiesterase beta subunit

arRP

PHYH

Refsum disease

PRPF3

phytanoyl-CoA 2-hydroxylase PRPF3

adRP

PRPF31

PRPF31

adRP

RDH5

11-cis retinol dehydrogenase

Fundus albipunctata

RDS

Peripherin

RP, pattern dystrophy

RGR

Retinal g-protein-coupled receptor Rhodopsin

arRP, adRP

Exact function unknown, may play a role in ciliogenesis Acts as a subunit in the major voltage-sensitive calcium channel in rod and cone photoreceptor terminals. Required for the calcium flux into photoreceptors (rods and cones) that is needed for sustaining the tonic neuro-transmitter release from presynaptic terminals. Required for formation/ maintenance of ribbon synapses Localizes to cilium, may mediate G-protein trafficking Participates in post-translational lipid modifications of proteins to enable membrane attachments that are essential in membrane trafficking. Acts as a geranylgeranyl transferase and appears required for specific Rab pathways. Codes for the alpha subunit of the cyclic nucleotidegated channels in cones Codes for the beta subunit of the cyclic nucleotide gated channels in rods Codes for the beta subunit of the cyclic nucleotidegated channels in cones Homologous to crumbs in the Drosophila, where it plays a role in cell–cell interactions and photoreceptor polarity Plays a role in the photoreceptor phototransduction cascade. Forms a complex with the beta and gamma subunits and acts to convert cGMP to GMP May play a role in trafficking of ribbon-synaptic vesicle complexes and renewal of the outer photoreceptors disks Predicted secreted protein important for development of ON bipolar cell signaling pathways Catalyzes the conversion of L-ornithine and a 2-oxo acid to L-glutamate 5-semialdehyde and an L-amino acid Plays a role in the photoreceptor phototransduction cascade. Forms a complex with the beta and gamma subunits and acts to convert cGMP to GMP Plays a role in the photoreceptor phototransduction cascade. Forms a complex with the alpha and gamma subunits and acts to convert cGMP to GMP Catalyzes the first step in the alpha-oxidation of phytanic acid Forms part of a spliceosome complex for mRNA processing Forms part of a spliceosome complex for mRNA processing 11-cis RDH is found in the RPE, where it catalyzes the final step in the biosynthesis of 11-cis retinaldehyde, the visual chromophore for both rods and cones Interacts with Rom-1 as the major morphogen for disk formation and to stabilize photoreceptor disks Expressed in the RPE and Mu¨ller cells and plays a role in retinoid recycling Mediates the detection of photons through light-induced isomerization of 11-cis to all-trans retinal, which triggers a conformational change leading to G-protein activation and release of all-trans retinal

CNGB1 CNGB3

RHO

arRP Achromatopsia

adRP, arRP CSNB

Continued


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Table 2

Continued

Gene symbol

Protein

Diseases

Function

RLBP1

Cellular retinaldehydebinding protein 1-like protein 1

arRP, retinitis punctata albescens, Bothnia dystrophy, Newfoundland rod–cone dystrophy

ROM

Rom-1

Digenic RP

RP1

RP1

adRP

RP2

RP2

xlRP

RPE65

RPE65

arRP, LCA

RPGR

Retinitis pigmentosa GTPase regulator Usherin

xlRP

Plays a role in visual pigment regeneration: carrier for endogenous 11-cis-retinol and 11-cis-retinal, major 11-cis-retinol acceptor in the isomerization step of the rod visual cycle, stimulating isomerization of all-trans- to 11-cis-retinol, facilitates oxidation of 11-cis-retinol to 11-cis-retinal by 11-cis-retinol dehydrogenase (RDH5) Interacts with peripherin to stabilize photoreceptor disks Localizes to photoreceptor cilium, may play a role in transport of proteins between inner and outer segments Exact function unknown. Stimulates GTPase activity of tubulin and may function to link cell membrane with cytoskeleton Expressed in the RPE and acts in retinoid metabolism to isomerizes all-trans-retinal ester to 11-cis retinol Exact function unknown. Localizes to cilium and may act to maintain protein polarization across the cilium Exact function unknown, Interacts with collagen IV and fibronectin and may be required for stable integration into the basement membrane

USH2A

Usher syndrome type II, arRP

Figure 3 Fundus photographs of a 16-year-old patient with Leber congenital amaurosis. There is waxy pallor of the optic nerve, severe vascular attenuation, RPE atrophy most notable in the macula, and bone spicule pigmentations.

severe vision loss, nystagmus, unrecordable ERGs, and poorly responsive pupils (amaurosis). Mutations in at least 16 genes have been found to cause LCA and most of these are inherited in an autosomal recessive fashion. Juvenile RP/SECORD is thought to be caused by less severe mutations in the same set of genes as LCA, and more mild mutations in some of these genes have been implicated in recessive RP (Figure 3). Classification by Fundus Appearance The classic fundus appearance in RP is described below (see the subsection titled ‘Fundus findings’). Deviations from this classic fundus appearance have given rise to

several alternative terms for pigmentary retinopathies including: inverse RP, concentric RP, sector RP, retinitis punctata albescens, fundus albipunctatus, RP with preserved peri-arteriolar RPE, pigmented perivenous retinochoroidal atrophy, and retinitis sine pigmento. Some of these terms are falling out of usage as genetic characterization of the disease is becoming more common. There have been many cases of unilateral RP described and it has been proposed that this could be caused by a somatic mutation. However, there is yet to be a histologically confirmed case of RP caused by a somatic mutation. Most of these cases likely represent other diseases that can mimic RP and cause a pigmentary retinopathy.

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Classification by Functional Loss Historically, adult RP was categorized as either type I (rod dysfunction) or type II (rod and cone dysfunction) based on psychophysical testing. These classifications were further refined on the basis of electrophysiological findings to include the categories: rod–cone dystrophy, cone–rod dystrophy, or cone dystrophy. Most forms of RP are rod–cone dystrophies in which rod photoreceptor death occurs first and is later followed by subsequent cone photoreceptor death. This article will use the term RP to describe the most common form, namely rod–cone dystrophy. The forms of RP that cause cone and cone–rod dystrophy will be denoted accordingly.

Mechanism of Disease RP and its allied disorders are caused by mutations in over 100 genes and likely the same number remains to be elucidated. Ultimately, these mutations lead to photoreceptor death by apoptosis. It is still not fully understood how mutations in genes, which code for an array of functionally different proteins, result in a common pathway to photoreceptor death. Mutations can result in decreased expression of a given protein, cause loss of function of that protein, or imbue a gain of function. In autosomal recessive forms of RP, there is a loss of expression or function when both copies of a given gene are mutated. In contrast, autosomal dominant forms of RP are thought to be caused by gain-of-function mutations, where the mutated protein becomes toxic or interferes with the function of the remaining normal forms of that protein (dominant negative effect). In autosomal dominant RP, the most common mutations are found in RHO. These dominant mutations can lead to forms of RHO that do not inactivate properly or are not transported to the outer segment. An example of one well-studied mutation that causes autosomal dominant RP is the P23H mutation in the RHO gene. This mutation results in misfolding of the protein such that it is sequestered in the endoplasmic reticulum and is never transported to the outer segment. The misfolded proteins accumulate creating aggregations that activate an unfolded protein response. Dysregulation of these responses may lead to photoreceptor death although the exact mechanism has yet to be determined.

Clinical Presentation Symptoms Most frequently, the earliest symptom of RP is night blindness that precedes visual-field change and, in some, retinal pathology. In children, parents may comment that

their child is afraid or becomes distressed in the dark. Older children often comment on poor vision compared with their fully sighted peers. In adults, difficulties with night driving are frequent. The second cardinal symptom of RP is progressive peripheral visual-field loss. Since central vision is spared early in the course of the disease, some patients do not notice this loss of visual field until the degeneration has become quite advanced. Another common symptom of RP is problems with dark adaptation, such as difficulties adjusting to dim illumination when entering a movie theater. Additionally, as the disease progresses, patients can develop photophobia. Color vision is typically normal early in the disease but with progression, blue–yellow defects become apparent. Flashes of light, or photopsias, are experienced by most patients. Patients with cone and cone–rod dystrophies can present with early photophobia, decreased central vision, and impaired color vision. These patients will typically have worse visual acuity than patients with rod–cone dystrophies due to earlier involvement of macular cones. Refraction Refractive errors in patients with RP have been studied and, on average, these patients are more myopic and have a greater degree of astigmatism than those in the general population. By contrast, patients with early-onset forms of RP, such as LCA or SECORD, tend to have hyperopic refractions. Anterior Segment and Cataract The external ocular exam and anterior segment are typically unremarkable in nonsyndromic forms of RP. However, there does appear to be a higher rate of keratoconus and glaucoma in patients with RP. Posterior subcapsular cataracts are common and often can become visually significant. Cataract extraction is beneficial in patients with RP if the cataract is thought to be the vision-limiting factor. Fundus Findings The characteristic signs on fundus examination include midperipheral atrophy of the pigment epithelium, intraretinal pigment accumulation (bone spicules; Figure 1), retinal vessel attenuation, and waxy pallor of the optic nerve. A yellowish ring of peripapillary atrophy is sometimes seen in patients with RP as well as with optic nerve head drusen. A minor number of vitreous cells are commonly observed in patients with RP. Cystoid macular edema is common in patients with RP and can often result in significantly decreased vision. Rarely, patients can develop a Coats-like retinopathy.


Diagnostic Tests for RP

methods for monitoring progression of the disease. Decreased visual-field sensitivity results from photoreceptor loss (Figure 5). The earliest change seen as measured by kinetic perimetry is concentric constriction or decreased sensitivity with static perimetry in diffuse disease and relative midperipheral scotomas seen in the in regional disease. As these midperipheral scotomas or regions of decreased sensitivity enlarge and deepen, severe tunnel vision results. Eventually, macular function fails and visual field becomes difficult or impossible to measure by conventional perimetry. Although visual function may be reduced to light perception only, it is rare for patients to become completely blind. With the exception of female carriers in X-linked RP, visual-field loss is usually symmetrical. Marked asymmetry should raise concern for diseases that mimic RP (Figure 6). The rate of visual-field loss has been shown to be exponential. This rate is thought to be similar for the different forms of inheritance once correction has been made for the critical age of onset. Massof and Finkelstein found that patients lost about 50% of their visual field every 4.5 years. The superior visual field, which corresponds to the inferior retina, is often more affected than inferior visual fields. Based on this finding, it has been suggested that increased levels of light may play a role in accelerating retinal degeneration and this in turn may play a role in the forms of RP with greater damage in the inferior retina.

Dark Adaptation Dark adaptation can be a useful test in patients with RP. Patients who manifest with a rod–cone dystrophy will usually have a detectable increase in final dark-adapted thresholds and show delayed dark-adaptation curves. Prolonged dark adaptation is especially common among patients with RHO mutations. Elevations of the early cone segment of the dark-adaptation curve may be particularly noticed by patients, more so than elevations of the rod segment (Figure 4). Visual Fields Visual fields are not only useful for making the diagnosis of RP, but are also one of the most useful objective 7

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ERGs play a crucial role in the diagnosis of RP because these electrophysiological recordings are sensitive enough to detect decreased photoreceptor function early in the disease when fundus findings and visual fields may be minimally altered. In addition, ERGs are particularly

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Figure 6 Kinetic visual fields obtained from patient with retinitis pigmentosa. Note the relative preservation of inferior fields, which correlated with preserved superior retina. From Weleber, R. and Evan, K. G. (2006). Retinitis pigmentosa and allied disorders. In: Ryan, S. J. (ed.) The Retina, 4th edn., vol. 1, chap. 17, pp. 395–498. Philadelphia, PA: Elsevier.

useful to assess visual function in preverbal infants and children. Almost all patients with symptomatic RP will have detectable changes on the ERG at the time of diagnosis. While the ERG is useful for the diagnosis of RP, visual fields are better for monitoring of the course of the disease. In severe cases of RP, such as LCA, the ERG may be not recordable. Patients with RP can show decreased amplitude and timing of the major components of the ERG. Caution must be taken when interpreting decreases in the amplitude of an ERG because poor contact of the electrodes, deviations of the eye, and high myopia can affect the amplitude of the signal. When present, delayed timing tends to be a more robust indicator of dysfunction. By analyzing the different components of the ERG, different forms of RP can be classified. Degeneration of the rod and cone photoreceptors leads to a decrease in the amplitude of different waveforms of the ERG and can also increase the timing or latency of the peaks of these waveforms. The most common forms of RP manifest as a rod–cone dystrophy and the first detectable changes will be apparent on the scotopic ERG. Decreases in the b-wave amplitude and timing of the peak of the b-wave are indicative of early rod photoreceptor death. Further loss of rod cells leads to further decreases in the b-wave amplitude and decreased amplitude of the a-wave responses at higher intensities. Patients with a cone–rod dystrophy have normal, or lesser defect of b-wave responses to dim scotopic stimuli, but typically have more markedly abnormal ERGs to 30-Hz flicker or single-flash stimuli measured under photopic conditions (Figures 7–9).

Fundus Photography/Fluorescein Angiography Documentation by fundus photography can assist in monitoring changes in patients with RP. Fluorescein angiography in patients with RP will demonstrate hyperfluorescence in areas of RPE atrophy and can highlight areas of cystoid macular edema. However, fluorescein angiography has largely been supplanted by optical coherence tomography (OCT) for detecting cystoid maculopathy. In addition, concerns about light exposure accelerating certain forms of RP in animal models have prompted many ophthalmologists to exercise caution in obtaining excessive photographs. Optical Coherence Tomography OCT provides a noninvasive cross-sectional image of the retina. It is very useful in patients with RP when there is a question of cystoid macular edema. The ability to detect cystoid macular edema by OCT often obviates the need to get a fluorescein angiogram.

Differential Diagnosis It is important to realize that RP is not the only cause of a pigmentary retinopathy but many other diseases can mimic RP. Significant asymmetry or the onset of symptoms in an elderly patient should raise suspicion for one of the diseases that mimics RP. Trauma to the eye can disrupt the retina and result in pigment migration of the RPE into the retina with the formation of bone spicules. By a similar mechanism,


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Figure 7 ERGs recorded from a patient with autosomal recessive RP (left column) compared to a control patient (right column). This patient is demonstrative of a rod–cone dystrophy. There is a flat response to the dim blue flash under scotopic conditions, which specifically stimulates rods. The bright flash under scotopic conditions normally elicits mixed responses from both rods and cones. In this case, the response is severely attenuated and the small amount of signal is likely coming from the cone system. Under light-adapted conditions (photopic single flash and 30-Hz flicker), which selectively stimulate the cones, the response is only slightly decreased consistent with the categorization of a rod–cone dystrophy.

ophthalmic artery occlusions and old retinal detachments can present with a pigmentary retinopathy. Additionally, infections caused by syphilis, toxoplasmosis, and herpes viruses can lead to a pigmentary retinopathy. Congenital rubella infection can often be misdiagnosed as RP or Usher syndrome because these patients present with deafness and a fine, speckled pigmentary retinopathy. The key to differentiating patients with rubella from those with RP is that patients with rubella retinopathy will have normal or near normal responses by ERG (Figure 10). Diffuse unilateral neuroretinitis (DUSN) is caused by a chronic infection with a nematode. In the early stages, this disease can be distinguished due to the appearance of crops of yellowish, deep choroidal infiltrates, neuroretinitis, and sometimes, visualization of the worm itself. However, late in the disease, with the exception of being unilateral, the fundus appearance is identical to RP with the fundus showing bone spicules, vascular attenuation, and optic atrophy (Figure 11). Inflammatory diseases that cause a posterior uveitis can cause chronic changes that mimic the pigmentary changes of RP. Some examples include sarcoidosis, birdshot choroidoretinopathy, serpiginous retinopathy, Behcet disease, and acute zonal occult outer retinopathy (AZOOR).

Certain drugs can cause pigmentary retinopathies and their usage must be excluded prior to making a diagnosis of RP. One example is thioridazine (Mellaril), an antipsychotic drug, which mimics RP by causing decreased night vision, RPE atrophy, and a pigmentary retinopathy. Hydroxychloroquine (Plaquenil) is used to treat systemic lupus erythematous and rheumatoid arthritis and when taken for extended period of time or at higher doses can lead to central vision loss. Other drugs that have been found to cause pigmentary retinopathies include chlorpromazine, chloroquine, and quinine. Autoimmune retinopathy is an incompletely understood disease resulting from antibodies to retinal antigens and can present with many of the same features of RP, such as decreased vision, visual-field loss, and decreased ERGs. Unlike the other diseases that mimic RP, autoimmune retinopathy does not present with a pigmentary retinopathy and, in many cases, the fundus appearance can be normal or only show vascular attenuation. A subset of cases of autoimmune retinopathy is associated with carcinomas in other parts of the body. Two examples of this entity are cancer-associated retinopathy (CAR), which often arises from small cell carcinoma of the lung and melanoma-associated retinopathy (MAR). CAR patients may test positive for antibodies directed against retinal

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Figure 8 ERGs recorded from a patient with peripherin/RDS null mutation (left column) compared to a control patient (right column). This patient is demonstrative of a rod–cone dystrophy where the rods and cones are equally affected. It is of importance that peripherin is expressed in both rods and cones. There is a severely diminished response to the dim white flash under scotopic conditions, which specifically stimulates rods. The bright flash under scotopic conditions normally elicits a mixed response from both rods and cones. In this case, the response is severely attenuated. Under light-adapted conditions (photopic single flash and 30-Hz flicker), which selectively stimulate the cones, the response is also severely decreased consistent with the categorization of an equal rod–cone dystrophy.

antigens, such as anti-recoverin or anti-enolase, while MAR patients may test positive for antibodies directed against bipolar cells.

Prognosis in RP It is uncommon for a patient with RP to lose all light perception. A study by Grover et al. in 1999 showed that only 0.5% of patients over the age of 45 had no light perception and 50% retained vision better than 20/40. In the typical rod–cone dystrophies, visual loss usually starts in the midperiphery with central visual acuity being spared for many years. Most patients will eventually qualify as being legally blind, often secondary to decreased visual fields prior to being disqualified on the account of decreased central visual acuity. RP is a slowly progressive disease and many patients will eventually experience decreased central acuity, most often from decrease in cone photoreceptor density, macular edema, epiretinal membranes, or retinal pigment defects. However, when there is an unexpected decrease of central acuity, the development of cataracts or cystoid macular edema should be suspected because these sequelae are more amenable to treatment.

Current Treatments Currently, there is no known cure for most forms of RP, although some treatments have been shown to slow down the progression of the disease and future therapies are promising. Treatable Forms of RP A few rare forms of RP are amenable to specific treatments. It is important to rule out these treatable forms of RP because prompt therapy can prevent further damage. The treatable forms of RP include abetalipoproteinemia and adult Refsum disease. Resources/Support for Patients with RP The diagnosis of RP can be both frightening and confusing to patients. In addition to having their questions answered by the physician, patients can benefit by meeting with a genetic counselor who can take a detailed family history and answer questions about heritability. Patients should be referred to the many organizations or websites dedicated to providing support for this disease, such as the Foundation Fighting Blindness.


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Figure 9 ERGs recorded from a patient with autosomal recessive RP (left column) compared to a control patient (right column). This patient is demonstrative of a cone–rod form of RP. There is a mildly diminished response to the dim blue flash under scotopic conditions, which specifically stimulates rods. The bright flash under scotopic conditions normally elicits a mixed response from both rods and cones. In this case, the response is only moderately attenuated. Under light-adapted conditions (photopic single flash and 30-Hz flicker), which selectively stimulate the cones, the response is severely decreased consistent with the categorization of a cone–rod dystrophy.

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Figure 10 (a) An example of rubella retinopathy. Note the fine, mottled pigmentary changes and normal-appearing nerve and vessels. Unlike RP, ERG testing in this patient would be expected to be normal. (b) Example of a pigmentary retinopathy caused by syphilis.

Optimizing Remaining Vision It is important that patients with RP have an up-to-date refraction to optimize remaining vision. Patients with significantly decreased visual acuity greatly benefit from a referral to low vision services. A variety of different magnifying devices are available to assist patients with RP. For example, night-vision devices can assist with navigation in dim conditions, although a bright, wide, beam flashlight may be a more cost-effective solution. Various

magnifiers and closed-circuit televisions (CCTVs) are available to enhance reading vision. Cataractogenesis is frequent in patients with RP. Functional visual improvement can be achieved by cataract surgery in carefully selected individuals with suitable remaining retinal function. The risks of surgery are increased in these individuals who should be counseled specifically regarding the risks of postoperative cystoid macular edema.

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Figure 11 (a) Normal fundus from a patient with diffuse unilateral subacute neuroretinitis (DUSN). (b) The affected eye in the same patient. Note that appearance is identical to RP, demonstrating waxy pallor of the nerve, vascular attenuation, bone-spicule pigmentation, and RPE atrophy.

Cystoid macular edema in RP has been treated with carbonic anhydrase inhibitors such as oral acetazolamide (Diamox) or topical dorzolamide (Trusopt). These medicines can improve vision in some RP patients with cystoid macular edema; however, their efficacy can decrease with time and many patients cannot tolerate the side effects induced by these medicines.

Vitamin A Observational studies of patients with RP taking vitamin A and vitamin E supplementation demonstrated a slower decline in cone ERGs than expected and led to a randomized, controlled, double-masked trial, to assess if these supplements could slow down retinal degeneration. Additional subgroup analyses in this study suggested that oral supplementation with 15 000 IU of vitamin A modestly slowed down the loss of ERG amplitude over a 5-year period in certain individuals with RP. Currently, vitamin A supplementation is frequently recommended but its use is not universal. High doses of vitamin A supplementation have also been associated with elevated liver enzymes, elevated triglycerides, and an increased risk of osteoporosis. It seems prudent to check annual liver function tests and triglyceride levels for all patients and bone density scans in older patients taking vitamin A supplementation. Vitamin A should be avoided in children, pregnant women, and those with decreased liver function. Additionally, from a mechanistic disease perspective, vitamin A should be avoided in forms of RP caused by mutations in the gene ABCA4 due to evidence in animal models of accelerated retinal degeneration. ABCA4 codes for the ABCR protein, which is important for transport of vitamin A-derived all-trans-retinal from the disk to the photoreceptor cytoplasm. Mutations in this gene are responsible for Stargardt macular dystrophy and rarely can also cause autosomal recessive RP.

Docosahexanoic Acid Docosahexanoic acid (DHA) is an important omega-3 fatty acid that comprises 30–40% of fatty acids in the retina. The exact role of DHA is not known, but it has been proposed to play a role maintaining membrane fluidity, mediating 11-cis retinal transport, and acting as a precursor for neuroprotective factors. Studies in patients with X-linked RP suggested that decreased levels of DHA correlate with decreased ERG responses and have prompted studies to evaluate if supplementation with DHA might slow down retinal degeneration. Two prospective, randomized, double-masked studies (one in patients with X-linked, the other in patients with all forms of RP) failed to show a significant benefit of DHA. However, considering the low risk of adverse effects, many centers do recommend DHA supplementation to patients with RP. Neuroprotection/CNTF A relatively new strategy for the treatment of RP is to prevent photoreceptor loss by the delivery of neuroprotective factors. Numerous studies in animal models have documented the successful rescue of photoreceptor degeneration by neurotrophic factors. A delivery system for one of these factors, human ciliary neurotrophic factor (CNTF), has been developed using encapsulated cell technology. These devices use RPE cells that have been transfected to express CNTF and are enclosed by a semipermeable membrane which allows nutrients to diffuse in, but prevents immune attack on the cells. Phase I studies, implanting this device in patients with RP, have been completed without any major adverse events. Phase II studies, which will be able to better assess any visual improvement, are underway. Gene Therapy Replacement of defective genes in autosomal recessive forms of RP holds much promise. Currently, three groups


have used an adeno-associated viral vector to deliver normal copies of the all-trans-retinol isomerase (RPE65) in patients with LCA. The treatment has thus far been well tolerated and some patients have demonstrated improvement in subjective vision and to some more objective tests such as pupillary reflexes. Gene therapy holds much promise for treating RP, but several challenges remain. RP and its allied disorders are caused by mutations in over 100 genes. Designing a multitude of vectors for each of these genes poses an arduous challenge. Autologous RPE Transplantation Replacement of RPE cells has been attempted using suspensions or sheets of cultured RPE cells or autologous grafts. Engraftment of these injected cells has been demonstrated in animal models. Rescue of photoreceptor degeneration has been achieved suggesting that transplanted RPE cells may modulate photoreceptor death. However, in spite of early photoreceptor rescue in these animal models, long-term restoration of vision has been disappointing. Stem-Cell-Based Therapies Cell-based therapy using stem cells is currently being intensely explored for rescuing vision. There are two fundamentally different strategies: one is to limit the progress of photoreceptor loss by introducing cells before such loss has progressed too far; the other is to replace lost photoreceptors. Stem cells are multipotent cells capable of self-renewal and have the potential to develop into many specific cell types. Their capacity for proliferative expansion to a large scale and their ability to produce a number of growth factors make them attractive candidates to be used to replace or repair damaged cells in adult organisms. Microelectrode Implants One novel concept for treatment of RP is to bypass the loss of the photoreceptors by electronically stimulating the retina, optic nerve, or visual cortex using microelectrode implants. Expression of Photosensitive Proteins A very recent approach for treating RP has been the strategy to bypass photoreceptor loss by using viral vectors to express light-sensitive proteins, such as channel rhodopsin, into postreceptor ganglion cells. Early experiments in small animals have demonstrated successful responses from ganglion cells using this strategy. Much work remains to be done, including how to obtain high

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enough expression to provide adequate sensitivity for useful vision.

Conclusions RP is a significant cause of vision loss in adults and children. Diagnosis is best made by careful history and clinical examination combined with retinal electrophysiology and psychophysical testing. For some individuals, genetic testing can identify causative mutations. While there is currently no cure for RP, many treatment options are emerging and future therapies are promising. See also: Adaptive Optics; Injury and Repair: Prostheses; Primary Photoreceptor Degenerations: Terminology.

Further Reading Berson, E. L., Rosner, B., Sandberg, M. A., et al. (1993). A randomized trial of vitamin A and vitamin E supplementation for retinitis pigmentosa. Archives of Ophthalmology 111: 761–772. Berson, E. L., Rosner, B., Sandberg, M. A., et al. (2004). Clinical trial of docosahexaenoic acid in patients with retinitis pigmentosa receiving vitamin A treatment. Archives of Ophthalmology 122: 1297–1305. Fishman, G. A., Farber, M. D., and Derlacki, D. J. (1988). X-linked retinitis pigmentosa. Profile of clinical findings. Archives of Ophthalmology 106: 369–375. Grant, C. A. and Berson, E. L. (2001). Treatable forms of retinitis pigmentosa associated with systemic neurological disorders. International Ophthalmology Clinics 41: 103–110. Grover, S., Fishman, G. A., Anderson, R. J., et al. (1999). Visual acuity impairment in patients with retinitis pigmentosa at age 45 years or older. Ophthalmology 106: 1780–1785. Hamel, C. P. (2007). Cone rod dystrophies. Orphanet Journal of Rare Diseases 2: 7. Hartong, D. T., Berson, E. L., and Dryja, T. P. (2006). Retinitis pigmentosa. Lancet 368: 1795–1809. Heckenlively, J. R. (1988). Retinitis Pigmentosa. Philadelphia, PA: Lippincott. Hoffman, D. R., Locke, K. G., Wheaton, D. H., et al. (2004). A randomized, placebo-controlled clinical trial of docosahexaenoic acid supplementation for X-linked retinitis pigmentosa. American Journal of Ophthalmology 137: 704–718. Radu, R. A., Yuan, Q., Hu, J., et al. (2008). Accelerated accumulation of lipofuscin pigments in the RPE of a mouse model for ABCA4mediated retinal dystrophies following vitamin A supplementation. Investigative Ophthalmology and Visual Science 49: 3821–3829. Sieving, P. A., Caruso, R. C., Tao, W., et al. (2006). Ciliary neurotrophic factor (CNTF) for human retinal degeneration: Phase I trial of CNTF delivered by encapsulated cell intraocular implants. Proceedings of the National Academy of Sciences of the United States of America 103: 3896–3901. Weleber, R. G. and Gregory-Evans, K. (2006). Retinitis pigmentosa and allied disorders. In: Ryan, S. J. (ed.) The Retina 395–498. Philadelphia, PA: Elsevier.

Relevant Websites http://www.ncbi.nlm.nih.gov – Online Mendelian Inheritance in Man (OMIM). http://www.sph.uth.tmc.edu – Retinal Information Network (Retnet).

Primary Photoreceptor Degenerations: Terminology M E Pennesi, P J Francis, and R G Weleber, Oregon Health and Sciences University, Portland, OR, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Allied disorders – Retinitis pigmentosa (RP) is often grouped with a class of more stable, inherited retinal disorders collectively referred to as RP and allied disorders. Some of these allied disorders cause similar clinic findings as RP, e.g., nyctalopia (night blindness), but usually do not show progression and deterioration with time. An example is congenital stationary night blindness (CSNB), which can present with nyctalopia and decreased rod and cone function on the electroretinogram (ERG). Unlike RP, most patients with CSNB have stable visual function. X-linked CSNB is caused by mutations in nyctalopin (NYX) and L-type voltage dependent calcium channel (CACNA1F). Although the majority of mutations of rhodopsin causes typical RP, rare mutations, such as G90D in rhodopsin, produce night blindness with such mild progression late in life that they have been called stationary night blindness. Another allied disorder is achromatopsia, which is caused by mutations in cyclic nucleotide-gated channel subunits (CNGA2, CNGB3) or guanine nucleotide alpha-binding protein 2 (GNAT2). Achromatopsia is associated with severely decreased central and color vision, photophobia, and nystagmus. These symptoms are similar to those that can be seen with some cone–rod dystrophies. Indeed, later in life some modest foveal atrophy can occur and cases of progressive cone–rod dystrophy have been associated with mutations of some of the achromatopsia genes. However, unlike cone–rod dystrophies, which invariably progress, achromatopsia is, in the vast majority of cases, stationary. Cone dystrophy – Cone photoreceptors are affected and rod photoreceptors are minimally affected or spared in cone dystrophy. Many cases of early cone dystrophies with time will develop significant rod abnormalities. Cone–rod dystrophy – Cone–rod dystrophy, as a group, involves both photoreceptors with cones affected more than rods. Certain forms of RP present with greater cone than rod involvement on ERG and these patients have been termed to have cone–rod RP. However, in cone–rod dystrophies as a group the primary defect lies in cones and secondaryrod loss

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occurs with time. Most investigators consider primary cone–rod dystrophy separate from RP. Extrinsic factor – An agent external to the organism that contributes to or is causative of a disease state. This can include drugs, foods, normal nutrients (excess or deficiency), toxins, inhaled chemicals, infectious agents, and exposures to radiation such as light, sound, and high-energy particles. Intrinsic factor – An agent that is inherent to the organism that contributes to or is causative of a disease state. Mixed intrinsic and extrinsic etiology for a secondary photoreceptor degeneration – This occurs when a person has a genetic variant that creates a toxic metabolite in the presence of an extrinsic molecule that would normally not be encountered. Mixed model of primary and secondary photoreceptor degeneration – This is considered when a genetic alteration within the photoreceptors is insufficient to cause photoreceptor degeneration by itself, but predisposes to degeneration in the presence of an extrinsic or intrinsic agent. A second mode of combined primary and secondary photoreceptor degeneration is when one group of photoreceptors, such as the rod photoreceptors, undergoes a primary degenerative process that is due to a mutation in a gene that is expressed in those photoreceptors and precipitates apoptosis, which leads to a secondary degenerative process, in this example cones, due to alterations in the cellular environment induced by death of neighboring cells. Primary retinal degeneration – This occurs when cells in the retina, usually photoreceptors, die secondary to a process that originates within the retina itself. An example of a primary retinal degeneration is RP, which is caused by mutations in genes that encode proteins important for retinal function. A disease can be classified as a primary retinal degeneration if the genetic defect is such that correction of expression of the normal gene product in the photoreceptors is required to correct the abnormality and arrest the degeneration. Primary retinal degeneration with secondary photoreceptor degeneration – This occurs when photoreceptor degeneration is the result of mutation(s) of a gene that exists in other retinal cells, for


example, retinal pigment epithelial (RPE) cells. Correction of the genetic defect would require modification of the effects of those other retinal cells (e.g, RPE cells). Retinal atrophy – A broad term encompassing not only processes that occur with retinal degenerations, but also abnormal retinal tissue or cellular loss due to developmental defects and malnutrition. Retinal degeneration – A process whereby cells in the retina undergo cell death by apoptosis. Most retinal degenerations affect both rod and cone photoreceptors, but some disorders reflect damage that occurs principally in other cell types, e.g., the RPE in Stargardt’s disease and other ABCA4-related retinopathies. Secondary degeneration of the RPE is also common. Transsynaptic degeneration of higherorder cells, bipolar and ganglion cells, can also occur. The general term retinal degeneration should be distinguished from the more specific term, photoreceptor degeneration. Retinal dystrophy – A broad term that not only encompasses retinal degenerations, but also includes abnormal retinal function due to developmental defects and malnutrition. Retinitis pigmentosa (RP) – A heterogeneous group of diseases that result in degeneration of the rod and cone photoreceptors and secondarily the RPE. This degeneration usually leads to a loss of night vision due to the early degeneration of rods, constricted visual fields, decreased responses on ERG, and ultimately a decrease in visual acuity once macular cones begin to degenerate. Typical fundus findings include midperipheral atrophy of the pigment epithelium, bone spicule pigments, retinal vessel attenuation, and waxy pallor of the optic nerve. The term RP usually refers to only rod–cone dystrophies; however, cone–rod dystrophies and cone dystrophies are sometimes grouped under this term. Rod–cone dystrophy – A retinal dystrophy in which the rod photoreceptors are affected more than the cones. Most forms of RP manifest as rod–cone dystrophies. Secondary photoreceptor degeneration of the extrinsic type – A secondary photoreceptor degeneration of the extrinsic type exists if, despite the underlying molecular defect, one could avoid the photoreceptor degeneration by preventing an individual’s exposure to an extrinsic agent or condition (e.g., toxin, drug, infectious agent, light, and trauma). Secondary photoreceptor degeneration of the intrinsic type – If one can prevent photoreceptor degeneration by correcting or reversing a systemic or

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ocular metabolic or immune process, then it is a secondary photoreceptor degeneration of the intrinsic type.

Histological and Fundus Features of Retinitis Pigmentosa The fundus exam in retinitis pigmentosa (RP) reveals midperipheral atrophy of the pigment epithelium, bone spicule pigments greater in the periphery than centrally, retinal vessel attenuation, and waxy pallor of the optic nerve. Bone Spicule Pigmentation Bone spicules are intraretinal accumulations of melanin pigment that result from the migration of retinal pigmented epithelial (RPE) cells into the retina after photoreceptor death. These spicules are commonly found in a perivascular pattern and may encircle and occlude these vessels, and are a typical feature of RP. However, there are cases of RP that do not present with bone spicules as well as other disease processes, such as trauma and infection, can result in an appearance that mimics RP by presenting with bone spicules (Figure 1). Waxy Pallor of the Optic Nerve Much as the name implies, waxy pallor of the optic nerve refers to the funduscopic appearance of the optic nerve seen in many patients with RP. When retinal photoreceptors die, Mu¨ller cells and astrocytes in the retina undergo gliosis to form scar tissues. It is thought that this process may lead to the waxy pallor of the optic nerve (Figure 2). Peripapillary/Optic Nerve Head Drusen Peripapillary drusen/optic nerve drusen are found more commonly in patients with RP. Peripapilliary drusen are histologically different from the drusen found in macular degeneration and are found near or within the optic nerve head. They are thought to result from accumulations of materials in the axons of ganglion cells by axoplasmic stasis and can become calcified with time. Such drusen can cause isolated and asymmetrical visual-field defects, which can be slowly progressive (Figure 3). Bull’s Eye Maculopathy A bull’s eye maculopathy results from photoreceptor loss and retinal thinning in a parafoveal distribution. It is often apparent on color fundus photos, but can be visualized on

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fluorescein angiography as well. Bull’s eye maculopathies are most commonly not only seen in cone–rod dystrophies, but can also be seen in other diseases such as Stargardt’s macular dystrophy, Batten’s disease, and with hydroxychloroquine (Plaquenil) toxicity (Figure 4).

Coats-Like Response

Figure 1 A fundus photo of a patient with retinitis pigmentosa demonstrating intraretinal pigment accumulations (white arrows), also known as bone spicules, which are a common finding in retinitis pigmentosa. They are caused by the migration of retinal pigment epithelial cells into the retina after photoreceptor degeneration. Modified from Weleber, R. and Evan, K. G. (2006). Retinitis pigmentosa and allied disorders. In: Ryan, S. J. (ed.) The Retina, 4th edn., vol. 1, chap. 17, pp. 395–498. Philadelphia, PA: Elsevier.

Coats disease is characterized by peripheral retinal telangiectasias, which are dilations of retinal blood vessels, and is usually seen in young males. Rarely, patients with RP can develop localized areas of vascular telangiectasias with exudation similar to those seen in Coats disease. Extravasation of fluid from these vessels can lead to an exudative retinal detachment. This process is termed a Coats-like response (Figure 5).

Classification of RP by Fundus Pattern Classic Pattern for RP The classic pattern on fundus exam in RP reveals midperipheral atrophy of the pigment epithelium, bone spicule pigments in the periphery greater than centrally, retinal vessel attenuation, and waxy pallor of the optic nerve (Figure 6).

Inverse RP

Figure 2 An example of the waxy nerve pallor seen in patients with RP. Also note the vascular attenuation and sheathing.

(a)

The term inverse RP refers to a funduscopic pattern where the central retina exhibits more pigmentary changes and RPE atrophy than the periphery. This term is falling out of favor, as it is now understood that this pattern is often seen with advanced cone and cone–rod dystrophies, as well as some instances of secondary pigmentary retinopathies.

(b)

Figure 3 (a) Patient with retinitis pigmentosa – note the lack of a normal physiological cup, which raises the concern for buried drusen. (b) The same patient seen years later who demonstrates peripapillary drusen (arrows).


Concentric RP Concentric RP is a defined by a subgroup of patients who show a consistent pattern of centripetal vision loss from the far periphery toward the center. This distinguishes it from the classic pattern of RP, which starts in the midperiphery and progresses both outward and inward. Histological studies have shown an abrupt transition between diseased and normal areas of retina.

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early disease. The pattern is usually symmetrical between the two eyes. Mutations in rhodopsin (RHO) have been associated with sector RP. These patients are often less symptomatic and their electroretinograms (ERGs) are much less reduced than patients with diffuse or more widespread RP. Early generalized RP can mimic sector RP and, therefore, the diagnosis of sector RP must remain provisional for at least 10 years (Figure 7).

Sector RP

RP Sine Pigmento

Sector RP is a term that refers to the fundus appearance where abnormal pigmentation and atrophy is confined to only one area of the retina, usually an arc inferior to the macula. Debate exists whether this term is specific for certain gene defects or is a stage in evolution of what will become a more generalized process with time. Autosomal recessive or autosomal dominant regional disease can be characterized by such a sectorial pattern at least in

The term RP sine pigmento is applied to the early stages of RP where the classical findings of visual-field loss, decreased ERGs, and vascular attenuation may be present, but there is a lack of bone spicules on the fundus exam. This is thought to represent an early manifestation of the disease and most patients will eventually develop bone spicules, although often not until later stages. Another term in use is pauci-pigmentary retinopathy. In the case of patients who develop symptoms later in life and have a normal, or minimally abnormal, fundus appearance, a work-up for autoimmune retinopathy should be considered.

Tapetal-Like Reflex/Sheen Tapetal-like sheen is a yellowish-white metallic reflex that can be seen in young males with X-linked RP, women who are carriers of this disease, and patients with cone–rod dystrophy. As the disease advances and RPE atrophy progresses, this reflex can fade. This finding is not pathognomonic of RP because it can be seen in other allied disorders (Figure 8).

RP with Preserved Peri-Arteriolar RPE Figure 4 A fundus photo demonstrating a Bull’s eye maculopathy (dark surrounded by light area).

(a)

RP with preserved peri-arteriolar RPE (PPARPE) has been found in families with mutations in the crumbs

(b)

Figure 5 Both (a) and (b) show fundus photos from a patient with RP and a Coat’s-like response. Note the yellowish subretinal exudates.

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homolog 1 gene (CRB1). These patients present with the typical findings of RP, but for reasons not understood, have preservation of the RPE near arterioles.

Pigmented Paravenous Retinochoroidal Atrophy In pigmented paravenous retinochoroidal atrophy (PPRCA), degeneration and accumulation of bone spicules are limited to the areas around the retinal veins. These patients are often asymptomatic, but with careful testing, scotomas corresponding to the areas of degeneration can be identified. Typically, the ERG is minimally reduced and disease is thought to be, at most, slowly progressive. Although PPRCA has been thought not to be inherited, mutations in the CRB1 gene have been found in families demonstrating dominant inheritance (Figure 9).

Fundus Albipunctata Fundus albipunctata is an autosomal recessive disease that does not result in a retinal degeneration like RP, but can cause a congenital stationary night blindness. It is more accurately classified as an allied disorder. It is caused by mutations in the retinol dehydrogenase 5 gene (RDH5), which results in problems regenerating visual pigment. As a result, these patients have severely decreased rod recovery with prolonged dark adaptation. The fundus exam shows many discrete yellowish-white dots at the level of the RPE. Atrophic lesions in the macula can occur in many patients in later years (Figure 10). Retinitis Punctata Albescens Retinitis punctata albescens is an autosomal recessive disease caused by mutations in the retinaldehyde-binding protein 1 gene (RLBP1). Similar to fundus albipunctata, this disease presents with severe night blindness and small, discrete, yellowish-white lesions in the fundus, but can have a pigmentary retinopathy as well. Later stages of RPA may develop diffuse disease similar to advanced RP. Gyrate Atrophy

Figure 6 Fundus photo of a typical patient with RP. Note the pallor of the optic nerve, the vascular attenuation, atrophy of the pigment epithelium, and bone-spicules. Reproduced from Weleber, R. G., Butler, N. S., Murphey, W. H., Sheffield, V. C., and Stone, E. M. (1997). X-linked retinitis pigmentosa associated with a two base-pair insertion in codon 99 of the RP3 gene RPGR. Archives of Ophthalmology 115: 1429–1435.

Gyrate atrophy is an autosomal recessive form of diffuse choroidal atrophy caused by mutations of the gene (OAT ) for ornithine-@-aminotransferase (OAT). The deficiency of this enzyme results in elevated plasma and tissue levels of ornithine, which exert a cytotoxic effect on the RPE, possibly by endpoint inhibition of a common intermediate for proline synthesis, L-D1-pyrroline-5-carboxylic acid (P5C), which is normally formed from ornithine by OAT and from glutamic acid by P5C synthase. The early stage of gyrate atrophy is associated with sharply demarcated areas of peripheral chorioretinal atrophy. Later stages develop more diffuse and generalized total vascular choroidal atrophy. Dietary restriction of arginine, the precursor for ornithine, can be beneficial. Additionally, a rare subset of patients has been shown to respond with lowered

Figure 7 An example of sector RP. From Weleber, R. and Evan, K. G. (2006). Retinitis pigmentosa and allied disorders. In: Ryan, S. J. (ed.) The Retina, 4th edn., vol. 1, chap. 17, pp. 395–498. Philadelphia, PA: Elsevier.


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ornithine levels to treatment with vitamin B6 (pyridoxine HCL), which acts as a co-factor for the defective enzyme. Disorders such as gyrate atrophy blur the distinction between primary and secondary retinal degenerations leading to the realization that it is difficult to draw the line between RP and allied diseases (Figure 11). Choroideremia

Figure 8 An example of tapetal-retinal sheen seen in a carrier of X-linked retinitis pigmentosa.

Choroideremia is an X-linked disease, which is caused by a mutation in the CHM gene and leads to progressive degeneration of the retina, RPE, and choroid. The CHM gene encodes the homolog of the Rab escort protein 1 (REP1) which is thought to be important in the function of a Rab geranylgeranyl transferase. Choroideremia can often be mistaken for X-linked RP, as the two diseases can share several features including: nyctalopia, retinal RPE atrophy, pigmentary changes, and decreased ERGs and X-linked inheritance. Unlike the waxy nerve pallor seen in RP, patients with choroideremia will often have a normal-appearing nerve and relative preservation of the macula and peripapillary retina (Figure 12).

Syndromic Forms of RP Abetalipoproteinemia

Figure 9 Example of pigmented paravenous retinal choroidal atrophy.

Figure 10 A fundus photograph of fundus albipunctata. Note the characteristic punctate white dots.

Also known as Bassen–Kornzweig syndrome, abetalipoproteinemia results from a deficiency of beta lipoproteins, which are necessary for normal absorption of fat-soluble vitamins from the gut, leading to poor absorption of vitamins A, D, E, and K. The syndrome is characterized by low levels of fat-soluble vitamins, ataxia, acanthocytosis,

Figure 11 Gyrate atrophy. Note the scalloped peripheral areas of chorioretinal degeneration as well as central atrophy.

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protein product, frataxin. The resulting disease causes muscle weakness, ataxia, cardiac hypertrophy, deafness, and retinal degeneration. Vitamin E Deficiency Mutations in the alpha-tocopherol transferase protein gene lead to vitamin E deficiency and cause a Friedreich-like ataxia associated with RP. Treatment with oral vitamin E has been shown to halt both the neurological and visual manifestations of this disease. Incontinentia Pigmenti (Bloch–Schulzberg Syndrome)

and RP. Treatment with combined vitamin A and vitamin E has been shown to prevent or slow retinal degeneration and, in rare patients, reverse the dark adaptation and ERG defects.

Incontinentia pigmenti (Bloch–Schulzberg syndrome) is an X-linked dominant disorder caused by mutations in the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) essential modulator gene, NEMO, and is usually lethal in males. Affected females demonstrate abnormal teeth and nails, hyperpigmentation of the skin, and central nervous system defects. Retinal findings in these patients include peripheral telangiectasias, hypopigmentation of the fundus, and a pigmentary retinopathy.

Alstro¨m Syndrome

Joubert Syndrome

Alstro¨m syndrome is an autosomal recessive disease characterized by deafness, obesity, diabetes, cardiomyopathy, and RP. This syndrome shares many overlapping features with Bardet–Biedl syndrome (BBS) and, indeed, much like the genes implicated in BBS, the gene involved in this syndrome, Alstro¨m syndrome 1 (ALMS1), is thought to play an important role in the structure and function of the cilium.

Also known as cerebellooculorenal syndrome, Joubert syndrome is an autosomal recessive disease caused by mutations in 11 different genes. One causative gene, the centrosomal protein 290 (CEP290), has also been associated with Senior–Loken syndrome and nonsyndromic Leber congenital amaurosis. Patients with Joubert syndrome have hypoplasia of the cerebellar vermis, and also renal problems and retinal dystrophy. The hypoplasia of the cerebellar peduncles of the midbrain creates a characteristic radiological finding on CT scans termed the molar tooth sign.

Figure 12 An example of choroideremia demonstrating large areas of chorioretinal atrophy with some macular sparing.

Bardet–Biedl Syndrome The details on Bardet–Biedl syndrome are discussed elsewhere in this encyclopedia. Chronic Progressive External Ophthalmoplegia/ Kearns–Sayre Syndrome Chronic progressive external ophthalmoplegia (CPEO) and Kearns–Sayre syndrome are mitochondrial myopathies that cause progressive muscle paralysis and pigmentary retinal degeneration. Kearns–Sayre syndrome is associated with sudden cardiac death. Friedreich’s Ataxia This is an autosomal recessive neurodegenerative disease caused by a trinucleotide repeat expansion in an intron of the frataxin gene (FXN), leading to silencing of the gene’s

Mucopolysaccharide Disorders Mucopolysaccharide (MPS) disorders can also present with a pigmented retinopathy, some examples of which are Hurler syndrome (MPS type IH), Scheie syndrome (MPS type IS), and Hunter syndrome (MPS type II). Neuronal Ceroid Lipofuscinosis (Batten Disease) Neuronal ceroid lipofuscinosis (Batten disease) is a group of autosomal recessive neurodegenerative diseases that result from the accumulation of lipofuscin. This disease is characterized by vision loss, seizures, progressive motor and cognitive dysfunction, and retinal degeneration. Eight genes (CLN1–CLN8) have been associated with this disease.


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Infantile Refsum Disease

Dark Adaptation

This disease is caused by defective peroxisomes and patients with this disease present with RP, hearing loss, hepatomegaly, and mental retardation. Unlike the adult form of the disease where specific peroxisomal enzymes are defective, the infantile form is characterized by a complete defect in perixosomal biogenesis. Levels of phytanic acid, very long chain fatty acids, and pipecolic acids are elevated. The disease is ultimately fatal.

Dark adaption is measured with a Goldmann–Weekers dark adaptometer. Each eye is tested separately by first bleaching the rod and cone photopigments with an intense light and then measuring the brightness of a second light needed to achieve a threshold response. A dark adaptation curve can be drawn by repeating this threshold measurement over time after the bleaching light has been turned off. The normal recovery curve can be separated into two segments. The first segment occurs as cones recover from the bleaching light. The rods are much slower to recover and contribute to the second segment of the curve.

Adult Refsum Disease This disease is an autosomal recessive peroxisomal disorder characterized by elevated levels of phytanic acid, which lead to RP, anosmia, deafness, ataxia, cardiac arrhythmias, skeletal anomalies, and polyneuropathy. Mutations in the gene PAHX, which encodes phytanoylCoA 2-dydroxylases, have been shown to be responsible for some cases of adult Refsum disease. Treatment for adult Refsum disease includes plasmapheresis and dietary restrictions of phytanic acid and its precursors. Senior–Loken Syndrome This syndrome is an autosomal recessive disease that also falls under the umbrella of ciliopathies and can share some of the same features as Joubert syndrome (see the section titled ‘Syndromic forms of RP’). This disease is characterized by Leber’s congenital amaurosis and nephronophthisis (cystic kidneys). Spinocerebellar Ataxia Type 7 This is an autosomal dominant neurodegenerative disease caused by a trinucleotide expansion in the ataxin 7 gene (ATXN7). The disease is characterized by cerebellar ataxias, dysphagia, dysarthria, and a cone–rod dystrophy.

Visual Fields The details on visual fields are discussed elsewhere in this encyclopedia.

ERG Terminology The ERG is a fundamentally important test for studying RP and allied disorders. Different forms of RP can be classified based on the ERG response. Just as the electrocardiogram (EKG) measures the electrical activity of the heart through surface electrodes placed on the chest, the ERG measures the electrical activity generated by the retina to flashes of light through electrodes placed on the eye. The standard ERG is most commonly measured using an electrode embedded in a contact lens. Reference electrodes are placed on the forehead and ears. The International Society of Clinical Electrophysiology in Vision (ISCEV) has developed standardized methods for eliciting and recording the ERG.

Usher Syndrome The details of Usher Syndrome are discussed elsewhere in this encyclopedia.

Visual Testing in RP Terminology of Light Adaptation The retina can respond to an astonishing 9 log units of light. Rods and cones differ in their sensitivity, temporal characteristics, and response to background lights. Depending on the intensity of a stimulus and background light present, different classes of cells respond. Scotopic refers to conditions under which only rods are functional. Mesopic refers to conditions under which both rods and cones are functional. Photopic refers to conditions under which only cones are functional.

Full-Field ERG With the full-field ERG, flashes of light are delivered into a Ganzfeld diffuser, which ensures a uniform distribution of the light to the retina. It is important to realize that the full-field ERG measures a summed response from all of the cells in the retina. Therefore, the visual acuity and the ERG response may not correlate. For example, a patient could have a small scar in the center of the fovea that significantly decreases the vision, but the recordings of the ERG could be normal; the opposite can also be true. A patient could have an extinguished ERG due to peripheral degeneration, but still maintain 20/20 visual acuity due to preservation of the central fovea. For this reason, it is important to correlate visual acuity and ERG recordings along with visual-field results.

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100 µV

b-wave

(c)

b-wave

b-wave

50 ms

50 µV

200 µV

a-wave (a)

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50 ms

a-wave (d)

20 ms

Figure 13 Components of a normal ERG. (a) Scotopic response to bright flash, which stimulates both rods and cones. Note the mixed a-wave, b-wave, and oscillatory potentials (superimposed on b-wave). (b) Scotopic response to a dim white flash, which stimulates only the rods. Note the rod-driven b-wave. (c) Photopic response to a 30-Hz flash, which isolates cones. (d) Response to a single flash under photopic conditions, which also isolates cones. Note the cone a-wave, b-wave, and oscillatory potentials (superimposed on b-wave).

Multifocal ERG The full-field ERG can demonstrate a large disparity between the ERG signal and the visual acuity. To better detect localized retinal dysfunction in the macula, the multifocal ERG (mfERG) is very useful. The mfERG uses an m-sequence-derived check board pattern to selectively stimulate and isolate electrical activity from the retina. Specifically, the mfERG tests the macula under mesopic or photopic conditions, and therefore is primarily indicative of macular cone function. Rod-Isolated ERG Response The ERG can be recorded under dark-adapted (scotopic) conditions or light-adapted (photopic) conditions. When dim flashes are delivered under scotopic conditions, only the rod photoreceptors are stimulated. Such a flash elicits a positive electrical potential termed the b-wave. The scotopic b-wave arises from responses elicited by the rod bipolar cells. As the intensity of the flash increases, the b-wave grows in amplitude and, at higher intensities, a negative potential generated by the rod photoreceptors emerges and is termed the a-wave. Oscillations imposed on the b-wave are generated by higher-order retinal circuitry and are termed the oscillatory potentials.

The amplitude from baseline to the peak of the a-wave is termed the saturated a-wave amplitude and the time to the peak of the a-wave is the implicit time. The b-wave amplitude is measured from the trough of the a-wave to the peak of the b-wave.

Cone-Isolated ERG Response The electrical activity of the cone photoreceptors can be isolated in two ways. The first is to measure the response to 30-Hz flicker flashes for which the cones have sufficient time to recover but the rods do not. The repetitive stimulus elicits a sinusoidal-like response. The peak-to-peak amplitude and the time to peak of this response relative to the stimulus can both be measured. The second method is to turn on a background light, which saturates the rod photoreceptors but minimally affects the cones (photopic conditions). Flashes under these conditions will elicit a cone-driven a-wave and b-wave as well as oscillatory potentials (Figure 13). See also: Anatomically Separate Rod and Cone Signaling Pathways; Non-Invasive Testing Methods: Multifocal Electrophysiology; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration.

Mixed Rod–Cone ERG Response At even higher intensities, both the rod and cone photoreceptors are stimulated to generate a mixed scotopic response. Contributions from the cones contribute to the growing a-wave, while activity of the cone bipolar cells contributes to the b-wave. Once the intensity of the light has become intense enough to saturate the photoreceptors, the a-wave will cease to increase in amplitude.

Further Reading Berson, E. L., Rosner, B., Sandberg, M. A., et al. (1993). A randomized trial of vitamin A and vitamin E supplementation for retinitis pigmentosa. Archives of Ophthalmology 111: 761–772. Berson, E. L., Rosner, B., Sandberg, M. A., et al. (2004). Clinical trial of docosahexaenoic acid in patients with retinitis pigmentosa receiving vitamin A treatment. Archives of Ophthalmology 122: 1297–1305.

Primary Photoreceptor Degenerations: Terminology Fishman, G. A., Farber, M. D., and Derlacki, D. J. (1988). X-linked retinitis pigmentosa. Profile of clinical findings. Archives of Ophthalmology 106: 369–375. Grant, C. A. and Berson, E. L. (2001). Treatable forms of retinitis pigmentosa associated with systemic neurological disorders. International Ophthalmology Clinics 41: 103–110. Grover, S., Fishman, G. A., Anderson, R. J., et al. (1999). Visual acuity impairment in patients with retinitis pigmentosa at age 45 years or older. Ophthalmology 106: 1780–1785. Hamel, C. P. (2007). Cone rod dystrophies. Orphanet Journal of Rare Diseases 2: 7. Hartong, D. T., Berson, E. L., and Dryja, T. P. (2006). Retinitis pigmentosa. Lancet 368: 1795–1809. Heckenlively, J. R. (1988). Retinitis Pigmentosa. Philadelphia, PA: Lippincott. Hoffman, D. R., Locke, K. G., Wheaton, D. H., et al. (2004). A randomized, placebo-controlled clinical trial of docosahexaenoic acid supplementation for X-linked retinitis pigmentosa. American Journal of Ophthalmology 137: 704–718. Radu, R. A., Yuan, Q., Hu, J., et al. (2008). Accelerated accumulation of lipofuscin pigments in the RPE of a mouse model for

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ABCA4-mediated retinal dystrophies following vitamin A supplementation. Investigative Ophthalmology and Visual Science 49: 3821–3829. Sieving, P. A., Caruso, R. C., Tao, W., et al. (2006). Ciliary neurotrophic factor (CNTF) for human retinal degeneration: Phase I trial of CNTF delivered by encapsulated cell intraocular implants. Proceedings of the National Academy of Sciences of the United States of America 103: 3896–3901. Weleber, R. G. and Gregory-Evans, K. (2006). Retinitis pigmentosa and allied disorders. In: Ryan, S. J. (ed.) The Retina, pp. 395–498. Philadelphia, PA: Elsevier.

Relevant Websites http://www.ncbi.nlm.nih.gov – National Center for Biotechnology Information, OMIM. http://www.sph.uth.tmc.edu – The University of Texas School of Public Health, Retinal Information Network (Retnet).

Proliferative Vitreoretinopathy P Hiscott, University of Liverpool, Liverpool, UK; Royal Liverpool University Hospital, Liverpool, UK D Wong, University of Hong Kong, Hong Kong, People’s Republic of China ã 2010 Elsevier Ltd. All rights reserved.

Glossary Cytokeratins – A family of proteins found in the cytoskeleton (intermediate filaments) of epithelial cells. Glial fibrillary acidic protein – A protein found in the cytoskeleton (intermediate filaments) of glial cells. Immunohistochemistry – The localization of antigens, especially proteins, in tissue sections by antigen-antibody reactions. The reaction sites are visualized by a label such as a chromogen (typically a brown or red dye). Myofibroblast – A fibroblast-like cell with some of the features of smooth muscle, including contractile properties.

Introduction In 1983, the Retina Society Terminology Committee published a landmark paper in which the term proliferative vitreoretinopathy (PVR) was proposed for a condition that had been recognized as the major cause of failure of retinal detachment surgery. Previous names for this disease included preretinal organization, massive vitreous retraction (MVR), massive preretinal retraction (MPR), and massive periretinal proliferation (MPP) – terms that highlighted some of the main clinical features of the disorder. In this condition, following a retinal detachment, cells leave their normal location in the retina and migrate to the retinal surfaces. Here, the cells proliferate to form membranes. Although many of these membranes consist of only a thin layer of (glial) cells and produce no clinical problems or symptoms, in about 10% of retinal detachment patients the membranes develop into thicker scar-like tissues that are able to contract. It is this ability of the membranes to contract that leads to the clinical picture of PVR.

Definition PVR is strictly defined as a complication of rhegmatogenous retinal detachment that is characterized by the formation of membranes on both surfaces of the detached retina and on the posterior surface of the detached vitreous gel.

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Location of PVR Membranes The membranes on the vitreous surface of the retina are usually called epiretinal membranes and these are the most common membranes of PVR. Membranes that form beneath the detached retina (i.e., between the neuroretina and the retinal pigment epithelium) are termed sub- or retro-retinal membranes. Membranes on the posterior surface of the vitreous gel are known as posterior hyaloid membranes and typically are continuous with epiretinal membranes in PVR (hence they tend to have a similar composition). The expression periretinal membrane is sometimes used to encompass all membranes around the retina. In addition, membranes can extend into the vitreous base and anteriorly over the pars plicata to the back of the iris: a condition known as anterior PVR. There is also evidence that PVR can have a distinct component within the neuroretina itself – a situation that may be called intraretinal PVR (iPVR). It is clear that cellular proliferation can occur at several of the above sites in the same eye. It is also important to recognize that membrane formation can occur in a wide variety of diseases other than retinal detachment. For example, the ischemic retinopathies can also lead to epiretinal and posterior hyaloid membranes although the membranes in these conditions are usually heavily vascularized and thus differ from PVR membranes (see below).

The Significance of Membrane Formation in PVR In PVR, the membranes may contract so that they exert traction on adjacent tissues. Epiretinal membranes tend to produce tangential traction on the retinal surface. The effect of this retinal traction is to cause retinal folding and/or (re)detachment of the sensory retina (Figure 1). Such an effect can be localized, as for example when an epiretinal membrane over the macular (epimacular membrane) causes folding of the macular (macular pucker) or in a peripheral membrane causes a star fold (Figure 2). On the other hand, epiretinal membranes may be diffuse, rather than localized, covering much of the retinal surface. The traction from epiretinal membranes can be mild, causing no more than subtle surface wrinkling of the inner retina. Moderate traction can cause marked folds of the retina. Severe traction can be associated with


displacement of retinal vessels and ectopia of the fovea. The effect of the traction is, however, not just two dimensional (2-D). The newer generation of Optical Coherence Tomography systems provides high-resolution crosssectional views or reconstructed 3-D images of the retina (Figure 2). These images clearly show that the traction may be superficial but sometimes the whole thickness of the retina is involved. Thus, these signs can indicate the development of PVR. Clinically, we often see the effect of traction rather than the epiretinal membranes themselves: on the attached retina, epiretinal membranes can be difficult to visualize (Figure 1). Nonetheless, the epiretinal membranes are present and exerting isometric’ traction on the retina. The effect of this traction may only become apparent once the retina becomes detached, at which point one may observe star folds or retinal breaks with rolled edges when the traction in the tissue is focal. In addition,

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when the epiretinal membrane is diffuse, the detached retina will appear to have reduced mobility. Anterior PVR membranes will draw the pre-equatorial retina anteriorly toward the ciliary processes, whereas epiretinal membranes in the post-equatorial retina tend to contract the retina into a cone configuration. The combination of the anterior and posterior traction systems on a totally detached retina will give rise to a so-called closed funnel (Figure 3). Subretinal membranes, particularly in the form of bands, are apt to elevate the neuroretina like a sheet on a washing line or, in the case of a circular band, like a napkin in a napkin ring. Posterior hyaloid membranes can produce circumferential or, if the vitreous base is involved, anteroposterior traction. Particularly in eyes that have undergone previous vitrectomy or trauma, proliferative tissue can extend into the vitreous base and anteriorly toward the pars plicata of the ciliary body, the iris and even as far as the pupil margin. Traction in this latter situation may lead to traction on the ciliary body (hypotony can result) and posterior displacement of the iris.

The Cells Involved in PVR It is now accepted that PVR combines reaction to damage by astrocytes of the central nervous system (gliosis) with fibrosis. In this context, the gliosis involves Mu¨ller cells and retinal astrocytes, while the fibrosis includes metaplasia or transdifferentiation of the retinal pigment epithelial (RPE) cells. Although retinal glia and RPE cells are thus major players in PVR, it is also apparent that a variety of other cell types are involved in the disease.

Figure 1 Fundus photograph showing detached, inferior retina with diffuse PVR.

Figure 2 OCT image of retina showing a thin epiretinal membrane and subjacent retinal distortion.

Figure 3 Section through an eye with PVR and a closed-funnel retinal detachment. Retinal folds can be seen. The eye is aphakic and there are anterior PVR membranes that also involve the iris.

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Glial Cells There is compelling evidence from a variety of morphological, immunohistochemical, in vitro, and experimental studies that astrocytes and Mu¨ller cells are involved in PVR membranes, particularly in epiretinal and, to a lesser extent, subretinal membranes.

Within PVR epiretinal membranes, glial cells often form layers in the tissue (Figure 4). These layers are adherent to fibrous components of the membranes and, in both human and experimental PVR, the cells can sometimes be traced through defects in the retinal inner limiting lamina into the retina itself. These observations

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Figure 4 (a–h) Sections through neuroretina (nr) with PVR membranes in enucleated eyes. The sections have been stained with the immunohistochemical method for glial elements with an antibody to glial fibrillary acidic protein (see insets in (a–c) , and also (e) and (g)) or for RPE elements with antibody to cytokeratin 7 ((c), (d), (f), and (h)) or to a range of cytokeratins (inset in (h)): red-brown reaction product, hematoxylin counterstain. (a) The retina is gliotic and there is an epiretinal membrane ‘e’ composed of a glial monolayer. The internal limiting lamina is marked (arrows). Inset: this epiretinal membrane is composed of a double layer of glia and there is some distortion of the internal limiting membrane (arrowheads). (b) The epiretinal membrane ‘e’ is composed of glial and nonglial cells. Again, the underlying retina is gliotic. (c and d) Epiretinal RPE cells are seen. The subjacent retina is gliotic (inset in (c)). The internal limiting lamina is marked (arrows). Parts (e) and (f) show the same area of the same specimen: the epiretinal membrane contains both glial (e) and RPE (f) components. The internal limiting lamina is marked (arrows). Parts (g) and (h) show the same area of the same specimen: the retina is folded, disorganized, and gliotic. There is a PVR membrane containing fibroblastic RPE cells and a few glia. Some of the fibroblast-like cells do not label for glial or RPE markers, even with an antibody that reacts with a range of cytokeratins (inset in (h)): their origin is unclear. Scale bars: (a–f) 50 mm; (g and h) 100 mm.


have led to the concept that glial cells traversing the vitreoretinal interface serve to anchor the epiretinal membrane to the retina and that the epiretinal glia may form a substrate or scaffold that other cells might use to produce the rest of the epiretinal tissue. In support of this notion, experimental models have demonstrated that glial cells can break through the retinal inner limiting lamina early in the formation of epiretinal membranes and spread across the retinal surface to form sheets that other cells may adhere to. Failure of glial sheets to become populated by other cells may account for arrest of the disease process at an early stage and the production of asymptomatic or nonproblematic subtle membranes on either surface of the neuroretina. RPE Cells The same sorts of methods that were used to detect glia in PVR membranes have been employed to demonstrate RPE cells. Indeed, it is now widely accepted that RPE cells are major components of PVR membranes and the presence of large numbers of RPE cells in epiretinal membranes is one of the distinguishing factors between PVR membranes and epiretinal membranes caused by other diseases. Early in the development of PVR, RPE cells leave their normal location at the chorioretinal interface and either migrate or are swept with subretinal fluid movements to the surfaces of the detached retina. Here, the cells may attach to early membranes (Figure 4). In the case of epiretinal membranes, RPE cells may adhere to glia that have already arrived, while in subretinal membranes it has been suggested that the cells may in addition settle on fibrin deposits. Some RPE cells may also migrate into or through the neuroretina. RPE cells have a remarkable propensity to undergo metaplasia or transdifferentiation. As a result, an RPE cell may change from a polarized, sedentary pigmented cuboidal epithelial cell to a nonpigmented fibroblastic cell, a migratory macrophage-like cell, a cell in a gland-like structure or even a bone-forming cell (Figure 4). In PVR membranes, RPE cells most often adopt fibroblastic or macrophagic phenotypes, though they may attempt to (re-)form a polarized monolayer as well. Fibroblastic Cells PVR membranes often have a substantial fibrous element that consists of fibroblastic cells in extracellular matrix. Many of these cells are of RPE origin (Figure 4). Others may be derived from perivascular sources such as adventitial cells of larger retinal vessels. There has been much interest in the role of fibroblastic cells in PVR membranes for two reasons. First, they are believed to be responsible for generating most of the tractional forces within the tissue. Second, the cells are

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thought to be responsible for the production of the bulk of the extracellular matrix in the membranes. There is still debate about how these cells may produce traction: theories include smooth muscle-like contraction of the (myofibroblastic) cells and cell–matrix interactions that cause shortening of matrix elements. Macrophages In addition to macrophagic RPE cells, macrophages of hematogenous origin are involved in PVR. Macrophages are especially abundant in membranes arising in the presence of some tamponade agents (see below). Irrespective of their origin, macrophages are believed to have a number of important roles in PVR. In the early stages of the disease, they produce mitogens, chemotactic agents, and growth factors that probably have a role in cell recruitment to and proliferation in the membranes. Growth factors and enzymes produced by macrophages later in the disease process may be involved in matrix synthesis and remodeling. Vascular Elements Blood vessels are found in around 10–20% of PVR membranes but, in contrast to periretinal membranes of conditions such as central retinal vein occlusion and proliferative diabetic retinopathy, blood vessels usually do not form a major component of the tissue. However, vessels are probably more abundant in anterior PVR membranes (Figure 5). Other Cells T lymphocytes, including CD4 and CD8 positive cells, have been found in PVR membranes. Some of these cells express interleukin-2 receptor, suggesting that they are activated and capable of promoting the cellular events in the tissue. Ciliary body epithelial cells have been reported in the membranes of anterior PVR (Figure 5). Hyalocytelike cells have been described in epiretinal membranes generally and it has been suggested that hyalocytes may give rise to some of the fibroblastic and macrophagic cells in the epiretinal tissues. Recently, ganglion cell neurites have been observed in experimental and human epi- and subretinal membranes. They are co-localized with glia, suggesting active outgrowth into the periretinal tissues.

The Extracellular Matrix in PVR Membranes PVR membranes contain a matrix that, in terms of composition, is similar to the one seen in a healing skin wound (Figure 6). These components include structural proteins

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Figure 5 Anterior PVR membrane extending over distorted ciliary epithelium (CE) in an enucleated eye. (a) Stained with the immunohistochemical method for the endothelial marker CD34: note that there are blood vessels in the membranes (arrows). (b) Stained with the immunohistochemical method for cytokeratins: note that in addition to cells in the membranes (arrowheads), the CE normally expresses cytokeratins too. Thus, it is possible that at least some of the epithelial cells in anterior PVR membranes are of CB rather than RPE origin (hematoxylin counterstain, scale bar: 200 mm).

such as collagens and elastic fiber precursors, adhesive glycoproteins like fibronectins and laminins, glycosaminoglycans, matricellular proteins such as tenascins and thrombospondins, and matrix enzymes like matrix metalloproteinases together with their inhibitors. Much of the matrix in PVR is produced locally by the membrane cells themselves, though there is evidence that some components enter from elsewhere. For example, some blood derivatives like plasma fibronectin are found in PVR membranes. Irrespective of origin, the extracellular matrix in PVR membranes increases with time and there is a corresponding decrease in cellularity of the tissue. Indeed, this change in cellularity together with the contractile nature of the tissue and the presence of fibroblastic cells in matrix gave rise to comparisons between PVR membranes and healing wounds. As in healing wounds generally, the matrix is more than a passive space filler: there is good evidence that PVR matrix is an important regulator of cell behavior (notably migration and proliferation) in the tissue. For example, there have been a number of reports demonstrating that cells in PVR membranes express a range of cell-surface receptors for the various matrix components around them (e.g., integrins) and experimental studies implicating the importance of such receptors in contraction in PVR models. Moreover, early matrix may have adhesive properties that aid cohesion of the developing tissue. There is also evidence of matrix remodeling in more established membranes. Longstanding PVR membranes are often densely fibrous. It is also worth noting that, when surgically excised, PVR membrane specimens often contain internal limiting membrane from the retina. This material tends to become convoluted in the tissue (Figure 6), presumably as a result of tractional forces upon it.

Pathogenesis and Natural History

Figure 6 Section through a surgically excised PVR epiretinal membrane. The tissue contains a fibrous element (stars). Convoluted retinal internal limiting membrane is also present (arrows). Periodic acid Schiff reagent, hematoxylin counterstain. Scale bar: 50 mm.

Although the pathogenesis of PVR is not fully elucidated, our understanding of the disease is at the point where logical therapies can be designed and implemented. Fundamental to the development of PVR is retinal detachment, itself dependent upon degenerative changes in the vitreous and retina. Vitreous degeneration (syneresis) is a normal aging process that can be accelerated by conditions such as myopia or trauma and results in the formation of fluid and formed components. Attachments between formed vitreous and retina may permit rotational tractional forces, such as dynamic traction from saccadic eye movements, to be transmitted between the two structures. In turn, a retinal tear may form and fluid vitreous pass through the hole in the retina to give rise to a (rhegmatogenous) retinal detachment. There is experimental evidence that some changes associated with PVR can occur within a day of retinal


detachment. In retinal detachment, together with entry of fluid vitreous to the subretinal space, there is breakdown of the blood–retina barrier with accumulation of plasma proteins in the vicinity of the retina and influx of blood-borne inflammatory cells. Thus, there is aggregation of hematogenous and locally derived proteins and cytokines, including plasma glycoproteins, and growth and differentiation factors. Many of the components of this collection have chemotactic and/or mitogenic properties for RPE and glial cells. In fact, RPE cells can be observed to detach from Bruch’s membrane and may be transported to the surfaces of the detached retina while glia may breech the retinal inner limiting lamina (see above) in retinal detachment. The above reaction to retinal detachment might set the scene for PVR formation, as we have seen only a minority of retinal detachment patients develop the condition. It thus appears that additional factors are required and, indeed, a number of clinical risk factors have been identified (see below). These include factors that would be expected to elevate the concentrations of chemotactic and mitogenic chemical mediators and increase the influx of inflammatory cells to the retinal surfaces, such as hemorrhage, multiple surgical interventions, and large surface area of RPE cells exposed to detachment. Cells displaced to the surfaces of the detached retina can be seen to adhere to each other as well as the retinal surface. These very early membranes lack matrix but they do possess adhesive glycoproteins like fibronectins. Moreover, a number of matricellular proteins including thrombospondin 1 are also present. It has been hypothesized that fibronectin and thrombospondin 1 can form a provisional matrix in healing wounds. Thus, it is possible that they provide some integrity to developing PVR membranes as well as an adhesion substrate for the cells. Once in the developing membranes, the cells proliferate. Proliferation is assumed to increase the amount of membrane tissue and it can be detected as long as a year after the onset of the disease. In this respect, PVR differs from skin wounds where proliferation is restricted to a short wave early in the process. It is thought that cells also migrate along the retinal surfaces toward developing membranes, perhaps in response to local chemotactic agent production in the new tissue. In addition to cell recruitment, cell migration in PVR membranes might also be a mechanism by which tractional forces are generated (motile cells impart a force on their substrate). Thus, it may be that cell migration, myofibroblastic contraction, and cell–matrix interactions (see above) can all be involved in membrane contraction. The buildup of extracellular matrix with time in PVR membranes is matched by a reduction in cellularity of the tissue (Figure 6). Cell loss in PVR membranes probably occurs through apoptosis and nonapoptotic pathways. Ultimately, untreated PVR membranes become paucicellular and fibrous in nature.

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Intraretinal PVR The concept that retina-shortening cellular changes may occur within the neuroretina itself in PVR is relatively recent. Nevertheless, it is now clear that several cell types, including Mu¨ller cells and astrocytes, not only become reactive but also do replicate in the retina in PVR and it is thought that this gliosis contributes to retinal shortening. RPE cells are also involved in this process, although their numbers appear to be small compared to the numbers in periretinal membranes. Moreover, gliosis, with or without epiretinal and/or subretinal membranes, can cause marked retinal distortion and localized retinal thickening that can lead to the formation of a focal mass (Figure 7). Indeed, similarities in the microscopic appearances of localized PVR masses and vasoproliferative tumors of the retina, including the presence of RPE cells in both lesions (Figure 8), have led to speculation that some vasoproliferative tumors may be part of the spectrum of PVR.

Incidence and Risk Factors It is often stated that PVR afflicts around 10% of all patients with retinal detachment. However, it is clear that some patients are at much greater risk of developing the condition than others. The most important risk factor for postoperative PVR seems to be preoperative PVR. A variety of clinical risk factors have been identified or suggested, including size and number of retinal holes, extent and duration of the detachment, the presence of blood and/or intraocular inflammation, aphakia, preoperative choroidal detachment, early stages of the disease or poor visual acuity prior to initial surgery, and multiple previous attempts at re-attaching the detached

Figure 7 Gliotic, disorganized, thickened retina that has full-thickness folds in association with epiretinal membrane (E), in a section of an eye removed for complications of PVR. The retinal changes give rise to the formation of a localized mass (hematoxylin and eosin; scale bar: 500 mm).

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Figure 8 Section showing part of a vasoproliferative tumor (reactive retinal glioangiosis) in an eye removed for complications of retinal detachment. The lesion contains scattered RPE cells (arrowheads), revealed (red) by immunohistochemical staining for cytokeratin 7 (hematoxylin counterstain). A few of these cells contain melanin pigment (arrows). Staining of the lesion for glial cells (inset: immunohistochemical staining red, hematoxylin counterstain) confirms that the retinal tissue is gliotic and disorganized. Scale bar: 200mm

retina. Methods employed during the primary retinal detachment surgery may also increase the risk of PVR. Thus, there is evidence that cryopexy, choice of tamponade agent, and vitrectomy impact on the risk of developing PVR.

Clinical Classification of Proliferative Vitreoretinopathy There are several attempts to classify PVR according to the clinical features of the retinal detachment. These classifications are descriptive and help surgeons communicate, especially with regard to surgical approaches in the management of the condition. Thus, they are useful for surgical planning and are not based on pathobiology. PVR classifications are also not prognostic. They do not correlate well with visual prognosis or anatomical success with treatment. The classification is also not related to the stages of the disease. It is just the clinical picture at one snapshot in time. Despite their many shortcomings, clinical PVR classifications are widely used in clinical trials and for clinicopathological correlates. Several classifications have been suggested, based on the clinical manifestations of the disease. For the most part, there is commonality between these systems with regard to the earlier or milder stages of the disease, whereas the schemes tend to diverge in their classification of later or more advanced stages of PVR. Within these systems, stage A (minimal) is usually regarded as the presence of vitreous haze and pigment

clumps, whereas stage B (moderate) is typically recognized as wrinkling of the retinal surface, decreased vitreous mobility, and increased retinal stiffness. With respect to the more severe or later stages of PVR, some schemes separate the advanced stages by the number of quadrants of the retina involved. Thus, for example, the Retina Society Terminology Committee classification of 1983 associates stage C (marked) disease with fixed retinal folding and adds a number to reflect the number of quadrants involved (e.g., C-3 is fixed folds involving three quadrants of retina). In this system, stage D (massive) reflects the involvement of all four retinal quadrants. In addition, stage D is graded 1–3 depending on how extensive the folding of the retina is (D-1 being an open funnel of totally detached retina, D-3 being a closed funnel so that the optic nerve head cannot be seen: Figure 3). Other schemes classify the more severe stages of PVR into anterior and posterior groups, according to the location of the disease with reference to the retinal equator. In these systems, the number of retinal quadrants involved by the disease is again used so that PVR involving fixed folds in, say, two quadrants of retina posterior to the equator would be classed P2 or CP2 (stage D is generally discarded in these schemes). The schemes employing anterior and posterior also add a contraction type, depending on the extent of epiretinal membrane (focal or diffuse), the presence of subretinal membrane, or posterior hyaloid/vitreous base proliferation.

Management There is much interest in preventing proliferation of membranes after retinal detachment surgery by treating high-risk patients with combinations of agents. One such combination is the antiproliferative drug 5-fluorouracil and low-molecular-weight heparin (which binds growth factors). This combination has been shown to reduce the incidence of PVR in high-risk retinal detachment patients undergoing vitrectomy. Once established, PVR membranes are removed by microsurgery so that the retina can be reattached. Again, there is much interest in the use of pharmacological measures to stop membrane recurrence after PVR surgery. For example, there is evidence that daunomycin used preoperatively can reduce the requirement for repeat surgery in these patients. Tamponade agents to maintain retinal attachment are frequently employed during and after surgery for retinal detachment and PVR. These agents incorporate gases (e.g., air) and liquids (e.g., silicone oil). Liquids have a tendency to emulsify in the eye, particularly if they are of low viscosity. The result is the formation of droplets of various sizes in the vitreous cavity or even elsewhere in the eye (such as in the aqueous if tamponade gains access


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Figure 9 Photomicrographs from sections of an eye removed following liquid tamponade use in the treatment of PVR. Parts (a) and (b) show the vitreoretinal interface with numerous vacuolated cells that label for the macrophage marker CD68 (b), but not the glial marker glial fibrillary acidic protein (a). Similar cells are seen in the drainage angle (c) and in the anterior chamber and iris (d). The features are consistent with macrophage reaction to emulsified liquid tamponade agent (hematoxylin and eosin (c); immunohistochemical staining, red reaction product (a), (b), and (d); scale bars (a), (b), and (d): 100 mm; (c) 200 mm).

to the anterior chamber: Figure 9). Thus, emulsification of tamponade agent may impact on the pathology of PVR membranes. It appears that the droplets can stimulate a foreign body-type reaction and attract macrophages to the retinal surface (Figure 9). New membranes may develop and these tissues characteristically have the microscopic appearances of PVR membranes containing granulomata to emulsified oil.

years has greatly increased the final anatomical success rate. Disappointingly, this success rate has not been translated into visual improvement. In retinal detachment patients, the fellow eye is also likely to be involved in sight-threatening pathology so that many PVR patients end up with visual impairment in both eyes.

Outcomes

Despite intense research over the last 25 years that has improved our understanding of the condition, PVR remains the major cause of failure after retinal detachment surgery. Nevertheless, continuing advances in both surgical and pharmacological manipulation of the disease, based on an expanding knowledge of PVR pathobiology, can be expected to reduce the impact of the disease in the future.

Successful treatment in PVR has often been measured in terms of final retinal reattachment rates. The assumption is that effective anti-PVR therapy would lead to an increased rate of successful reattachment. This assumption may or may not hold true. It is often missed or untreated holes that lead to retinal redetachment and not necessarily PVR, which may or may not be controlled by the anti-PVR drugs. Indeed, a totally ineffective antiPVR treatment may be compatible with anatomical success so long as the epiretinal membranes do not act on the retina to produce another retinal break or cause tractional retinal detachment. Hence, anatomical success rate is a poor proxy for PVR control. Another important point is that anatomical success does not guarantee visual recovery. The advancement of surgery in the last few

Conclusions

See also: Rhegmatogenous Retinal Detachment.

Further Reading Asaria, R. H., Kon, C. H., Bunce, C., et al. (2001). Adjuvant 5-fluorouracil and heparin prevents proliferative vitreoretinopathy: Results from a randomized, double-blind, controlled clinical trial. Ophthalmology 108: 1179–1183.

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Charteris, D. G. (1995). Proliferative vitreoretinopathy: Pathobiology, surgical management, and adjunctive treatment. British Journal of Ophthalmology 79: 953–960. Colthurst, M., Williams, R. L., Hiscott, P., and Grierson, I. (2000). Biomaterials used in the posterior segment of the eye. Biomaterials 21: 649–665. Fisher, S. K., Lewis, G. P., Linberg, K. A., and Verardo, M. R. (2005). Cellular remodeling in mammalian retina: Results from studies of experimental retinal detachment. Progress in Retinal and Eye Research 24: 395–431. Heimann, K. and Wiedemann, P. (1989). Proliferative Vitreoretinopathy. Heidelberg: Kaden. Hiscott, P. and Mudhar, H. (2008). Is vasoproliferative tumour (reactive retinal glioangiosis) part of the spectrum of proliferative vitreoretinopathy? Eye 23: 1851–1858. Hiscott, P. and Sheridan, C. (1998). The retinal pigment epithelium, epiretinal membranes and proliferative vitreoretinopathy. In: Marmor, M. F. and Wolfensberger, T. J. (eds.) Retinal Pigment Epithelium – Function and Disease, pp. 478–491. New York: Oxford University Press. Hiscott, P., Morino, I., Alexander, R., Grierson, I., and Gregor, Z. (1989). Cellular components of subretinal membranes in proliferative vitreoretinopathy. Eye 3: 606–610. Hiscott, P., Sheridan, C., Magee, R., and Grierson, I. (1999). Matrix and the retinal pigment epithelium in proliferative retinal disease. Progress in Retinal and Eye Research 18: 167–190.

Kampik, A., Kenyon, K. R., Michels, R. G., Green, W. R., and de la Cruz, Z. C. (1981). Epiretinal and vitreous membranes. Comparative study of 56 cases. Archives of Ophthalmology 99: 1445–1454. Kirchhof, B. and Wong, D. (eds.) (2005) Vitreo-Retinal Surgery. Essentials in Ophthalmology. Berlin: Springer. Machemer, R. and Laqua, H. (1975). Pigment epithelium proliferation in retinal detachment (massive periretinal proliferation). American Journal of Ophthalmology 80: 1–23. Pastor, J. C., de la Ruá, E. R., and Martıń, F. (2002). Proliferative vitreoretinopathy: Risk factors and pathobiology. Progress in Retinal and Eye Research 21: 127–144. The Retina Society Terminology Committee (1983). The classification of retinal detachment with proliferative vitreoretinopathy. Ophthalmology 90: 121–125. Wiedemann, P., Hilgers, R. D., Bauer, P., and Heimann, K. (1998). Adjunctive daunorubicin in the treatment of proliferative vitreoretinopathy: Results of a multicenter clinical trial. Daunomycin study group. American Journal of Ophthalmology 126: 550–559.

Relevant Website http://www.youtube.com – Number of videos concerning PVR and its management.

Retinal Cannabinoids S Yazulla, Stony Brook University, Stony Brook, NY, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Age-related macula degeneration (AMD) – It comprises a variety of diseases, mainly of the elderly, that involves loss of vision in the central region of the retina. Endocannabinoids – Natural chemicals in the body that are mimicked by the active component of marijuana. Monoamine oxidase (MAO) – An enzyme that degrades dopamine. Snellen acuity – A test of visual acuity that uses a standard sized ‘‘E’’ in four orientations. Transient receptor potential type vanilloid 1 receptor (TRPV1) – An ionotropric receptor that increases intracellular calcium either by entry through the plasma membrane or from intracellular stores. It is activated by noxious heat, capsaicin, and the endocannabinoid, anandamide. Vernier acuity – A test of visual acuity that tests the ability to detect displacement of two lines end-to-end.

Marijuana and the Endocannabinoids The active component of the marijuana plant Cannabis sativa, D9-tetrahydrocannabinol (THC), mimics endogenous chemicals, endocannabinoids (eCBs) that activate membrane receptors. eCBs include a variety of amide, ester, and ether derivatives of arachidonic acid. The most widely studied of these are arachidonoyl ethanolamide (anandamide, AEA) and sn-2 arachidonoyl glycerol (2-AG) (Figure 1). Other eCBs have been identified with varying degrees, or no affinity for cannabinoid receptors, and also compete with AEA and 2-AG for metabolizing enzymes. In this way, they modulate activity by competition at the receptors or by affecting substrate availability for metabolism. Synthesis and Release Unlike water-soluble transmitters, AEA and 2-AG are lipophilic and not stored in synaptic vesicles. Rather, membrane phospholipids are metabolized on demand to liberate AEA and 2-AG by calcium-dependent phospholipases. The precursor of AEA is N-arachidonyolphosphatidyl ethanolamine (NAPE), formed by calcium-dependent transfer of

arachidonic acid (AA) from arachidonoylphosphatidylcholine to phosphatidylethanolamine (PE). There are multiple pathways for AEA liberation from the membrane. First, NAPE is hydrolyzed by phospholipase D (PLD) to release AEA and phosphatidic acid. Second, NAPE is hydrolyzed to N-acyl-lyso-PE by phospholipase A1/A2; then, AEA is released by lysophospholipase D. Third, phospholipase C (PLC) cleaves NAPE to generate phosphoanandamide, which is dephosphorylated to liberate AEA. The PLC pathway may be involved in the on-demand synthesis of AEA rather than in maintaining basal tissue levels of AEA. The primary pathway for 2-AG synthesis involves hydrolysis of diacylglycerols (DAG) by DAG lipase isozymes, DAGLa and DAGLb. DAGs may be produced by the PLC b-catalyzed hydrolysis of phophotidylinositol or hydrolysis of phosphatidic acid by a phosphohydrolase. AEA and 2-AG freely diffuse within the membrane where they interact with the active sites of degradative enzymes and receptors. AEA binds reversibly to serum albumin, and it is likely that such binding is critical for the movement of AEA and 2-AG in blood, the extracellular matrix, and the cytoplasm. The presence and localization of AEA and 2-AG are inferred from the distribution of receptors, synthesizing and inactivating enzymes as well as physiological effects on identified cells. Inactivation AEA and 2-AG are inactivated following intracellular accumulation by fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MGL), cyclooxygenase-2 (COX-2), and lipoxygenase (LOX). AEA and 2-AG are hydrolyzed by FAAH into AA and ethanolamine or glycerol, respectively. 2-AG, but not AEA, is hydrolyzed by MGL. Following hydrolysis of AEA or 2-AG, AA is incorporated into membrane phospholipids. COX-2 oxidizes arachidonic acid, AEA, and 2-AG to prostamides or prostaglandin glyceryl esters, leading to prostaglandins. In addition, oxidation of AA by LOX produces 12-(S)-hydroperoxyeicosatetraenoic acid (15-(S)-HPETE), 5-(S)-HETE, and leukotriene B4, all of which are agonists of TRPV1 receptors (Figure 2).The effects of AEA and 2-AG are modulated by the balance of metabolic enzymes that is specific to each cell type. Receptors Effects of cannabinoids are mediated by metabotropic (G-protein-coupled receptors (GPCRs)) and ionotropic

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Figure 2 This schematic illustrates some of the metabolic pathways for the degradation of AEA and 2-AG. In the dominant pathways (bold arrows), AEA and 2-AG are hydrolyzed to arachidonic acid (AA), and then rapidly incorporated into membrane phospholipids via N-acyltransferase (NAT) and acyl-Coenzyme A synthetase. Lesser pathways (shaded arrows) involve oxidation by cyclooxygenase-2 (COX-2) of AEA, 2-AG, and AA to prostaglandins (PGE2 and PGD2). Additionally, AA may be oxidized by lipoxygenase (LOX) to 12-(S)- and 15-(S)-HPETE and 5-(S)-HETE. Hollow arrows show that AEA and 2-AG are endoligands for CB1, CB2, and PPAR receptors, while AEA also activates TRPV1 receptors. Metabolites of COX-2 oxidation activate EP2 receptors, and metabolites of LOX oxidation activate TRPV1 receptors.

(ion channel) receptors (Figure 2). In general, activation of cannabinoid 1 receptors (CB1Rs), via heterotrimeric guanosine-5’-triphosphate (GTP)-binding proteins Gi/o (Gi/o), modulates voltage-gated Kþ and Ca2þ conductances, resulting in a reduction of neurotransmitter release, particularly g-aminobutyric acid (GABA) and glutamate. CB2 receptors, which also signal through Gi/o, are expressed in cells of the immune system and the central nervous system (CNS), particularly in astrocytes. There is evidence for additional cannabinoid receptors, perhaps GPCR 55. AEA, but not 2-AG, activates the ionotropic transient receptor potential type vanilloid 1 receptor (TRPV1) that increases intracellular calcium

either by entry through the plasma membrane or from intracellular stores. Prostamides and prostaglandin glycerol esters, produced by eCB oxidation by COX-2, bind to a variety of prostaglandin receptors. eCBs are ligands for peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor superfamily that are involved in lipid metabolism, insulin sensitivity, regulation of inflammation, and cell proliferation. Distribution and Function CB1Rs are the most numerous GPCRs in the brain. eCBs, their receptors, and metabolizing enzymes are enriched in


brain regions associated with the physiological and psychomotor effects of cannabis. AEA and 2-AG have short- and long-term effects on synaptic plasticity and neuroprotection. The effects depend largely on retrograde transmission in which postsynaptic dendrites release an eCB that binds to presynaptic CB1Rs to reduce transmitter release. Retrograde release of eCBs is evoked by two mechanisms. In a voltage-dependent mechanism, depolarization of postsynaptic dendrites by L-glutamate opens voltage-gated calcium channels. The increase in intracellular Ca2þ activates Ca-dependent PLD to release an eCB. A second mechanism involves activation of heterotrimeric GTP-binding protein Gq/11 (Gq/11) coupled metabotropic receptors, usually group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, and muscarinic receptors (M1 and M3). By enzymatic cascades that may or may not release calcium from intracellular stores, eCBs are released from the plasma membrane. The eCB-induced reduction of presynaptic glutamate and GABA release contributes to synaptic plasticity, while the reduction of glutamate release inhibits excitotoxicity following ischemia. Evidence implicates 2-AG more so than AEA in plasticity, while both AEA and 2-AG are involved in neuroprotection.

Cannabinoids and Ocular Tissues Marijuana induces conjunctival vasodilation and reduces intraocular pressure (IOP), but is not a mydriatic. These effects are mediated locally by eCBs as demonstrated in the ciliary body, iris, choroid, and trabecular meshwork in mammalian tissues. THC, as low as 10–12 M, increases monoamine oxidase (MAO) activity in the bovine trabecular meshwork, choroid, and ciliary processes but not in the iris. Hydrolysis of anandamide has been measured in the porcine iris, choroid, lacrimal gland, and optic nerve. CB1 mRNA and CB1R-immunoreactivity (IR) have been detected in the ciliary body, trabecular meshwork, and conjunctival epithelium of rat, mouse, bovine, and human. AEA and 2-AG have been measured by gas chromatography in human ocular tissues. The content of eCBs varies in certain disease states, suggesting the importance of eCBs in maintaining ocular homeostasis. For example, 2-AG levels are lower in the ciliary body of patients with glaucoma. However, in diabetic retinopathy there are higher levels of 2-AG only in the iris, and increased levels of AEA in the retina, ciliary body, and cornea. Eyes of patients with age-related macula degeneration (AMD) also show increases of AEA in the retina, choroid, ciliary body, and cornea. Topically applied AEA reduces IOP by activation of CB1R and activation of the prostaglandin E 2 receptor (EP2R) after conversion of AEA to prostamides (see Figure 2). Administration of either AEA or THC to human nonpigmented epithelium (NPE) cells

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induces COX-2 expression, indicating a relationship among prostaglandins, COX-2, and eCBs in lowering IOP. In addition, EP2 receptors have been localized in the NPE of mouse, porcine, and human ciliary body.

Cannabinoids – Retinal Anatomy Early studies of the effects of cannabis on vision were performed in concert with the effects of alcohol in order to examine the influence on visual motor behaviors as they related to driving. Anecdotal reports also came from studies citing side effects of cannabis when used as an analgesic. Effects on vision are subtle and include blurred and double vision, a reduction in vernier and Snellen acuity, alterations in color discrimination, an increase in photosensitivity and an increase in recovery from foveal glare. It is unlikely that all of these effects of marijuana are due to cortical or preretinal sites because processes of light–dark adaptation take place in the retina. Knockout mice that lack CB1Rs or FAAH are not blind, but the effects on vision have not been studied. Biochemical Assay The first evidence for cannabinoids in the retina was the demonstration that THC induced an increase in MAO activity, indicating a role in dopaminergic transmission. Later, FAAH-mediated hydrolysis of 3H-AEA was shown in homogenates of porcine, bovine, and goldfish retinas. AEA and 2-AG were detected in mammalian retina by gas chromatography. Release of AEA from bovine retinal extracts in a physiological buffer demonstrated that the extracts contained the metabolic machinery necessary for eCB release, the precursor NAPE, and PLD. Localization – Cannabinoid Receptors CB1Rs have been localized by immunohistochemistry in the retinas of numerous species, including human, monkey, mouse, rat, chick, salamander, and goldfish. Despite differences in detail, there is a common theme. In general, the most prominent label is in cells of the through pathway: photoreceptors, bipolar cells, and ganglion cells. Cone pedicles in all species contain CB1Rs. Rod spherules appear to be labeled in all species except goldfish. Ultrastructural analysis has been performed exclusively on goldfish cones. CB1R-IR is on plasma membrane at the perimeter of the pedicle as well as within the invagination. CB1R-IR is not immediately apposed to the synaptic ribbon, but is at some distance from it. Regarding bipolar cells in mammals, CB1R-IR is restricted to rod bipolar cells as confirmed by double labeling with antisera against PKC. In goldfish, there is a higher proportion (3:1) of CB1R-IR in ON bipolar cells compared to OFF bipolar

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cells. This difference holds for mixed rod–cone bipolar cells as well as for cone bipolar cells. CB1R-IR, on the bipolar cell synaptic terminal membrane, is not adjacent to the synaptic ribbons. Rather, the CB1R-IR is always some distance removed from the ribbon, the same as observed for the cone pedicles. Regarding rat horizontal cells, CB1R-IR is confined to the cell bodies and is not present on the dendrites, unlike bipolar cells. CB1R-IR is also found on a population of large amacrine cells, identified in rat as a rare type that is immunoreactive for PKC and GABA. In goldfish, CB1RIR is on presynaptic membrane of amacrine cell boutons. These boutons appear throughout the depth of the inner plexiform layer and are presynaptic to bipolar cell terminals and small processes derived from ganglion cells. It is likely that these CB1R-immunoreactive processes are from a single type of diffuse amacrine cell. CB1R-IR is on Mu¨ller’s cells in goldfish but not in any other preparation. There are inconsistent reports of CB1R-IR in mammalian astrocytes, microglia, and oligodendrocytes. Activation of CB1Rs inhibits excitatory amino acid transport and induces glutamate release from astrocytes in the mammalian brain. CB1R and CB2R are involved in gliotic responses to injury. The interaction of eCBs and glia has not been investigated in the retina. CB2 mRNA was described in all cellular layers of the rat retina; this could include glial labeling, particularly Mu¨ller’s cells. Localization – Metabolizing Enzymes There is relatively little information regarding the distribution of eCB metabolizing enzymes in the retina. The distribution of FAAH-IR in the rat and mouse is quite different from that in the fish. FAAH-IR, in rat and mouse, is most prominent in medium size and large ganglion cells, while weaker FAAH-IR is observed in the soma of horizontal cells, large dopaminergic amacrine cells, dendrites of starburst amacrine cells, and Mu¨ller’s cells. FAAH-immunoreactive bipolar cells in rat and mouse are exclusively cone bipolar cells, in contrast to CB1RIR that is exclusively in rod bipolar cells. In goldfish, FAAH-IR is present over cone photoreceptors, Mu¨ller’s cells, and some amacrine cells, not ganglion cells as in mouse and rat. The distribution of FAAH-IR as it relates to FAAH activity was studied in goldfish retina. 3H-AEA is hydrolyzed by FAAH with 3H-AA rapidly incorporated into membrane phospholipids. Silver-grain deposition represents the trapping of 3H-arachidonic acid in the plasma membrane. FAAH-IR and specific 3H-AEA uptake showed the same pattern over cone photoreceptors, Mu¨ller’s cells, and some amacrine cells. The codistribution of FAAH-IR and 3H-AEA uptake indicates that the bulk clearance of AEA from the extracellular

space in the retina occurs as a consequence of a concentration gradient across the plasma membrane created by FAAH activity. AEA is a ligand for the TRPV1 receptor whose binding site is on an intracellular domain. As FAAH and TRPV1 are integral membrane proteins of the endoplasmic reticulum and plasma membrane, respectively, FAAH activity may regulate the levels of AEA for TRPV1 activation. Also, following the hydrolysis of AEA by FAAH, LOX metabolites of AA could activate TRPV1. AEA then could act as an intracellular mediator by being produced from and/or degraded by the same neurons that express TRPV1 receptors. Supporting anatomical evidence for this scheme was provided first in goldfish in which co-localization of TRPV1-IR with FAAH-IR occurs in three types of amacrine cells, two of which are GABAergic. These cells ramify in interplexiform layer sublaminae a and b, indicating a general function in the OFF, ON, and ON/OFF pathways. The role would depend on the downstream cascade following the increase in calcium concentration. MGL-IR co-localizes with FAAH-IR over medium size and large ganglion cells and with CB1R-IR in all rod bipolar cells in rat and mouse. In rat retina, COX2-IR is constitutive in horizontal, amacrine, and ganglion cells. Following transient ischemia, COX-2 is upregulated in these cell types and induced in Mu¨ller’s cells. This pattern in rat differs from mouse in which COX-2 is restricted to bipolar cell bodies and their axons. COX-2 bipolar cells are a mixed group, with 65% rod bipolar cells and 35% cone bipolar cells. Also rod bipolar cells are of two types: 68% contain COX-2-IR and 32% do not. The cannabinergic system in vertebrate retinas, as indicated by CB1R-IR, FAAH-IR, COX-2-IR, and MGL-IR, is concentrated in the through pathway of photoreceptors, bipolar cells, and ganglion cells (Table 1).These cells for the most part use L-glutamate as their neurotransmitter. Cells of the inhibitory lateral pathways, horizontal cells and amacrine cells, do not feature as prominently. Exceptions are some horizontal cells, dopaminergic amacrine cells, and cholinergic starburst amacrine cells that label weakly for FAAH-IR. Bipolar cells tend to be of the on type and differentiated by rod and cone input. CB1R-IR and MGL-IR are restricted to rod bipolar cells, FAAH-IR to cone bipolar cells, and COX-2-IR to subtypes of rod and cone bipolar cells.

Cannabinoids – Retinal Physiology Effects on Transmitter Release Stimulation of CB1Rs via Gi/o reduces voltage- and Ca2þ-evoked release of [3H]-noradrenaline and [3H]dopamine in guinea pig retina. Agonists of CB1Rs, but not CB2Rs, inhibit Kþ- and ischemia-evoked [3H]


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Table 1 The general distribution of CB1 receptors, FAAH, MGL, and COX-2 immunoreactivities in the retina of a variety of species as determined by immunohistochemistry Species

CB1

FAAH

Fish

Cones

Rat/Mouse

Cones 25% OFF BC / 100% ON BC Diffuse AC Mu¨ller’s cells Rods/cones

Salamander Chick Monkey

Rod BC PKC AC Ganglion cells Rods/cones/ganglion cells Rods/cones/ganglion cells Rods/cones/ganglion cells

MGL

COX-2

Rod BC

Rod/cone BC

TRPV1 AC Mu¨ller’s cells HC (weak) Cone BC DA AC/ACh AC (weak) Ganglion cells

Ganglion cells

BC – bipolar cells, AC – amacrine cells, DA – dopamine. Specific details may be found in the citations indicated for each species. D-aspartate release from isolated bovine retina. Uptake of [3H] D-aspartate identifies high-affinity uptake sites for L-glutamate and L-aspartate in photoreceptors, a small percentage of ganglion cells and Mu¨ller’s cells. The rank order of potency for the CB1 agonists differs for Kþ- and ischemia-evoked release. As photoreceptors are more resistant to ischemia than ganglion cells, the difference in the rank order could reflect the relative potencies of the agonists on CB1Rs on these cell types.

Effects on Ganglion cells CB1R-mediated activity was demonstrated in rat retinal ganglion cells by [35S]GTPgS autoradiography and reverse transcription polymerase chain reaction (RT-PCR). Voltageactivated Ca2þ currents in cultured rat ganglion cells are suppressed by cannabinoid agonist, WIN 55,212-2, an effect that is blocked by CB1 antagonists, SR141716A and AM281. The presence of CB1R function on rat retinal ganglion cells appears unusual in that CB1Rs tend to be at presynaptic boutons. One possibility is that CB1Rs are present on associational ganglion cells, whose axons and axon collaterals do not leave the retina. Rat and mouse ganglion cells also contain FAAH and MGL, putting them in position to regulate AEA and 2-AG as potential retrograde transmitters for suppression of bipolar cell and amacrine cell activity. Effects on Bipolar Cells CB1-mediated inhibition of L-type calcium (Ica) and delayed rectifier (IK(V)) currents has been reported for ON-bipolar cells of salamander and goldfish. As yet there are no data on OFF-bipolar cells. The voltageactivation range of the currents is not altered, but simply scaled down over the entire activation range. Goldfish mixed rod–cone (Mb) bipolar cells also have D1 dopamine

receptors that enhance ICa and IK(V) via G protein Gs. CB1R agonists and dopamine oppose each other to modulate IK(V) of Mb bipolar cells. Co-application of WIN 55,212-2 (0.1–0.25 mM) reversibly blocks the enhancement induced by 10 mM dopamine even though low concentrations of WIN 55,212-2 have no effect when applied alone. The effects of dopamine and cannabinoid agonists on IK(V) occur within the physiological range of Mb bipolar cell function (–25 to 0 mV). IK(V) would be activated during the on portion of the response and, as a counter current, would modulate the peak:plateau ratio of the response. CB1R activation should make the Mb bipolar cell on response more tonic by suppressing the hyperpolarizing effect of IK(V), whereas D1 receptor activation should make the on response more phasic by enhancing IK(V). The effect on ganglion cells should be relatively tonic responses in scotopic (dark-adapted) conditions and relatively phasic responses in photopic (light-adapted) conditions. CB1-induced suppression of calcium currents should reduce transmitter release and reset sensitivity to further increments.

Cannabinoids and Photoreceptors Voltage-Gated Currents CB1-mediated modulation of photoreceptor membrane currents has only been reported for tiger salamander and goldfish. The voltage-activation ranges of these currents are not affected. Salamander rods and cones responded differently to WIN 55,212-2. IK is suppressed in single cones and rods, whereas ICa is suppressed in cones but enhanced in rods. The differential effect on ICa and IK in rods would increase transmitter release, resulting in the reduction of sensitivity, which is an apparent counteradaptive effect. Goldfish cones show a biphasic response

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to WIN 55,212-2: an enhancement of IK, ICl, and ICa via Gs at concentrations 1 mM. The data obtained with retrograde suppression, to be described below, suggest that the enhancement produced by WIN 55,212-2 may be due to agonist-specific trafficking in which binding of agonists to CB1Rs favors coupling to different G proteins. For example, WIN 55,212-2 increases intracellular calcium by Gq/11 coupling in human trabecular meshwork cells, while other CB1 agonists, including THC, 2-AG, CP55940, and methanandamide couple to Gi/o but not to Gq/11.

The caution is that the data obtained with WIN 55,212-2 may or may not apply to other agonists or the eCBs. An effect of evoked released of eCBs in the retina was demonstrated in goldfish. Retrograde suppression of membrane currents in goldfish cones in a retinal slice was achieved by applying a puff of saline with 70 mM KCl or an mGluR1 agonist, RS-3,5-dihydrophyenylglycine (DHPG), through a pipette at an Mb bipolar cell body while recording IK(V) from cone inner segments under whole-cell voltage clamp (Figure 3(a)). Retrograde inhibition of IK(V) was reversible and stable over several hours

IK(V)

50 nA 50 ms C1

OPL K+puff

K+1

C2

K+2

(b) Control #1 Control #2 K+ #2 K+ #1

IPL

100 nA 10 ms

(a)

(c)

1.0

0.9

0.8

1.00 0.95 0.90 0.85 0.80 0.75

0.7 0.1 (d)

Relative Amp of IK(V)

Relative Amp of IK(V)

1.05

0.70 1.0

10

100

Time (s) after a single K+ puff

0 (e)

20 40 60 80 100 120 140 160 180 200 220 K+-puff duration (ms)

Figure 3 Properties of the retrograde responses of cones. (a) An illustration of the method used to detect retrograde responses in goldfish cones in a retinal slice. Whole cell recordings of IK(V) were obtained from long-single cones (long arrow). A puff pipette, containing 70 mM KCl, was positioned slightly upstream and at the cell body of an Mb bipolar cell (short arrow). Thin arrows indicate the synaptic terminals of Mb bipolar cells. OPL – outer plexiform layer, IPL – inner plexiform layer. Calibration bar ¼ 20 mm. (b), (c) Sequential and overlay of raw records of IK(V) from a single cone evoked by a 50-ms depolarizing pulse to þ54 mV from a holding potential of –70 mV. The records have not been normalized. A 50-ms Kþ puff was delivered twice. IK(V) in response to Kþ puff #1 was reduced compared to that evoked for the prepuff control #1. The cone was allowed to recover for 30 min after Kþ puff #1. IK(V) returned to control amplitude (C2, control #2). The Kþ puff #2 produced an equivalent reduction in IK(V). (d) Time course (log scale) of the reduction of IK(V) in response to a single 50-ms puff of Kþ shows a latency of about 200 ms following the puff, a peak response at about 500 ms, and a gradual return to control level by 5 min. (e) Effect of Kþ puff duration on IK(V). These data were obtained from a single cone over 4 h. After a prepuff control value of IK(V) was obtained, a 25-ms Kþ puff was administered and the effect on IK(V) was determined. The cell was allowed to recover for 30 min and another prepuff control and a Kþ puff of a longer duration was administered. This sequence was followed for all puff durations. Thus, the value plotted for each puff duration is relative to its own prepuff control. There was no effect with a puff of 25 ms duration. Near maximal suppression of IK(V) at about 25% was achieved with a puff of 50 ms and there was little additional effect with puffs as long as 200 ms. Reproduced from Fan, S. F. and Yazulla, S. (2007). Retrograde endocannabinoid inhibition of goldfish retinal cones in mediated by 2-arachidonoyl glycerol. Visual Neuroscience 24: 257–267.


(Figures 3(b) and 3(c)). It had a latency of about 200 ms after a Kþ puff, was reduced on average by 25%, and had a halftime of 3.4 min to recover (Figure 3(d)). Retrograde suppression of IK(V) was unaffected by a combination of the GABA receptor antagonist, picrotoxin, and a-amino-3-hydroxyl-5-methyl4-isoxazole-propionate (AMPA) glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but blocked completely by the CB1 antagonist, SR141716A, indicating mediation by CB1 receptors. Experiments with the FAAH inhibitor (URB597), a COX-2 inhibitor (nimesulide), and a blocker of 2-AG synthesis (Orlistat) indicated that 2-AG, rather than AEA, is the retrograde eCB. Two conditions evoke 2-AG release from Mb bipolar cells, strong depolarization and activation of mGluR1, corresponding to voltage-dependent and voltageindependent mechanisms. Rods and cones release glutamate at a steady rate under any ambient illumination; this rate is increased by decrements of light intensity and

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decreased by increments in light intensity. Voltagedependent release of 2-AG would occur following depolarization of the Mb bipolar cell in response to a light flash. The retrograde suppression of glutamate release from cones would be a positive feedback that would amplify the reduction in cone transmitter release initially caused by increasing light intensity. This may not be physiologically relevant because the halftime to recover from a 50-ms stimulus is several minutes. The long halflife of the suppressive effect should make this mechanism insensitive to rapid changes in intensity. The voltage-independent mechanism that provides negative feedback on glutamate release may be more functional. Hypothetically, glutamate, released during ambient illumination, stimulates mGluR1a on Mb bipolar cells tonically to maintain a steady release of 2-AG via a Gq/11 mechanism. The degree of feedback inhibition of glutamate release from cones varies inversely with ambient illumination; the

Control –2.8 log –2.1 log

Control

WIN 0 mV

WIN

–20 mV

WIN

WIN

Control Control

–1.4 log WIN

Control Control

–40 mV

WIN Control

–0.7 log WIN

–60 mV

WIN

Control

–80 mV

0.0 log

WIN

100 pA

Control

WIN Control 5 mV

200 ms

200 ms (a)

(b)

Figure 4 Effect of WIN 55,212-2 on the responses of goldfish cones in an isolated retinal preparation to flashes of light. (a) Voltagelight responses of an L-cone under current clamp to a 200-ms light stimulus of increasing intensities (log unit changes, top to bottom) for Control conditions and after 8 min in 10 mM WIN 55,212-2. Indicated at the left are relative stimulus intensities. The response amplitudes in the control and WIN conditions differed from each other by about 10%. To facilitate comparison, the traces were normalized and superimposed. Except for the dimmest intensity (-2.8 log), there was a speeding up of the response to light offset and an enhancement of the overshoot at two intermediate intensities. There was no effect on the response to light onset or on the plateau phase of the response. The 5 mV calibration refers to the control response. (b) Current-light responses of an L-cone at different holding potentials to a 200-ms light stimulus of approximately half-maximal intensity in control and 10 mM WIN 55,212-2. The timing of the light stimulus is indicated at the bottom of the figure. The amplitude of the light response decreased with decreasing holding potential because the holding potential approached the reversal potential of the photocurrent. The response amplitudes in the control and WIN conditions differed from each other by 5–20%. To facilitate comparison, the traces were normalized and superimposed. Speeding up of the response to light offset in response to WIN 55,212-2 is apparent at all holding potentials. There was no effect of WIN 55,212-2 on the response to light onset or plateau phases of the light response. The holding potential did not change the kinetics of the light responses. The 100 pA calibration refers to the control response. Modified from Struik, M., Yazulla, S., and Kamermans, M. (2006). Cannabinoid agonist WIN 55212-2 speeds up the cone light offset response in goldfish. Visual Neuroscience 23: 285–293.

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dimmer the background, the stronger the negative feedback. As background is increased, feedback is reduced. Thus, ambient illumination produces an eCB tone that maintains transmitter release from cones within narrow limits. In this way, the ability of the cone to respond to increases and decreases of light intensity is maintained regardless of background. Retrograde transmission occurs even though Mb bipolar cells were hyperpolarized by glutamate acting on either the excitatory amino acid transporter (EAAT) or mGluR6 receptor. The retrograde effect was suppression of currents and not biphasic as expected from data obtained with low concentrations of WIN 55,212-2. It was this finding that led to the idea that the enhancing effect of WIN 55,212-2 on goldfish cones and salamander rods was due to agonist specific trafficking.

WIN 55,212-2 Affects the Cone Light Response WIN 55,212-2 affects not only cone membrane currents, indicative of presynaptic modulation, but also the response of cones to light. Goldfish cones in an isolated retina preparation were stimulated by light in combination with voltageand current-clamp protocols (Figure 4). WIN 55,212-2 (10 mM) has no effect on the absolute sensitivity of the cones or the kinetics of the onset response. However, the light offset response is faster and the depolarizing overshoot is enhanced. This effect is seen at all but dim intensities (Figure 4(a)) and is independent of holding potential (Figure 4(b)). This is found under current-clamp as well as under voltage-clamp conditions, indicating modulation of the cyclic guanosine monophosphate (cGMP)-gated channels in the cone outer segment rather than by voltagedependent currents. The effects of WIN 55,212-2 are not blocked by SR141716A, indicating that CB1Rs are not involved. Given a train of flashes, the photocurrent recovers more quickly with WIN 55,212-2, such that the peak-topeak response to succeeding flashes is increased. This effect, combined with the shortened recovery time to the offset of bright flashes, could increase contrast detection or critical flicker frequency. A concern is whether the effect of WIN 55,212-2 on the photoresponse would be observed with other CB1 agonists or eCBs because the effect of WIN 55,212-2 is not mediated by CB1 receptors. In summary, cannabinoids presynaptically suppress the synaptic output of photoreceptors and on-bipolar cells. The effect is subtle as might be expected since smoking marijuana does not produce blindness. Evidence for effects of cannabinoids on amacrine cells is strongest for their suppression of dopamine release. Dopamine, a signal for light adaptation in the retina, antagonizes the action of cannabinoids in onbipolar cells. eCBs are critically involved in neuronal plasticity. This also appears to include light and dark adaptation, processes of neuronal plasticity that occur in the retina.

Cannabinoids – Development and Neuroprotection Studies regarding the effects of prenatal-marijuana use on children show deficits on visual habituation, tremors, and startle responses in neonates of 4–30 days old, but no effects on children of 1–6 years old. Problems with behavior, visual perceptual tasks, language comprehension, attention, and memory in 9-year-olds are attributed to effects on the prefrontal cortex, an area enriched in CB1Rs. Although CB1R localization and effects on GABA release have been studied in embryonic rat and chick retinas, no studies have investigated or commented on the effects of manipulating eCBs on retinal development. The end point of glaucoma is ganglion cell death by apoptosis that may be caused by optic nerve injury following compression or ischemia. CB1 agonists (THC and cannabidiol) as well as inhibition of FAAH protect ganglion cells from glutamate excitotoxicity and ischemia caused by increased IOP. In contrast, COX-2 contributes to neuronal cell death following ischemia or NMDAtoxicity in glial cells, retinal pigment epithelium (RPE), and ganglion cells, while COX-2 blockers prevent ganglion cell apoptosis. Despite progress on the interaction of eCBs, COX-2 metabolites, and EP2 receptors in neuroprotection in the brain, such information is lacking in the retina.

Conclusion The cannabinergic system is concentrated in the through pathway of the retina. Cannabinoids suppress dopamine release from amacrine cells and presynaptically inhibit potassium currents and glutamate release from cones and on-bipolar cells. How this relates to light and dark adaptation, receptive field formation, temporal properties of ganglion cell responses, and ultimately visual behavior needs to be addressed. eCBs are the most recently described neuromodulators to be studied extensively in neural and non-neural tissues. The existence of multiple eCBs, degradative enzymes, and receptors paints a picture of great complexity. They are important for their role in neuroplasticity and neuroprotection. Further study will verify the importance of eCBs in the retina as well. See also: Information Processing: Amacrine Cells; Information Processing: Bipolar Cells; Information Processing: Ganglion Cells; Information Processing: Horizontal Cells; Neurotransmitters and Receptors: Dopamine Receptors; Phototransduction: Adaptation in Cones.


Further Reading Fan, S. F. and Yazulla, S. (2007). Retrograde endocannabinoid inhibition of goldfish retinal cones is mediated by 2-arachidonoyl glycerol. Visual Neuroscience 24: 257–267. Glaser, S. T., Deutsch, D. D., Studholme, K. M., Zimov, S., and Yazulla, S. (2005). Endocannabinoids in the intact retina: 3H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide. Visual Neuroscience 22: 693–705. Iversen, L. L. (2000). The Science of Marijuana. New York: Oxford University Press. Nucci, C., Gasperi, V., Tartaglione, R., et al. (2007). Involvement of the endocannabinoid system in retinal damage after high intracellular pressure-induced ischemia in rats. Investigative Ophthalmology Visual Science 48: 2997–3004. Onaivi, E. S., Sugiura, T., and Di Marzo, V. (eds.) (2006). Endocannabinoids: The Brain and Body’s Marijuana and Beyond. Boca Raton: CRC Press. Straiker, A. and Sullivan, J. M. (2003). Cannabinoid receptor activation differentially modulates ion channels in photoreceptors of the tiger salamander. Journal of Neurophysiology 89: 2647–2654. Straiker, A., Stella, N., Piomelli, D., et al. (1999). Cannabinoid CB1 receptors and ligands in vertebrate retina: Localization and function of an endogenous signaling system. Proceedings of the National Academy of Sciences of the United State of America 96: 14565–14570.

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Struik, M., Yazulla, S., and Kamermans, M. (2006). Cannabinoid agonist WIN 55212-2 speeds up the cone light offset response in goldfish. Visual Neuroscience 23: 285–293. Tomida, I., Pertwee, R. G., and Azuara-Blanco, A. (2004). Cannabinoids and glaucoma. British Journal of Ophthalmology 88: 708–713. Yazulla, S. (2008). Endocannabinoids in the retina: From marijuana to neuroprotection. Progress in Retinal and Eye Research 27(5): 501–526. Yazulla, S., Studholme, K. M., McIntosh, H. H., and Deutsch, D. G. (1999). Immunocytochemical localization of cannabinoid CB1 receptor and fatty acid amide hydrolase in rat retina. Journal of Comparative Neurology 415: 80–90. Yazulla, S., Studholme, K. M., McIntosh, H. H., and Fan, S. F. (2000). Cannabinoid receptors on goldfish retinal bipolar cells: Electronmicroscope immunocytochemistry and whole-cell recordings. Visual Neuroscience 17: 391–401.

Relevant Websites http://cannabinoidsociety.org/ – This is the official website of the International Cannabinoid Research Society. It provides updates and background information on all aspect of the endocannabinoid field. http://webvision.med.utah.edu/ – This website from the University of Utah provides extensive coverage of retinal anatomy and physiology, particularly mammals.

Retinal Degeneration through the Eye of the Fly N J Colley, University of Wisconsin, Madison, WI, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary ABC-type multidrug transporter – A family of adenosine-triphosphate-binding cassette (ABC) transmembrane proteins that transports various molecules, including proteins, ions, sugars, and lipids, across extracellular and intracellular membranes using energy derived from adenosine triphosphate (ATP). Allele – One member of a pair of genes occupying a specific location on a chromosome (locus) that controls the same trait, for example, eye color. cGMP phosphodiesterase (PDE) – This enzyme is found in several tissues, including the rod and cone photoreceptor cells, and it belongs to a large family of cyclic nucleotide PDEs that catalyze the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) into AMP and GMP, respectively. Class B scavenger receptors – A family of proteins, which includes the scavenger receptor class B type I (SR-BI), and CD36, which are cell surface receptors that mediate lipid uptake. They are also thought to play an important role in vitamin A metabolism by mediating the uptake of carotenoids into cells. Cyclophilin – A protein that binds the immunosuppressant drug, cyclosporin, which is often used to suppress tissue rejection following an organ transplantation. The protein displays peptidyl prolyl cis–trans isomerase activity, which catalyzes the cis/trans isomerization of peptide bonds on proline residues and is thought to play a role in protein folding. Flippases – The enzymes located in the membrane that aid in the movement of phospholipid molecules between the two leaflets that comprise a cell’s membrane. The process requires energy derived from ATP. Homeodomain-containing transcription factors – A homeobox is a DNA sequence found within genes that regulates developmental processes in animals, fungi, and plants. A homeobox is about 183-DNA-bp long and it encodes a 61-amino-acid protein domain, called the homeodomain, which binds DNA and plays a key role in the regulation of gene expression. Paired domain – A conserved domain found in a set of transcription factor proteins which are important in regulating gene expression during development.

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Paired box (PAX) genes belong to the PAX family of transcription factors. Rab-GTPase – These are small guanine-nucleotidebinding proteins (G proteins) conserved from yeast to humans and are members of the Ras superfamily of small GTPases. They function in distinct steps in membrane trafficking pathways, including vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion events. Retinitis pigmentosa (RP) – A heterogeneous group of genetically inherited retinal degeneration disorders leading to progressive loss in vision. Many people with RP retain some sight all their life, others become legally blind in childhood, and some become legally blind in their 40s or 50s. The progression of RP is different in each case. Rhabdomere – The light-sensing organelle of the Drosophila photoreceptor cell. It is the functional equivalent of the outer segment of vertebrate rod and cone cells. A rhabdomere is made up of 60 000 tightly packed microvilli, and each microvillus is 50 nm in diameter and about 1–2 mm in length. Second-site modifier screens – The genetic screens that are designed to detect a mutation in a second locus (gene) that enhances or suppresses the effect of an existing mutation.

‘Prized pest’ You hover over the soft, brown bananas like a floater, in and out of my vision, you whose eyes are so much like my own. Who am I, dear one, to swat at you, send you swirling toward the ceiling when, really, we share the same humble beginnings. And you in your simplicity hold the key to my complexity. Let me set out a plate of the sweetest peaches, invite you to rest on the arm of my chair, however late it is. Marilyn Annucci


Each model organism used to study retinal degenerative diseases has the advantage that others lack. Frogs, fish, rats, and mice have all provided great insights, but it is the tiny fruit fly, Drosophila melanogaster, that has played a central role in elucidating the molecular genetics of eye development and the early identification of mutations that cause retinal degeneration. The first mutations in Drosophila known to cause retinal degeneration were identified in the 1960s by the pioneering studies of Bill Pak and co-workers. At that time, these findings were only of interest to a few investigators. It was thought that animals as different as flies and humans could not share a similar genetic makeup and, therefore, the amount of transferable knowledge would be limited. The revolutionary finding that put flies into the spotlight was the one showing that genes controlling pattern formation and development in flies could also do so in humans. In the 1980s, homeodomain-containing transcription factors were found to be essential during development in Drosophila for directing the production of appendages, such as the legs and the antennae. Almost identical homeodomain-containing genes were found in the genomes of a wide range of organisms, including humans and mice. This knowledge led to the conclusion that organisms as different as flies and humans contain nearly identical genes. A few years later, working on eye development, Walter Gehring’s lab cloned the eyeless gene in Drosophila. They discovered that eyeless is a transcription factor, containing a paired domain and a homeodomain, that directs eye formation. The eyeless gene is related to the mouse and human Pax6 genes (paired box), and the eyeless/Pax6 genes regulate a cascade of genetic processes involved in eye development. Mutations in these genes result in aniridia in humans, a Small eye (Sey) phenotype in mice and an eyeless phenotype in Drosophila. Aniridia is a congenital condition that is characterized by incomplete iris formation. Further, when expressed in flies, both the Drosophila eyeless gene and the mouse Pax6 gene (Small eye, or Sey) were able to direct the production of ectopic compound eyes. That Sey induced the formation of compound eyes and not mouse structures revealed that mice and flies share signaling components that are interchangeable. The proteins encoded by these genes share 94% identity in the paired domain and 90% identity in the homeodomain. It is remarkable that eyeless is not only essential for eye formation, but also its ectopic expression can override other developmental processes in a variety of tissues. For example, eyeless is capable of directing leg, wing, and antennal tissues to form eyes. As a result of these findings, eyeless/Pax6 was dubbed a master regulatory gene for eye formation during development. These landmark discoveries led to an explosion of exciting work in the 1990s that prompted a reassessment of the evolution of eyes. A complex network of eye determination genes direct eye formation, including another Pax6, twin

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of eyeless (toy), sine oculis (so), eyes absent (eya), and dacshund (dac). Counterparts of these genes play a role in mammalian eye development and have been implicated in a variety of human diseases. These studies reveal that even though the compound eye of the fly looks very different from mammalian eyes, both share similar signaling pathways that are able to substitute for each other to form an eye. Due to these elegant findings, it is now widely accepted that many genes are functionally equivalent between flies and humans. In addition, the same (or similar) mutations cause disease in both species. In fact, nearly three-fourths of all human disease genes have related sequences in Drosophila. Examples include gene mutations involved in retinal degeneration, deafness, skeletal malformations, cognitive impairment, cancer, immunity, alcoholism, cocaine and nicotine addiction, heart disease, metabolic and storage diseases, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. At the turn of the twentieth century in the hands of Thomas Hunt Morgan, the founding father of Drosophila research, Drosophila emerged as a powerful genetic workhorse. In 1910, Morgan identified the first mutation in Drosophila, which was a spontaneous white eye-pigment mutation that caused a normally red-eyed fly to be white eyed (Figure 1(a)). This first allele transformed our understanding of genetics and heredity. The white gene encodes a membrane-associated, adenosine triphosphate (ATP)-binding, ATP-binding cassette (ABC)-type multidrug transporter required for the transport of pigment precursors involved in eye pigment biosynthesis. In humans, mutations in the ABCA4 gene (also ABCR) account for approximately 3% of autosomal recessive retinitis pigmentosa (RP) and are linked to both recessive cone–rod dystrophy and recessive Stargardt macular dystrophy. Similar to the Drosophila white gene, the ABCA4 gene encodes a membrane-bound, ATP-binding transporter that in humans localizes to the rims of rod and cone outer segment disks. The ABCA4 transporter serves as a flippase in the retinoid cycle. When the ABC4 gene is mutated, toxic detergent-like by-products accumulate in the retinal pigment epithelium (RPE) leading to severe pathology. Therefore, the white gene, discovered at the turn of the century, was subsequently found to encode an ABC-type transporter required for eye pigment biosynthesis in Drosophila, and is related to another ABC-type transporter in the human eye that is involved in the retinoid cycle and several types of retinal diseases. Not only do we share many genes in common with flies, but we also share a great deal of the same metabolic and signaling pathways. Flies are now being used as genetic models for the National Aeronautics and Space Administration (NASA) astronauts and are providing vital information on how space travel and gravitational changes alter gene expression. Work on flies continues to reveal

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Figure 1 (a) Wild-type red-eyed fly, Canton S compared to a white-eyed mutant fly, w1118. (b) Cross section through the compound eye showing the R1-7 photoreceptor cells and their photosensitive rhabdomeres (R). The R8 photoreceptor cell is located below the plane of the section. (c) The adult Drosophila visual system showing the two compound eyes and the three simple eyes (ocelli) located on the top of the head (arrows). (d) A higher magnification of a rhabdomere showing the microvilli. The rhabdomeres are made up of about 60 000 microvilli and are 50 nm in diameter and 1–2 mm in length. (e) A newly eclosed ninaE I17 mutant fly, showing the reduced size of the rhabdomeres. ninaE I17 is a null allele, so the flies completely lack Rh1 rhodopsin expressed in the R1-6 photoreceptor cells. (f) Six-day-old ninaE I17 fly, showing that the rhabdomeres of the R1-6 photoreceptor cells are almost completely gone, but the R7 cell rhabdomere remains.

general principles that are fundamental to a wide spectrum of biological processes. Studies in Drosophila have led to conceptual and technical breakthroughs in the areas of development, gene expression, learning and memory, sleep, alcoholism, cocaine and nicotine addiction, ecology and evolution, olfaction, taste, mechanotransduction, vision, hearing, aging, pigmentation, biological clocks and circadian rhythms, courtship and mating behaviors, and human disease and the development of new pharmaceuticals. In addition to sharing genes and signaling pathways with humans, flies are a powerful model for providing insights into human health and disease for other reasons. In spite of their small size, flies display complex rituals such as courtship behavior, so questions related to the genetic basis of complex behavior are tractable in the fly.

The fruit fly uses the same or similar genes to develop from a fertilized egg to an adult, but they do it in the short time of about 11 days. A female will lay hundreds of eggs, allowing large numbers of genetically identical offspring to be obtained. Flies have a short life span of about 2 months, so the onset and progression of age-related retinal degeneration disorders or any other age-related degenerative process can be studied quite rapidly. The eye is not essential for viability or fertility of the flies, therefore genes encoding proteins that are uniquely required for visual function may be easily manipulated and studied. Large-scale mutagenesis screens have been carried out, producing hundreds of thousands of mutant flies whose phenotypes can be analyzed to identify genes required for vision. In addition, second-site modifier screens have been used to identify novel genes in


signaling pathways. Fly models can be used to dissect the cell biological basis and physiological basis of retinal degeneration, and therefore can be used to obtain insights into mechanisms of degenerative disorders. Just like in humans, electroretinogram (ERG) recordings can be carried out, and they have proved to be an indispensable means for uncovering visual system defective phenotypes that would otherwise have remained unnoticed. Transgenic flies can be easily produced and, as a result, mutant genes may be introduced and mutant phenotypes may be complemented with wild-type transgenes. Using specialized promoters, genes may be targeted to specific tissues and may also be overexpressed. The fly has a relatively small genome, made up of about 13 600 genes in four pairs of chromosomes. However, despite the dramatic differences in size and apparent complexity between humans and flies – we have less than twice as many genes as a fly – our genome is estimated to be made up of only 20 000–25 000 genes contained in 23 pairs of chromosomes. Therefore, despite the fly’s perceived simplicity, or our perceived complexity, our genetic makeup may not be all that different. Its versatility for genetic manipulation and convenience for unraveling fundamental biological processes continue to guarantee the fly a place in the spotlight for unraveling the basis of and therapeutic treatments for human disease.

The Compound Eye and Phototransduction The Drosophila compound eye is composed of approximately 800 individual eye units called ommatidia, each containing the outer, R1-6 photoreceptor cells that extend the full length of the retina and express the major rhodopsin in the eye, the blue-sensitive rhodopsin, Rh1 (Figure 2). Rh1 is encoded by the ninaE gene, and it displays 22% amino acid identity with human rhodopsin. The inner photoreceptor cells, R7 and R8, are arranged such that a subset of the R7 cells expresses the ultraviolet (UV)-opsin, Rh3. They pair with the R8 cells expressing the blue-sensitive opsin Rh5 (p-ommatidia), while the R7 cells expressing the UV-opsin Rh4 pair with the R8 cells expressing the green-sensitive opsin Rh6 (y-ommatidia). The R7 cells are located above their partner R8 cells (Figures 1(b) and 2). Above the photoreceptor cells are four cone cells and two lens components; the pseudocone (also called the crystalline cone) and the corneal lens. Two primary pigment cells surround the cones and each ommatidium is optically isolated by a sheath of secondary and tertiary pigment cells (Figure 2). The adult visual system also contains three simple eyes, ocelli, located on the top of the head (Figure 1(c)). The ocelli express the violet-sensitive, Rh2 opsin. Drosophila photoreceptor cells contain specialized portions of the plasma membrane,

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Figure 2 Schematic of an ommatidium. CL, corneal lens; PSC, pseudocone; PP, primary pigment cells; CC, cone cells; R1-6, R7, and R8 photoreceptor cells; SP and TP, secondary and tertiary pigment cells. Adapted from Tomlinson, A. and Ready, D. F. (1987). Cell fate in the Drosophila ommatidium. Developmental Biology 123: 264–275.

called rhabdomeres, which comprises approximately 60 000 tightly packed microvilli containing rhodopsin photopigments and other components of the phototransduction cascade (Figure 1(b) and 1(d)). The microvillar processes of the rhabdomeres are functionally similar to the phototransducing disk membranes present in the vertebrate photoreceptor outer segments (Figure 3). Phototransduction in Drosophila utilizes a signaling cascade in which light stimulation of rhodopsin leads to the activation of the heterotrimeric guaninenucleotide-binding G protein (Gq) and the stimulation of phospholipase C beta (PLC-b), leading to the opening of the cation-selective transient receptor potential (TRP) and TRP-like (TRPL) channels. The photoreceptors depolarize as intracellular calcium dramatically rises from about 100 nm to about 10 mM. In the rhabdomeres, calcium rises even higher, to about 1 mM, and it is required for amplification, rapid response kinetics, and light adaptation in Drosophila. Calcium is subsequently removed from the rhabdomeres by a combination of sodium/calcium exchange and diffusion into the cell body where calcium increases to about 10 mM. Calcium in the cell body is buffered by calcium-binding proteins and is removed by uptake into intracellular stores by the sarco/endoplasmic reticulum (ER) calcium ATPase. The Drosophila phototransduction cascade shares some similarities with the phototransduction cascade in mammalian rod and cone photoreceptor cells. Both cascades are initiated by light-activation of rhodopsin that in turn leads to the stimulation of heterotrimeric G proteins. Phototransduction in Drosophila as well as in humans is terminated when the protein arrestin binds to lightstimulated rhodopsin and blocks the binding of rhodopsin

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Figure 3 The Drosophila photoreceptor cell compared with human rod and cone photoreceptor cells. In Drosophila, the pseudocone cone (also called the crystalline cone) and the corneal lens are the lens elements, and they are secreted by the underlying cone cells. The Drosophila lens is comprised of droscrystallin, which is similar to insect cuticular proteins. R, rhabdomere; OS, outer segments; I, inner segments; N, nucleus. Photoreceptor cell drawings adapted from Chang, H. Y. and Ready, D.F. (2000). Science 290: 1978–1980.

to Gq. However, notable differences are that rod and cone channels are gated by cyclic nucleotides and they close in response to light, leading to a hyperpolarizing response. Although certain features of phototransduction in Drosophila differ from rod and cone phototransduction, Drosophila phototransduction shares many common features with the cascade in intrinsically photosensitive retinal ganglion cells (ipRGCs). These cells function in circadian rhythm entrainment and pupil constriction. The light response in ipRGCs is initiated with absorption of light by melanopsin, which is more similar to Drosophila rhodopsins than to the photopigments in rods and cones. Light-stimulated melanopsin is thought to activate a phophoinositide cascade leading to the opening of channels that display similar properties to TRP channels. Therefore, Drosophila photoreceptor cells and ipRGCs share similar phototransduction cascades, and studies in flies will continue to provide insights into ipRGC function.

Genetic Screens Identify Retinal Degeneration Loci Several forward genetic screens in Drosophila led to an explosion in the identification of many genes involved in retinal degeneration, and their counterparts in human disease. The approach has been to chemically mutagenize flies to disrupt photoreceptor cell function. The mutagenized flies are tested for function by ERG analysis, morphology by a deep pseudopupil (DPP), or Western blotting for the loss of candidate gene expression (such as arrestin). The ERG approach was pioneered by Bill Pak and co-workers in the 1960s and led to the isolation of over 200 ERG-defective mutants. In the 1980s, the development of gene-cloning techniques for Drosophila made it

possible to isolate the corresponding genes. For example, neither inactivation nor after potential E (ninaE), a mutant isolated in the original ERG screen was later found to harbor a mutation in the structural gene for the major rhodopsin in Drosophila, Rh1. In 1985, almost a decade prior to the cloning of eyeless, Drosophila rhodopsin, Rh1, was cloned and sequenced by two groups and found to display 22% amino acid identity with bovine rhodopsin. In addition, the first evidence that mutations in a rhodopsin gene led to retinal degeneration came from elegant studies in the 1980s in Drosophila. The use of the ERG in Drosophila was an effective strategy to identify mutants in phototransduction and also in retinal degeneration. The DPP is a sensitive phenotype in the eye that can be easily assessed in live flies. It is based on the precise packing of the photoreceptor cells. Any mutation leading to, even subtle, structural alterations in photoreceptor cells will cause attenuation in the DPP. For example, a reduction in rhodopsin levels in the R1-6 photoreceptor cells in ninaE mutants, leads to structural alterations in the photoreceptor cells (Figures 1(e) and 1(f )) and attenuation in the DPP. A variety of mutants were isolated by this method, including dominant alleles of ninaE (rhodopsin), and alleles of two chaperone proteins, ninaA (cyclophilin) and calnexin. Both the ERG and the DPP screens accelerated the pace of identifying mutations that cause retinal degeneration in Drosophila.

Retinal Degenerations in Flies and Humans Mutations in rhodopsin are the leading cause of blinding disease in RP. RP is a heterogeneous group of inherited disorders that is characterized by progressive retinal


degeneration and eventual blindness. RP may be inherited as an X-linked (about 5–15% of cases), autosomal recessive (50–60%), and autosomal dominant trait (30–40%). It affects one person in 4000 worldwide and is often restricted to the eye, but not always. In about 20–30% of the cases of RP, the genetic defects are not eye specific. There are approximately 30 syndromes that involve RP. One of the most common syndromes is Usher’s syndrome. This syndrome is characterized by vision and hearing impairment, and mutations in myosin VIIA are responsible for one form, Usher 1B syndrome. Interestingly, loss of myosin VIIA function leads to deafness in Drosophila. In flies, like in humans, there are many examples of mutations in which the phenotype caused by the mutation is restricted to the eye, whereas there are others that are not. For example, mutations in the gene encoding rhodopsin (ninaE) and the arrestin gene cause defects that are restricted to the eye. Mutations in genes such as retinal degeneration B (rdgB, encoding a phosphatidylinositol transport protein), involve olfactory as well as visual defects. Since the initial findings, in 1983, that mutations in Drosophila rhodopsin lead to retinal degeneration, over 100 mutations in human rhodopsin have been found to cause autosomal dominant RP (adRP). The first mutation identified in adRP patients, published by Dryja and coworkers in 1990, was a mutation that caused a proline residue located near the N-terminus of rhodopsin to be replaced by a histidine residue (Pro23His). A great majority of these mutants, including Pro23His, produce misfolded rhodopsin that is improperly transported through the secretory pathway. However, the mechanism by which the mutant rhodopsins cause dominant retinal degeneration was not known. In 1995, studies in Drosophila on rhodopsin mutations that act dominantly to cause retinal degeneration revealed that the retinal degeneration results from the interference in the maturation of normal rhodopsin by the mutant protein. These studies in Drosophila provided a mechanistic explanation for the cause of certain forms of adRP.

Mechanisms of Retinal Degenerations Light-Dependent Retinal Degenerations It is now widely appreciated that retinal defects and retinal degeneration can be triggered by mutations in almost every component of the photoreceptor cells. These mutations can be divided into two distinct classes. One class pertains to the unregulated activities of phototransduction and/or calcium toxicity. Mutations in this class lead to retinal degenerations that are dependent on or influenced by light stimulation of the cascade and the opening of the TRP and TRPL channels, and these are termed light dependent.

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For example, some mutations in rhodopsin itself or mutations in the arrestin gene lead to light-dependent retinal degeneration. Arrestin is required for deactivating rhodopsin, and loss of arrestin causes unregulated rhodopsin and hence excessive activation of phototransduction. It is also thought that the loss of arrestin causes decreased endocytosis of Rh1 and all of these defects lead to retinal degeneration. The precise spatial and temporal regulation of calcium is also essential for photoreceptor survival in flies and people. Prolonged elevation of cytosolic calcium or low levels of calcium can be toxic, leading to cell death and retinal degeneration. In Drosophila, mutations in arrestin, the Naþ/Ca2þ exchanger (calx ), the diacylglycerol kinase (retinal degeneration A, rdgA), and constitutively active TRP channels are all thought to trigger cell death by causing abnormally high levels of calcium. In humans, a lack of cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE), caused by mutations in PDE6A and PDE6B, leads to an elevation in the cGMP concentration in the outer segments, which in turn causes cGMP channels to be open, resulting in excessive levels of calcium. Defects in PDE6A and PDE6B cause recessive RP.

Light-Independent Retinal Degenerations A second class of retinal degenerations involves defects in rhodopsin maturation and does not require activation of phototransduction by light. These are termed light independent. In Drosophila, as in humans, Rh1 is synthesized and glycosylated in the ER, binds its vitamin-A-derived chromophore (11-cis 3-hydroxyretinal), at a lysine residue in the seventh transmembrane domain, is transported through the various compartments of the Golgi, and is delivered to its final destination for phototransduction. The mechanisms that regulate rhodopsin maturation, such as its folding, glycosylation, chaperone interaction, chromophore attachment, and transport, are key to photoreceptor survival in flies and humans. In flies, the transport of Rh1 from the ER to the rhabdomere requires the cyclophilin, NinaA. Cyclophilins are known to display peptidyl-prolyl cis–trans isomerase and are thought to play a role in protein folding during biosynthesis. Consistent with a role in protein folding, NinaA resides in the ER. In addition, NinaA is detected in secretory transport vesicles together with Rh1, and forms a specific and stable complex with Rh1, consistent with a broader role as a chaperone in the secretory pathway. Similarly, in mammals a cyclophilin-like protein (RanBP2/ Nup358) modulates protein biogenesis. The Drosophila cyclophilin, NinaA, is a chaperone that is specifically required for Rh1 biosynthesis and maturation. Another chaperone required for Rh1 biosynthesis in Drosophila is calnexin and mutations in ninaA (cyclophilin), ninaE (Rh1), and calnexin

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all lead to severe retinal pathology in flies. In mammalian photoreceptors, calnexin is also expressed in the ER. Although calnexin is not required for the expression of rod rhodopsin, cone M-opsin, or melanopsin (in the ipRGCs) in the mouse, it is required for proper retinal morphology. Rhodopsin in both mammals and flies undergoes N-linked glycosylation during biosynthesis, and in flies, elimination of the glycosylation site, asparagine 20 (N20I), results in the retention of rhodopsin in the secretory pathway. Moreover, in both mammals and flies, genes involved in rhodopsin chromophore biosynthesis and transport are critical to rhodopsin maturation and expression as well as photoreceptor function. Defects in chromophore production in the Drosophila mutants ninaB, ninaD, ninaG, and santa maria, cause a failure in Rh1 transport from the ER to the rhabdomere, resulting in a severe reduction in Rh1 and retinal pathology. The ninaB gene encodes an enzyme that catalyzes the conversion of carotenoids to retinal (b, b0 – carotene-15, 150 -monoxygenase) and ninaG encodes an enzyme that acts to convert retinal to 3-hydroxyretinal (oxidoreductase). Two additional Drosophila loci, ninaD and santa maria, are both similar to the mammalian class B scavenger receptors and play a role in transporting b-carotene to cells. In flies, b-carotene is required in the diet for the production of all-trans retinol, which is in turn converted to 11-cis 3-hydroxyretinal. Upon light stimulation 11-cis 3-hydroxyretinal is photoconverted to all-trans 3-hydroxyretinal. Mutations in chromophore biosythesis result in defective Rh1 maturation, low levels of Rh1, and retinal pathology, establishing the importance of vitamin A in the fly. Once rhodopsin exits the ER, it requires several Rabguanosine triphosphate (GTP)ases for vesicular transport through the secretory pathway in flies and in mammals. Rab-GTPases are conserved from yeast to humans and are members of the Ras superfamily of small GTPases. They function in distinct steps in membrane trafficking pathways including vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion events. In Drosophila, Rab1, Rab6, and Rab11 mediate vesicular fusion between the ER and the Golgi (Rab1), intra-Golgi (Rab6), and post-Golgi (Rab11) transport of rhodopsin in Drosophila. Defects in Rab function cause inadequate Rh1 transport and retinal pathology. Therefore, the mechanisms that regulate Rh1 maturation, such as its folding, chaperone interaction, and chromophore binding and transport are essential for photoreceptor health in flies and humans. Retinal Degenerations Caused by Mutations in Dual-Role Proteins Although most retinal degenerations are classified as either light dependent or light independent, there is a growing list of retinal degenerations that fall into both

classes. In these cases, the corresponding mutant proteins play dual roles. For example, as was described above, calnexin is a chaperone required for rhodopsin maturation. In addition, it is a calcium-binding protein for regulating calcium in photoreceptor cells. Mutations in calnexin lead to defects in Rh1 maturation and retinal degeneration. The degeneration due to defects in rhodopsin maturation is light independent, but calnexin mutants also display prolonged and elevated levels of calcium, following light stimulation. In the calnexin mutants, the retinal degeneration is enhanced by the stimulation of phototransduction by light. Therefore, calnexin plays a dual role: one in rhodopsin maturation and another in calcium modulation. Summary In the 1980s, Drosophila took on a surprising new role, as an animal model for retinal disease, when the genetic similarities and fundamental processes between flies and humans became apparent. It became clear that information obtained in flies was transferable to human blinding diseases. As a result, and since then, there has been an explosion in the use of Drosophila as an animal model for unraveling the molecular genetic basis of retinal degeneration disorders. Despite its perceived simplicity, the fruit fly is, indeed, a remarkably complex creature with a genetic makeup that is surprisingly similar to our own. Investigators continue to capitalize on a whole host of versatile genetic techniques together with the accessibility of the fly to dissect fundamental photoreceptor cell mechanisms in vivo. The short life span of the fly, only 2 months, allows for monitoring the onset and progression of retinal degeneration in a short time. These advantageous features place Drosophila at the forefront of current research efforts, aimed at unraveling the basis of and therapeutic treatments for retinal degenerative disorders.

Acknowledgments Our research, on retinal degeneration in Drosophila, is supported by funding from the National Eye Institute, the Retina Research Foundation, and the Retina Research Foundation/Walter H. Helmerich Research Chair. I gratefully acknowledge C. Vang, E. Rosenbaum and B. Larson for assistance with preparing the figures. For the poem, I thank M. Annucci, author of Luck (Parallel Press) and member of the Department of Languages and Literatures at the University of Wisconsin-Whitewater. See also: Circadian Photoreception; Circadian Rhythms in the Fly’s Visual System; Coordinating Division and Differentiation in Retinal Development; Embryology and Early Patterning; Evolution of Opsins; Ganglion Cell Development: Early Steps/Fate; Genetic Dissection of

Retinal Degeneration through the Eye of the Fly Invertebrate Phototransduction; Histogenesis: Cell Fate: Signaling Factors; Photoreceptor Development: Early Steps/Fate; The Photoreceptor Outer Segment as a Sensory Cilium; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Retinal Histogenesis; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration; Secondary Photoreceptor Degenerations; Xenopus laevis as a Model for Understanding Retinal Diseases; Zebra Fish as a Model for Understanding Retinal Diseases; Zebra Fish–Retinal Development and Regeneration.

Further Reading Bok, D. (2007). Contributions of genetics to our understanding of inherited monogenic retinal diseases and age-related macular degeneration. Archives of Ophthalmology 125: 160–164. Colley, N. J., Baker, E. K., Stamnes, M. A., et al. (1991). The cyclophilin homolog ninaA is required in the secretory pathway. Cell 67: 255–263. Colley, N. J., Cassill, J. A., Baker, E. K., et al. (1995). Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. Proceedings of the National Academy of Sciences of the United States of America 92: 3070–3074. Graham, D. M., Wong, K. Y., Shapiro, P., et al. (2008). Melanopsin ganglion cells use a membrane-associated rhabdomeric phototransduction cascade. Journal of Neurophysiology 99: 2522–2532.

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Greenspan, R. J. and Dierick, H. A. (2004). ‘Am not I a fly like thee?’ From genes in fruit flies to behavior in humans. Human Molecular Genetics 13(2): R267–R273. Halder, G., Callaerts, P., and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267: 1788–1792. Hardie, R. C. and Postma, M. (2008). Phototransduction in microvillar photoreceptors of Drosophila and other invertebrates. In: Allan, A. K., Basbaum, I., Shepherd, G. M., and Westheimer, G. (eds.) The Senses: A Comprehensive Reference vol. 1, pp. 77–130. San Diego, CA: Academic Press. Hartong, D. T., Berson, E. L., and Dryja, T. P. (2006). Retinitis pigmentosa. Lancet 368: 1795–1809. Pak, W. L. (1995). Drosophila in vision research. The Friedenwald lecture. Investigative Ophthalmology and Visual Science 36: 2340–2357. Reiter, L. T., Potocki, L., Chien, S., et al. (2001). A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Research 11: 1114–1125. Rosenbaum, E. E., Hardie, R. C., and Colley, N. J. (2006). Calnexin is essential for rhodopsin maturation, Ca2þ regulation, and photoreceptor cell survival. Neuron 49: 229–241. Rubin, G. M. and Lewis, E. B. (2000). A brief history of Drosophila’s contributions to genome research. Science 287: 2216–2218. Tomlinson, A. and Ready, D. F. (1987). Cell fate in the Drosophila ommatidium. Developmental Biology 123: 264–275. Wang, T. and Montell, C. (2007). Phototransduction and retinal degeneration in Drosophila. Pflugers Archiv 454: 821–847. Wernet, M. F., Celik, A., Mikeladze-Dvali, T., et al. (2007). Generation of uniform fly retinas. Current Biology 17: R1002–R1003.

Relevant Website http://www.sph.uth.tmc.edu – Genes and mapped loci causing retinal diseases: Homepage.

Retinal Ganglion Cell Apoptosis and Neuroprotection K M Coxon, J Duggan, L Guo, and M F Cordeiro, UCL Institute of Ophthalmology, London, UK ã 2010 Elsevier Ltd. All rights reserved.

Glossary Apoptosis – The process of programmed cell death, whereby the cell proceeds through a highly regulated series of morphological changes resulting in the controlled disassembly of the affected cell. Bax, Bak, Bad, and Bid – Proapoptotic proteins. Bcl-2 – B-cell CLL/lymphoma 2, an antiapoptotic protein. Excitotoxicity – The process by which raised levels of neurotransmitters trigger cell death. Glaucoma – A major cause of blindness worldwide, resulting from the loss of retinal ganglion cells, with raised intraocular pressure as a major modifiable factor. Neuroprotection – The use of therapeutic agents to prevent or reverse neuronal damage thereby retaining physiological function. Neurotrophic factors – The growth factors that promote the growth, differentiation, and survival of neurons. Retinal ganglion cells – The neurons that relay visual information from their cell soma located in the retina, through their axons which project along the optic nerve to the brain.

Introduction Retinal ganglion cells (RGCs) relay visual information from their cell soma located in the retina, through their axons which project along the optic nerve to the brain. Their loss is associated with various optic neuropathies such as Leber hereditary optic neuropathy, optic neuritis, anterior ischemic optic neuropathy, and glaucoma. The most prominent of these is glaucoma, which is a major cause of irreversible blindness worldwide. In this article, glaucoma is used to highlight the challenges involved in unraveling the complexities of RGC apoptosis and neuronal cell death in order to facilitate more effective treatment strategies.

Apoptosis Unlike necrosis, apoptosis is a regulated process of cell death leading to chromatin condensation, DNA

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fragmentation, oxidative damage, and autophagic degeneration, commonly proceeding through either extrinsic or intrinsic caspase-dependent pathways outlined in Figure 1. Identifying which aspect of apoptosis regulation is susceptible in glaucoma has important implications for the elucidation of pharmacological targets. Apoptosis in Glaucoma The exact mechanism triggering RGC apoptosis in glaucoma is still unidentified, although the major modifiable risk factor identified is that of raised intraocular pressure (IOP) and is commonly used to investigate apoptotic mechanisms. Mitochondria play a fundamental role in RGC apoptosis, with raised hydrostatic pressure inducing apoptosis that is at least partially dependent on mitochondria. More specifically, Bax, a regulator of membrane permeability, has been suggested to be essential in triggering apoptosis, with RGCs-expressing mutant Bax showing complete resistance to raised hydrostatic pressure, even after axonal loss. While the primary site of damage in RGCs appears to be the axon, leading to axonal degeneration, and a positive correlation between raised IOP and axon loss has been established, it does not inevitably lead to cell soma death. The contribution of axonal degeneration and secondary challenges to the cell soma are evaluated below.

Diagnosis and Measuring Glaucoma Progression Traditionally, glaucoma is screened by monitoring changes in IOP using tonometry, a technique lacking sensitivity in glaucoma detection, as damage to the RGCs can occur in the absence of raised IOP. This lack of sensitivity sparked the development and introduction of alternative screening methods. Examples of these are standard automated perimetry to measure visual-field loss, optical coherence tomography, allowing the quantification of the retinal nerve fiber layer (RNFL) thickness, and disk tomography to assess any structural damage to the optic nerve head (ONH). The major drawback common to these methods is the inability to detect the disease before considerable damage to the retina has occurred. It is estimated that death of 50% of the RGCs occurs before there is a significant-enough visual loss to diagnose glaucoma. The development of detection of apoptosing retinal cell (DARC), a method of detecting RGC apoptosis in glaucoma before the onset of visual loss, may be instrumental in early diagnosis and successful treatment

Retinal Ganglion Cell Apoptosis and Neuroprotection

735

Extrinsic D apoptotic

signals D

Intrinsic apoptotic signals

Bid

Caspase-8

mPTP

ROS ∇

Neuron

D

Bcl-2

Ψm

Cyt C

Apoptosome

smac Caspase-9 AIF

ROS

DNA degradation

IAPS Caspase-3 Apoptosis

Figure 1 The intrinsic and extrinsic pathways of apoptotic cell death. Apoptosis can be triggered extrinsically through the binding of a death ligand to death receptors at the cell surface. The recruitment of procaspase-8 to the death receptors by adaptor proteins such as FADD follows allowing the conversion of procaspase-8 to its active form, caspase-8. Caspase-8 can cleave procaspase-3 triggering caspase cascade and the activation of the various effecter caspases which degrade DNA and various proteins. It can also act through a mitochondria-dependent manner, cleaving Bid, facilitating its translocation to the mitochondrial membrane and thereby initiating release of death mediators. Various intrinsic signals are also able to mediate apoptosis, through disruption of mitochondrial activity causing a subsequent decrease in the mitochondrial membrane potential (Dcm) and the activation of the mitochondrial permeability transition pore (mPTP), or through the actions of proapoptotic proteins such as Bax, Bak, Bad, and Bid. Following loss of mitochondrial stability various death mediators are released including cytochrome C (Cyt C), which in conjunction with APAF-1 and procaspase-9 forms the apoptosome which activates caspase-9 and triggers a caspase cascade. The release of Smac facilitates this process by inhibiting the actions of IAPS, an inhibitor of the caspase cascade. Various compounds, such as apoptosis-inducing factor (AIF), are also released from the mitochondria and are able to translocate to the nucleus where they trigger chromatin condensation and apoptosis. Mitochondrial dysfunction also leads to the increased generation of reactive oxygen species (ROS), which can trigger cell death through the modification of various molecules, including lipids, proteins, and DNA.

of the disease as well as a high-throughput screening method for new neuroprotectants.

Current Treatments for Glaucoma Once diagnosed, the current treatments for glaucoma concentrate on lowering the raised IOP. First-line pharmacological therapies include prostaglandin analogs which increase aqueous humor outflow and beta-blockers to reduce aqueous humor formation. Alpha-agonists to increase the uveoscleral outflow of aqueous humor and carbonic anhydrase inhibitors suppressing enzymes involved in aqueous humor production are used as second-line treatments, while third- or fourth-line treatments rely on increasing trabecular outflow using

cholinergenic agonists and miotic agents. The disadvantages all the drugs share are the potential side effects and the requirement for topical administration up to four times daily. Furthermore, the incidence of poor compliance and persistence with the topical application is high and so the success of glaucoma management is limited. In addition, glaucoma appears to progress in many sufferers despite the continued use of pharmacological treatments. In these cases, surgery such as trabeculoplasty to reduce resistance to the outflow of aqueous humor by modifying the trabecular meshwork or trabeculotomy to remove part of the trabecular meshwork, allowing enhanced drainage of the aqueous humor, may prove successful in reducing IOP. As the degeneration of visual field in glaucoma is known to progress through RGC apoptosis, research is currently underway to develop neuroprotective treatments for glaucoma.

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Neuroprotection Neuroprotection is defined as the use of therapeutic agents to prevent or reverse neuronal damage, thereby retaining physiological function. For example, neuroprotective treatments are being researched for diseases which progress through the death of neurons of the central nervous system such as Alzheimer’s, Parkinson’s, and Huntington’s. The efficacy of these therapeutic agents in clinical trials has been somewhat controversial. A number of clinical trials performed on stroke patients testing different neuroprotective agents showed either little success or adverse side effects, while treating patients of spinal cord trauma with the neuroprotective agent methylprednisolone resulted in improved motor function. The variation in success of these agents is thought to be due to the mechanism of neuronal death in the different diseases. In strokes, neuronal injury occurs at the cell body, so irreversible cell death occurs instantly; therefore, treatment with a neuroprotective agent would be given too late. However, in spinal cord trauma, neuroprotective treatment may have the desired effect as injury occurs at the axon and death of the cell body results hours later. For this reason, the use of neuroprotection as a treatment for glaucoma, thought to be brought about by death of RGC axons, seems a promising option.

Research Models Research into the eye disease uses models which are important tools allowing researchers to monitor the progression of the disease and test new therapies. In order for these models to provide informative and transferable data, similarity to the human disease and reproducibility is required. A number of different models are used in the study of the disease, all of which have their strengths and limitations. In Vitro Glaucoma Models Initial experiments for neuroprotection in glaucoma are likely to be carried out in vitro. A number of ocular cells have successfully been cultured and present a costeffective alternative to animal models for studying the effects of apoptosis and neuroprotection. Cell cultures have the advantage of allowing rapid screening of potential therapies and observation of direct effects on the cultured cells, under strict environmental control. Cell culture also provides a greater understanding of how compounds function at a cellular level; however, the response of the isolated cells, maintained in an artificial environment, may differ from that in situ. For this reason and the contentious use of immortalized cells to study apoptosis, a number of in vivo glaucoma models have been developed.

In Vivo Glaucoma Models Pioneered in rat and more recently developed in monkey, the optic nerve crush is a well-calibrated and reproducible model of glaucoma. Damage to the optic nerve results in cell-body death and subsequent secondary injury to adjacent neurons, as seen in glaucoma. Alternatively, RGC apoptosis can be induced by raising the IOP and causing retinal ischemia, typically achieved through blockage of the aqueous humor outflow, including injection of hypertonic saline into the episcleral veins, cauterization of the episcleral veins, and laser photocoagulation of translimbus. As an alternative, excessive exposure to excitotoxins, such as glutamate or N-methyl-D-aspartic acid (NMDA) by intravitreal injections can be used to induce RGC apoptosis. A further model can be generated by laser coagulation at the retina where RGC apoptosis is induced adjacent to the site of laser contact. The DBA/J2 mouse is a genetically determined glaucoma model showing increased IOP, RGC apoptosis, optic nerve atrophy, and ONH cupping.

Mechanisms of Apoptosis and Development of Neuroprotective Agents Neurotrophic Factor Withdrawal Deprivation of neurotrophic factors (NFs) induces apoptosis, and has been suggested to play a role in glaucoma as outlined in Figure 2. Raised IOP is proposed to lead to a blockade of anterograde and retrograde transports, preventing transport of NFs. The neuroprotective effects of neurotrophins (NTs), a family of NTFs, are thought to be mediated through the activation of phosphoinositide (PI)(3)-kinase, the inhibition of which is sufficient to block the survival effects of NT. PI(3)K phosphorylates and activates Akt which is known to target several key apoptosis regulators, including Bcl-2 antagonist of cell death (Bad) and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB). The phosphorylation of Bad promotes its sequestration by the chaperone protein 14-3-3, while CREB is activated by Akt (also known as protein kinase B), leading to the upregulation of B-cell CLL/lymphoma 2 (Bcl-2). The actions of Akt have downstream consequences for mitochondrial function and both caspase activation and increased ROS production have been attributed to loss of NT-stimulated pathways. NFs as Neuroprotective Agents Brain-derived neurotrophic factor (BDNF) has been showed in a number of studies to have neuroprotective effects. Intravitreal injections of the NT administered to rats, following optic nerve transection and IOP-induced ischemia, increased the survival of RGCs. In addition to BDNF, ciliary neurotrophic factor (CNTF), glial-cell-line-derived neurotrophic factor (GDNF), and pigment-epithelium-derived


NT

737

NT NTR

NT 14-3-3 Akt

PI(3)K

Bad

∇

Neuron

CREB

mPTP

ROS Ψm

Bcl-2 Nucleus

Apoptosis NT

Figure 2 Apoptosis induced by neurotrophic factor withdrawal. Axotomy prevents the retrograde transport of both neurotrophins (NTs) and neurotrophic receptors (NTRs). NTs are able to suppress apoptotic signaling through the binding of NTR, the activation of phosphotidylinositol-3-kinase (PI(3)K), and subsequent activation of Akt. Akt promotes cell survival through multiple pathways, including the phosphorylation of Bad, which promotes its sequestration by the scaffold protein 14-3-3 and the activation of CREB, which promotes increased expression of Bcl-2. Bcl-2 is an essential antiapoptotic protein, which inhibits the release of death mediators, thereby preventing initiation of the caspase cascade.

growth factor (PEDF) have also demonstrated protection of RGCs in rat glaucoma models. The neuroprotective effects of the NFs are short-lived, with reduced survival of RGCs only weeks after a single injection. The longevity of the treatment has been addressed using a number of techniques. Injection of PEDF-peptide-loaded nanospheres into an ischemic rat model reduced RGC apoptosis over a longer period. The same technique was used to administer GDNF to the DBA/2J mouse glaucoma model and an ischemic rat model; again, increased survival of RGCs was observed over a longer period. An alternative method, meant to prolong the effects of the treatment, used an osmotic pump to administer NFs to an axotomized rat model. The technique was successful in reducing RGC apoptosis but most cells were dead within 1 month. As an alternative to prolonged administration of NTs, researchers in this field have also looked toward gene transfer as a way of maintaining the required level of the NFs for long-term RGC survival. Transformation of a rat glaucoma model with the BDNF gene extended the life of the axotomized RGCs for a similar period. This work was furthered in the same model by injecting BDNF while simultaneously expressing the gene encoding the BDNF receptor, TrkB, known to show reduced expression in glaucoma models. The results showed increased survival of 76% of RGCs over a prolonged period.

Excitotoxicity Glutamate is a neurotransmitter reported in raised concentrations in the vitreous of glaucoma patients and in animal models. RGCs are known to be highly susceptible to cell death through not only glutamate excitotoxicity and but also treatment with the glutamate analog, NMDA. This has lead to glutamate excitotoxicity being proposed as both a mechanism of primary insult upon RGCs and as a secondary insult following the death of RGCs and the release of further excitory amino acids and glutamate. At elevated levels, glutamate triggers the excessive activation of ionotrophic receptors, such as NMDA receptors, resulting in a subsequent influx of Ca2+ ions and an increase in oxidative stress leading to cell death as depicted in Figure 3. Ca2+ is shown to increase mitochondrial permeability to ions and solutes and thought to regulate the mitochondrial permeability transition pore (mPTP), which is associated with the release of proapoptotic factors such as cytochrome C (Cyt C). Ca2+ has also been proposed to activate nitrous oxide synthase (NOS), leading to the generation of pathological quantities of nitric oxide (NO) and the subsequent production of reactive NO free radicals capable of triggering cell death, the full implications of which are discussed later. Calpains are a family of cytoplasmic-calcium-activated cysteine proteases. Axotomy, ocular hypertension, and

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Glia Glutamate

14-3-3

Raised vitreal glutamate

CaN Bad

Calpain Bcl-2

Ca2+

Bid

NOS NO

mPTP ROS

Δψm Neuron

Apoptosis Figure 3 Excitotoxicity-induced apoptosis. Raised vitreal glutamate triggers the activation of ionotrophic calcium channels such as NMDA receptors. The resultant increase in intracellular calcium activates calpain and, in turn, calcineurin (CaN), which promote the translocation of Bad and Bid to the mitochondrial membrane, and the subsequent release of death mediators. Ca2+ also activates nitric acid synthase (NOS) which promotes mitochondrial dysfunction, ROS production, and activation of the mitochondrial permeability transition pore (mPTP). Increased cellular ROS and the release of death mediators, such as Cyt C and AIF, leads to apoptosis.

NMDA excitotoxicity all demonstrated calpain activity with inhibition showing reduced RGC loss. Increased Ca2+ in the retina correlates with increased calpain activity, which can activate the caspase cascade as well as lead to the phosphorylation of spectrin, Tau, and p35, and cleavage of known calpain substrates, including the autoinhibition domain of calcineurin (CaN). In addition to activation by calpains, CaN has also been shown to undergo activating cleavage by caspases and interestingly raised IOP, although the mechanism of the latter is unknown. Sustained Ca2+ has also been shown to lead to increased levels of CaN, which induces apoptosis through the dephosphorylation of Bad, releasing the proapoptotic protein from sequestration by the chaperon protein 14-3-3. Bad is then able to translocate from the cytosol to the mitochondria, allowing it to heterodimerize with Bcl-2 and B-cell leukemia XL (Bcl-XL), leading to the release of Cyt C and the triggering of apoptosis. A role for CaN is further supported by the observation that dosing of cells with glutamate led to the translocation of a green fluorescent protein (GFP) fusion protein of Bad from the cytosol to the mitochondria, and the prevention of this in cells containing an inactive mutant form of CaN. As mentioned previously, Bad phosphorylation is regulated by NT and treatment with NT is sufficient to confer

resistance to excitotoxic insult. This suggests that excitotoxicity rather than working alone could act in conjunction with withdrawal of NFs to facilitate RGC apoptosis. Recent findings, however, have thrown doubt upon the hypothesis of glutamate excitotoxicity. Crucially, the raised glutamate levels observed in humans and animals have failed to be reproduced in follow-up studies. In addition, NMDA was able to induce apoptosis in the absence of Bax, a proapoptotic factor, suggested to be essential in apoptosis of RGCs in glaucoma. An alternative explanation for increased intracellular calcium and the subsequent triggering of apoptotic pathways is the activation of stress-activated channels by raised hydrostatic pressure. NMDA-Antagonists and Neuroprotective Agents This breakthrough of excess glutamate resulting in RGC apoptosis has led to considerable interest in the use of NMDA-receptor antagonists as neuroprotective therapies, many of which have been identified and characterized. MK801, a noncompetitive NMDA-receptor blocker, has been shown to protect RGCs from apoptosis in a number of experimental models including increased IOP and following intravitreal injections of NMDA in


rats. Despite showing promise as a successful neuroprotectant in the animal models, MK801 was never used in clinical trials due to its adverse side effects of inducing vacuole formation in neurons and neuronal necrosis. Other NMDA-receptor antagonists were also studied for use as potential neuroprotectants. For example, dextromethorphan was shown to aid the recovery of retinal activity in rabbits suffering from IOP-induced ischemia and flupirtine had a similar effect on the same model. Riluzole, an agent used for treatment of amyotrophic lateral sclerosis, reduced neuronal death following retinal ischemia brought about by raised IOP in rats. The most promising neuroprotective agent to date, memantine, is similar to MK801 but lacks the neurotoxic effects. Successfully used in the treatment of Alzheimer’s disease (AD) and Parkinson’s disease, memantine has also been shown to protect neurons from apoptosis in many different glaucoma models. Disappointingly, a recent second phase III clinical trial of memantine has revealed the compound to have no positive effect on visual-field deterioration in glaucoma patients.

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increase in reactive oxygen species (ROS) and cell death. Inhibition of ROS demonstrated a 50% reduction in RGC loss as did increasing expression of superoxide dismutase (SOD), known to be reduced in rat models with raised IOP. The principal mechanism for ROS production is disruption of mitochondrial function, shown in Figure 4, leading to loss of mitochondrial membrane potential and subsequent release of death factors. In addition to raised IOP, light exposure has been suggested as a risk factor in glaucoma, through the increased generation of ROS. In addition, ROS can mediate apoptosis by reacting directly with various molecules including DNA, proteins, and lipids.

Mitochondrial Dysfunction and ROS Generation ROS generation, particularly through the inhibition of complexes I and IV of the electron transport chain, has been implicated in various mechanisms of apoptosis, with treatment of antioxidants preventing apoptosis by inhibiting ROS generation and Cyt C release. A wide variety of antioxidants have been suggested as neuroprotectants in glaucoma, including vitamin E, 3-methyl-1,2-cyclopentanedione (MCP), and melatonin. Derivatives of catechin have been suggested to be potent antioxidants, and intravitreal coaddition of epigallocatechin was shown to attenuate the

Reactive Oxygen Species Oxidative stress leading to neuronal apoptosis is an early event, occurring within hours of raised IOP, both in vitro and in vivo. Following atoxomy of RGCs, there is both an

Light stimulus

ROS

OONO–

I II

ROS

ROS

SOD

NO

Ca2+

IV Neuron

Δψm mPTP

Protein and DNA degradation

III

Apoptosis Figure 4 Reactive oxygen species (ROS) and mitochondrial dysfunction in apoptosis. Increased concentrations of ROS can lead to apoptosis through the modification of lipids, proteins, and DNA. Superoxide dismutase (SOD), a key enzyme involved in the removal of ROS, is reduced in glaucoma, potentiating the cells to increased damage by ROS. Inhibition of the electron transport chain by compounds, such as nitric oxide (NO) and peroxynitrite (OONO ), leads to reduced mitochondrial membrane potential (Dcm) and increased generation of ROS, as has exposure to light. NO and Ca2+ can also promote mitochondrial dysfunction through activation of the mitochondrial membrane permeability transition pore (mPTP), and the release of death mediators.

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retinal damage caused by treatment with the NO donor, sodium nitroprusside. Ubiquinone (CoQ 10), a member of the electron transport chain, has been demonstrated to prevent lipid peroxidation and DNA damage. Furthermore, in recent tests in rats, the intraocular administration of CoQ 10 afforded neuroprotective effects.

Antioxidants as Neuroprotective Agents The most promising antioxidant treatment to date is that of orally administered Gingko biloba extract, which contains a number of substances shown to be effective in preventing mitochondrial damage through oxidative stress. Visual improvement has also been demonstrated in a doublemasked long-term placebo-controlled study, with efficacy and safety reports suggesting a daily dose of 120 mg to be sufficient.

Protein Misfolding The observation that AD patients demonstrated RGC loss typically associated with glaucomatous changes, including

optic neuropathy and visual impairment, suggested possible mechanistic similarities. Supportive of mechanistic similarities between Alzheimer’s and glaucoma is the observations of abnormal and phosphorylated Tau, a major plaque component, in the retina of glaucoma patients. b-Amyloid (Ab), generated by the abnormal processing of amyloid precursor protein (APP), is another major component in Alzheimer’s plaques. APP and Ab are present in RGCs following elevation of IOP in rat models and in the DBA/2J mouse model. It has been shown in vivo that Ab is neurodegenerative and that disruption of these APP processing pathways is sufficient to reduce RGC apoptosis in glaucoma models in rats. In AD, mutations altering the function of APP processing proteins lead to increased Ab production and, subsequently, increased apoptosis. A complementary mechanism, shown in Figure 5, has been implicated for glaucoma whereby normally rapid anterograde transport is blocked resulting in increased somal concentrations of APP. This has been proposed to trigger the abnormal processing of APP by caspase-3 in RGCs in glaucoma models generating increased Ab in the retina and is supported by observations of decreased vitreal Ab (suggesting increased deposition) in glaucoma patients.

Aβ

MMP-9

ECM degradation

Ca2+mediated effects

I II Neuron

SOD

ROS

III Aβ

IV Aβ

Δψm mPTP

Caspase-3 APP

Apoptosis

Figure 5 Ab-induced apoptosis. The anterograde transport of amyloid precursor protein (APP) is blocked by axotomy, leading to increased somal concentrations of APP. At elevated concentrations, APP triggers the abnormal activation of caspase-3 which cleaves APP to yield Ab, which has both intracellular and extracellular actions in promoting apoptosis. Ab is shown to target the electron transport chain promoting increased reactive oxygen species (ROS) production and mitochondrial dysfunction. Additionally, Ab upregulates the activity of ionotrophic calcium channels, increasing intracellular calcium and therefore promoting calcium-mediated apoptosis. Metalloproteinase-9 (MMP-9) also shows increased activity in the presence of Ab, promoting increased extracellular matrix (ECM) degradation and apoptosis through anoikis.


Ab is believed to induce apoptosis through elevated intracellular calcium and increased oxidative stress, as seen in AD, where oxidative damage is observed before significant plaque formation. Increased oxidative damage is indicative of mitochondrial dysfunction and both APP and Ab have been shown to target mitochondria. At raised levels associated with glaucoma, APP has been shown to interact with mitochondria clogging them and preventing normal function. Ab has been implicated in the inhibition of ketoglutarate dehydrogenase and complex IV of the electron transport chain, inhibition of both of which leads to increased ROS generation. This process could constitute a positive-feedback loop accelerating cell death, as the presence of ROS has been shown to facilitate Ab production. Following mitochondrial dysfunction, apoptosis has ultimately been suggested to be mediated through the initiation of a caspase cascade. Reduction of Misfolded Proteins in Neuroprotection Recent in vivo studies have shown that a reduction in RGC apoptosis can be achieved by targeting the Ab

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pathway. Inhibition of b-secretase, responsible for Ab production, reduced plaque formation and RGC apoptosis. Increased plaque removal and inhibition of plaque formation had a similar effect. All of these mechanisms were shown to be effective in reducing RGC apoptosis, with the greatest benefit seen through the use of combination therapy, which resulted in a maximal reduction in RGC apoptosis of greater than 80%.

Glial–Neuronal Interactions Glial cells, comprised of astrocytes, microglia, and Mu¨ller cells, act as support cells for neurons, maintaining their regular function by providing both neurotrophins and sustenance while removing toxic neurotransmitters and ions. Glaucoma induces dramatic changes in the glia gene expression patterns, potentially converting them from supportive to neurotoxic, as summarized in Figure 6. In the scope of this article, the impact of distinct types of glial cells are not differentiated, but evaluated as a whole. Glial cell activation has been demonstrated in the glaucomatous optic nerve, in the retina of glaucoma

Glia

TNFα TN

Fα

NOS

α

TNFα

F TN

Glutamate NO NO NO

Caspase-8

Raised vitreal glutamate

Bid NO

mPTP ROS

Ca2+ mediated effects

Ψm

∇

Neuron

Apoptosis Figure 6 Apoptosis induced by glial–neuronal interaction. Glial cells release neurotoxic molecules such as TNF-a, glutamate, and nitric oxide (NO), which are known to induce apoptosis in RGCs. TNF-a is a death ligand and triggers caspase-8-mediated extrinsic apoptosis. Elevated intravitreal glutamate, due to increased secretion and decreased uptake by glia, leads to apoptosis through calcium-mediated mechanisms, while increased NO causes mitochondrial dysfunction and the subsequent release of death mediators as well as increased reactive oxygen species (ROS) and cytotoxic modifications to key cellular components.

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patients and in glaucoma models showing altered levels of secretion. Increased secretion of NT appears to represent an attempted neuroprotective function, supporting damaged neuronal cells, while other changes, such as decreased secretion of interleukin (IL)-6 and increased secretion of NO and tumor necrosis factor (TNF)-a, which have been implicated in RGC apoptosis. TNF-a, a trigger for extrinsic apoptosis, activates caspase-8 leading to activation of the caspase cascade, Bid activation, the loss of mitochondrial membrane potential, and the subsequent release of cell-death mediators such as apoptosis-inducing factor (AIF) and Cyt C. Increased secretion of TNF-a by glia has been shown to correlate with increased disease severity in glaucoma patients and is further accompanied by the upregulation of TNF receptor 1 (TNF-R1) in glial cells and in RGCs and their axons in glaucoma patients. TNF-a also represents a possible genetic component for glaucoma with the TNF-a-308 gene polymorphism identified in glaucoma patients suggesting a role for TNF-a signaling. Glial cell secretion of NO, potentially stimulated by TNF-a , is raised in glaucoma with elevated NO concentrations shown to trigger axonal degradation and cell death in RGCs. There are three isoforms of NOS: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Initially, iNOS received the most attention as it is induced under various stress conditions. It was suggested to be raised in the ONH of glaucoma patients and also shown to be raised in glaucoma rat model utilizing cauterization, where treatment with inhibitors was sufficient to abate apoptosis. However, validation of this work in alternative models failed to produce correlating results, with both a murine model and a rat glaucoma model of IOP raised through intravitreal injections, failing to show the involvement of NOS or a reduction in RGC apoptosis by NOS inhibitors. It appears that iNOS upregulation could be due to secondary factors caused by the cauterization process and not raised IOP. While the role of iNOS in glaucoma has been called into doubt, it does not undermine the potential importance of NO signaling. nNOS and eNOS have been showed to be expressed at low levels in normal eyes in glial cells in the ONH, but shows dramatically raised levels in the ONH of glaucoma sufferers. The main mechanism, through which NO triggers cell death, appears to be disruption of mitochondrial function. An interaction between NO and the mPTP directly facilitates the release of Cyt C and AIF, with mPTP inhibitors abating NO-induced neuronal apoptosis. Caspase-3 activation, however, has been shown without initial loss of mitochondrial membrane potential, suggesting activation may result from blockade of the electron transport chain and subsequent increased levels of ROS. Loss of mitochondrial membrane potential can be triggered as a downstream event, following prolonged inactivation of the electron transport chain.

NO binds complex IV of the electron transport chain, also known as cytochrome oxidase, reducing the enzyme’s affinity for oxygen. Prolonged exposure to NO or peroxynitrite (OONO ), a highly destructive molecule formed from ROS and NO, can lead to blockade of complex I of the electron transport chain. This facilitates further ROS production and OONO synthesis, which are capable of extensively modifying cellular components leading to apoptosis, while antioxidant treatment in NO-induced apoptosis was shown to abate caspase activation. Disruption of mitochondrial respiration has also been suggested to facilitate the disruption of Ca2+, associating NO with excitotoxicity-induced apoptosis. This link was strengthened by observations that nNOS-deficient mice were resistant to NMDA-induced RGC death. NO induced modest increases in the amplitude of Ca2+ channels and the induction of the Ca2+-mediated death effectors, the calpains. Extracellular Matrix Degradation Glaucomatous changes include extensive remodeling of the extracellular matrix (ECM), altering the levels of the various ECM components, including collagen I and IV, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), transforming growth factor beta 2 (TGF-b2), and laminin. MMP expression in particular has received attention, with expression in glaucoma patients being raised in comparison to normal patients. The condition of the ECM both regulates and is regulated by the expression and release of MMPs, with the subsequent reduced levels of laminin contributing to cell death, and significant RGC loss in the retina. Supportive evidence from the ability of TIMP-1 to abate neuronal apoptosis, as well as observations that MMP-9-deficient mice demonstrate reduced laminin degradation and increased resistance to neural trauma, further highlights the potential importance of MMPs in glaucoma. It was recently demonstrated in vivo that raised IOP induced remodeling of the ECM within the retina. Raised IOP was shown to correlate to decreases in laminin and TGF-b2 and increased MMP-9, TIMP-1, and RGC apoptosis. Loss of survival signals from the ECM is thought to induce a specific form of apoptosis called anoikis. Whether glaucomatous changes are initiated at the retina or ONH is still a matter of contention. Astrocytes in the ONH have been shown to be activated by raised IOP and produce MMPs that are able to remodel the ECM, possibly resulting in axonal compression and thereby facilitating apoptotic mechanisms associated with the blockade of anterograde and retrograde transport. In addition to raised IOP, potential apoptotic mechanisms such as Ab, NO, ROS, excitotoxicity, and TNF-a have all been implicated in triggering an increase in


MMP-9, suggesting that MMP-9 may represent an important downstream executor of apoptosis rather than a primary affecter.

Table 1 Comparison of the models used to test the different neuroprotective agents Neuroprotectant

Animal

Model

MK801

Rat

Detromethorphan Flupirtine

Rabbit Rabbit Rat

Riluzole Memantine

Rat Monkey Mouse Rat

Brain-derived neurotrophic factor (BDNF)

Rat

Ciliary neurotrophic factor (CNTF)

Rat

Glial-cell-line-derived neurotrophic factor (GDNF) Pigment-epithelium-derived growth factor (PEDF)

Mouse Rat Rat

Myelin basic protein (MBP) T-Iymphocytes

Rat

Myelin basic protein (MBP)

Rat

Copolymer 1 (Cop 1)

Rat

Monoclonal anti-Ab lgG Congo Red b-Secretase inhibitor Epigallocatechin (EGC) Ubiquinone (CoQ10) Vitamin E

Rat Rat Rat Rat Rat – Human Rat Human

Increased IOP NMDA injection Increased IOP Increased IOP Increased IOP NMDA injection Increased IOP Increased IOP DBA/2J Glutamate injection Increased IOP Optic nerve crush Increased IOP Optic nerve transection Increased IOP Optic nerve transection DBA/2J Increased IOP Increased IOP Optic nerve transection Optic nerve transection Spinal cord contusion Spinal cord contusion Glutamate injection Increased IOP Optic nerve crush Increased IOP Increased IOP Increased IOP NO donor Increased IOP Primary cultures Glaucoma patients Increased IOP Glaucoma patients

Neuroprotective Vaccine Studies using rat and mouse models of glaucoma have suggested a potential role for autoimmunity in the protection of neurons from secondary damage. Following the initial insult, nonspecific T-lymphocytes have been shown to accumulate at the primary lesion site. While nonspecific T-lymphocytes did not exhibit neuroprotection, effects were observed upon injection with myelin basic protein (MBP)-specific T-lymphocytes or immunization with MBP. Unfortunately, injections of anti-MBP-specific T-lymphocytes and MBP immunization induced the paralytic condition – experimental autoimmune encephalomyelitis (EAE). Copolymer 1 (Cop 1), a synthetic peptide based on MBP, was discovered to suppress EAE and shown in clinical trials to be beneficial to patients with multiple sclerosis. Further studies have revealed that immunization with Cop 1 increases RGC survival following optic nerve crush, glutamate injections, and increased IOP in rats. This research shows that there is potential for the development of a glaucoma vaccine.

Summary The primary mechanism of RGC loss in glaucoma is through apoptosis, which can be triggered through a variety of mechanisms both intrinsic and extrinsic. Apoptotic signaling shows a large level of redundancy with extensive cross talk. This redundancy poses a major problem in terms of neuroprotection with inhibition of one apoptotic pathway merely delaying apoptosis before mediation through an alternative pathway or the triggering of necrosis. It is still unclear as to the primary mechanism of apoptosis induction in glaucoma, although an increased understanding would aid a more effective development of neuroprotective strategies. Fundamental to the successful development of neuroprotective strategies is targeting the apoptotic pathway upstream and at multiple points to maximize effectiveness. The studies of neuroprotective agents on glaucoma models carried out to date have shown great promise with many different agents demonstrating efficacy in a large number of models, summarized in Table 1. Unfortunately, translation of this research from animals through to clinical trials has exposed complications with side effects and inefficacy, hindering progression. However, due to the complicated nature and a number of different overlapping pathways inducing apoptosis, the identification of a multitude of potential neuroprotectants has been possible. One major problem still faced in the analysis of these compounds is the difficulties monitoring efficacy in vivo.

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Gingko biloba

It is hoped that the development of the novel DARC technique for early diagnosis of glaucoma may also be a valuable tool in the analysis of potential neuroprotectants. See also: Information Processing: Ganglion Cells; IOP and Damage of ON Axons.

Further Reading Cheung, W., Guo, L., and Cordeiro, M. F. (2008). Neuroprotection in glaucoma: Drug-based approaches. Optometry and Vision Science 85(6): 406–416. Cordeiro, M. F., Guo, L., Luong, V., et al. (2004). Real-time imaging of single nerve cell apoptosis in retinal neurodegeneration. Proceedings of the National Academy of Sciences of the United States of America 101(36): 13352–13356.

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Grossmann, J. (2002). Molecular mechanisms of ‘‘detachment-induced apoptosis – anoikis’’ Apoptosis 7(3): 247–260. Guo, L. and Cordeiro, M. F. (2008). Assessment of neuroprotection in the retina with DARC. Progress in Brain Research 173: 437–450. Kuehn, M. H., Fingert, J. H., and Kwon, Y. H. (2005). Retinal ganglion cell death in glaucoma: Mechanisms and neuroprotective strategies. Ophthalmology Clinics of North America 18(3): 383–395, vi. Lebrun-Julien, F. and Polo, A. D. (2008). Molecular and cell-based approaches for neuroprotection in glaucoma. Optometry and Vision Science 85(6): 417–424. Lin, M. T. and Beal, M. F. (2006). Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443(7113): 787–795.

Lipton, S. A. (2003). Possible role for memantine in protecting retinal ganglion cells from glaucomatous damage. Survey of Ophthalmology 48(supplement 1): S38–S46. Wein, F. B. and Levin, L. A. (2002). Current understanding of neuroprotection in glaucoma. Current Opinion in Ophthalmology 13(2): 61–67. Weinreb, R. N. and Lindsey, J. D. (2005). The importance of models in glaucoma research. Journal of Glaucoma 14(4): 302–304. Yuan, J. and Yankner, B. A. (2000). Apoptosis in the nervous system. Nature 407(6805): 802–809. Zhong, Y. S., Leung, C. K., and Pang, C. P. (2007). Glial cells and glaucomatous neuropathy. Chinese Medical Journal (Engl) 120(4): 326–335.

Retinal Histogenesis J A Brzezinski, IV and T A Reh, University of Washington, Seattle, WA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Birthdate or born – The time when a progenitor permanently exits the cell cycle. Marking cells in their final division is referred to as birthdating. Cell fate determination – The process by which a progenitor is programmed to become a specific cell type and its functional maturation. In the retina, a progenitor needs to acquire competence, exit the cell cycle, become specified, and differentiate. Competence – The potential to adopt a certain cell fate(s). Differentiation – The functional maturation of a specified cell. Histogenesis – The development of a mature tissue from a naive progenitor population. Lineage – A cohort of cells derived from division(s) of a common progenitor. This is also referred to as a clone. Lineage tracing – The use of an indelible marker to label cells and all their descendants. Analysis at a later time point allows the inference of lineal relationships. This is also referred to as fate mapping. Multipotent progenitor/progenitor – A cell that has competence to adopt several different fates. These cells may or may not be proliferative. Progressive restriction model – A model of cell fate determination where multipotent progenitors lose the competence to adopt multiple cell fates over time. By this model, early progenitors have the competence to form all fates in a tissue. Serial competence model – A variation on the progressive restriction model whereby multipotent progenitors transiently gain and subsequently lose competence for a subset of fates in a tissue over time. By this model, progenitors do not have the competence to form late fates at the earliest time points. Specification or commitment – The point when a progenitor cell has irreversibly decided on a cell fate.

Birthdating An interesting property of retinal cells is that they do not continue to divide after they differentiate. This means

that at some point in development, a progenitor permanently exits the cell cycle, referred to as its birthdate. Investigators took advantage of this property and designed a clever pulse-chase experiment to investigate whether different cell types exited the cell cycle at characteristic times. Animals at various stages of development were given a pulse of 3[H]-thymidine (3HdT). The 3HdT is incorporated into replicating DNA during synthesis (S)-phase and any excess is cleared from the body quickly. The incorporated 3HdT is maintained in the newly synthesized DNA permanently. If the cell continues to divide, it will dilute the 3HdT signal by one-half each division. Next, autoradiography was conducted after the retina was fully formed (the chase). Cells that retained maximum labeling are those that exited the cell cycle (born) on the day of 3HdT administration. More recently, birthdating studies have been conducted with synthetic nucleotides, such as 5-bromo-2-deoxyuridine (BrdU), which can be detected by antibodies instead of autoradiography. About 50 years ago, Sidman used birthdating studies to test what order, if any, retinal cells were formed in the rodent retina. The observations of Sidman and future investigators revealed a stereotypical birth order that was broadly broken down into early and late groups (Figure 1). Retinal ganglion cells (RGCs) were born first, followed closely by horizontal cells, cones, and amacrines. The late cohort comprised rods, bipolar cells, and Mu¨ller glia. Birthdating has been conducted in several vertebrate species. While there are some small differences in the order, the first cells born in all species examined are RGCs. Although there is clearly an overall birth order, which is evident from the production onset of each cell type, there is also considerable overlap in the genesis of the cell types, such that multiple cell types are born on the same day of development (Figure 1). This overlap is also observed in species where retinal histogenesis is long, such as in monkey. These data implied that cell fate determination is not strictly regulated by measuring time (or cell cycles) during development. Nonetheless, the observance of a birth order indicated that there is a temporal input into cell fate determination. These studies raised several questions about retinal progenitors. Are all retinal cell types derived from the progenitors in the optic cup? Are there different progenitors for each retinal cell type? Is fate choice predetermined or stochastic? These questions were addressed by tracing the fate of individual progenitors.

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Retinal Histogenesis 120 100 con 80

hor rgc

60

ama 40

rod bip

20

mul 0 E12

P0 Age

(a)

P10

Cumulative percent of cells generated

100 Amacrine Müller Bipolar Rods

60 50%

40

20

0 (b)

Ganglion Horizontal Cones

80

0

50 100 Age at injection (postconception days)

150

Figure 1 Birthdating. (a) Data from the rat retina showing the birthdates for each cell type as a percentage of its total. The progression from early to late cell-type generation that was first observed by Sidman can be clearly identified. CON, cone; HOR, horizontal cell; RGC, retinal ganglion cell; AMA, amacrine cell; BIP, bipolar cell; and MUL, Mu¨ller glia. (b) Similar thymidine labeling study in monkey shows a similar, though not identical, pattern of birthdates for the various types of retinal cells. Despite the fact that the patterns of generation are not identical, the early and late fates are largely segregated. (a) Data from Rapaport, D. H., Wong, L. L., Wood, E. D., Yasumura, D., and LaVail, M. M. (2004). Timing and topography of cell genesis in the rat retina. Journal of Comparative Neurology 474(2): 304–324. (b) Modified from La Vail, M. M., Rapaport, D. H., and Rakic, P. (1991). Cytogenesis in the monkey retina. Journal of Comparative Neurology 309(1): 86–114.

Lineage Tracing To understand the behavior of progenitor cells, a way to trace the fate of individual progenitors was needed. Starting in the late 1980s, investigators designed elegant lineagetracing (fate-mapping) experiments to study the retina. In rodents, Turner, Snyder, and Cepko used replicationincompetent retroviruses encoding a marker gene (e.g., LacZ) to infect retinal progenitors at various time points. These retroviruses can only infect dividing progenitors. Since the virus integrates into the genome, the progenitor and its descendents become permanently labeled. By adjusting the titer of the virus, individual progenitor lineages (clones) can be mapped from mature retinas (Figure 2).

The first set of retroviral lineage-tracing studies examined postnatal infections of rat retinas. In postnatal day 0 (P0) infections, majority of clones contained rods and were predominantly small (one to four cells). A large number of clones contained only rods, whereas others contained rods, amacrines, bipolars, and/or Mu¨ller glia (Figure 2 and Table 1). There were few clones that did not contain any rods. Cell types born before P0 (e.g., cones, horizontals, and RGCs) were not observed in these clones, consistent with previous birthdating analyses. Clones generated from P2, P4, and P7 infections become progressively smaller. This reflects the progressive decrease in progenitor division that occurs in the first postnatal week. Clones were heterogeneous; some


contained multiple cell types and others one cell type or just a single cell. From all these time points, there was no obvious lineage hierarchy that could be constructed from the clone composition data. This showed that mammalian retinal cell fate determination is stochastic, or nondeterministic. Importantly, they observed two-cell clones that had different fates. This implied that fate choice is decided during or after the last cell division. To see if the progenitors in the optic cup can give rise to all the retinal cell types, the investigators generated clones from embryonic (E) time points when few cells have exited the cell cycle. For this reason, they injected their retroviruses into the subretinal space of E13 and E14

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mice in utero, a difficult procedure. The mice were allowed to mature and clone composition was examined. The clone size and composition were highly heterogeneous (Table 1). All seven cell fates were represented in these clones. Moreover, these clones never contained any other cell types (i.e., astrocytes, vascular endothelial cells, pigment epithelium, etc.). These early lineage traces showed that all seven retinal cell types derive solely from a retina-restricted progenitor pool. The heterogeneity in clone composition (there were few multicell clones alike) reinforced that fate choice is apparently stochastic. Consistent with retinal cell frequency (78% are rods in mice), nearly all clones contained one or more rods. Clone size varied from 1 to

Apical ONL

INL Basal

GCL Figure 2 Lineage analysis. Diagram showing the basic strategy to track the lineages of the retinal progenitor cells. A progenitor cell is labeled (red) at an early stage of development, by using either a retroviral vector with a reporter gene, or a direct injection of a tracer. The cell undergoes multiple rounds of division in this case, and when the retina is examined in the adult animal, the different types of retinal cells can be identified by their laminar position and their morphology. In this case, two rods, a bipolar cell, an amacrine cell, and a Muller glial cell were derived from the progenitor. See Table 1 for more examples of the types of clones found in these experiments. Table 1

Retroviral lineage tracing in rodents

Age of infection

E13

E14

P0

P2

P4

P7

Ave. clone size Max. clone size Cell fatesa Clone examples

46.4 217 r, b, a, c, m, g, h 1g 1c 1c, 1h 17r, 1c, 3b, 1m, 1a 43r, 1c, 7b, 2a 111r, 16b, 1m, 5a, 1g

26.3 234 r, b, a, c, m, g, h 2c 1c, 1g 8r, 1c 51r, 6b, 1m 164r, 2c, 1h, 12b 28r, 9b, 3a, 1g

2.5 22 r, b, a, m 4r 1a 3r, 1b 2r, 1b, 1a 2r, 1b, 1m 10r

1.6 7 r, b, m, a 2r 1b 1r, 1b 1r, 1m 1r, 1a 3r, 1m

1.4 7 r, b, m, a 1r, 1b 1r, 1m 2r 3r, 1b 1r, 1a 1r, 1b, 1a

1.1 2 r, m 1r 1m 1r, 1m 2r

a

Cell fates represented in clones (r, rod; b, bipolar; a, amacrine; c, cone; m, Mu¨ller; g, ganglion; h, horizontal) listed in decreasing frequency observed. In mice, the cell frequency in decreasing order is r, a, b, m, c, g, and h. Data from Turner, D. L. and Cepko, C. L. (1987). A common progenitor for neurons and glia persists in rat retina late in development. Nature 328: 131–136; and Turner, D. L., Snyder, E. Y., and Cepko, C. L. (1990). Lineage-independent determination of cell type in the embryonic mouse retina. Neuron 4: 833–845.

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234 cells and had a bimodal distribution. There were abundant small clones (20 cells). The largest clones are simply too big to have been generated by simple asymmetric (one progenitor, one neuron) divisions in the time allotted for development. Based on the distribution of clone sizes, it is possible that progenitors preferentially utilize symmetric divisions during retinal histogenesis. Lineage-tracing studies have been conducted in other vertebrates, yielding similar results. These lineage-tracing studies answered several questions. First, retinal progenitors in the optic cup are multipotent and give rise to all seven cell types. Second, there are no separate progenitors for each cell type. Nonetheless, rod-only clones were observed, raising the possibility of a rod-only progenitor. Since rods make up 78% of the mouse retina, small- to medium-sized rod-only clones are expected from multipotent progenitors. Fourth, cell fate choice is stochastic. Fifth, cell fate specification occurs during the last cell cycle or later. These data raised several more questions about the mechanisms of retinal histogenesis. Is fate determination a cell autonomous process, a cell nonautonomous process, or a combination of both? Do retinal progenitors have broad competence that is gradually lost, or are progenitors more limited in their cell fate choices during development? These questions have been addressed by manipulating the local cellular environment.

Environmental Challenge Several approaches have been undertaken to discriminate between cell autonomous and cell nonautonomous control of retinal cell fate determination. Most of these experiments involve challenging retinal progenitors with different environments. Unlike the previous birthdating and lineage-tracing studies, these environmental challenge experiments are harder to interpret and, at times, yield conflicting results. One type of experiment was designed to answer the question: What is the default state of retinal progenitors? For these experiments, retinal progenitors were labeled with 3HdT and dissociated to single cell density to examine their potential for differentiation in isolation. Reh and Kljavin saw that when rat progenitors were isolated from early stages of development, the majority of the progenitors differentiated into RGCs, while progenitors isolated from late stages of development differentiated into cell types normally generated late in development, like rods. A somewhat different result was obtained from similar studies in the chick embryo by Adler and colleagues, suggesting that cones were the default cell fate. However, subsequent studies in chick revealed a ganglion cell bias for the progenitors isolated from the earliest stages of

retinal development. Together, these results led to the concept of a rolling default, or shifting competence in the progenitors; in other words, there is an intrinsic bias to the types of neurons generated by the progenitors that shifts progressively over developmental time. This idea has been recently supported by Notch-signaling studies. The Notch receptor is active in progenitor cells throughout development, and inhibition of signaling leads to premature progenitor differentiation. Inhibition of Notch in early progenitors leads to the overproduction of RGCs and cones, whereas inhibition of Notch function later in development results in overproduction of rods, but not of RGCs or cones. These results show that retinal progenitors change their competence intrinsically over time. In the next type of experiment, investigators asked whether inductive interactions among the retinal cells played a role in their cell fate determination. Studies from Drosophila eye imaginal disk had shown an important role for cell–cell interactions in directing the ommatidial progenitors to their individual identities. In that tissue, the data were best fit by a sequential cell induction model in which the first cells, the R8 photoreceptors, induce the recruitment of the next type of photoreceptor, and so on. To test whether similar sequential inductions occurred in vertebrate retinas, investigators used heterochronic co-cultures, surrounding early embryonic progenitors with late-generated retinal cells. The groups of Raff and Reh co-cultured early embryonic rodent retinal cells with an excess of postnatal retinal cells. In both sets of experiments, the early progenitors were more likely to develop into rhodopsin expressing (rod photoreceptors) cells when compared to early progenitors cultured alone (which developed primarily early retinal fates). This increase was not seen when the challenging (older) cells were derived from the brain instead of the retina. Watanabe and Raff also found that the increase in rods was observed when the two cell populations were separated by a cell-impermeable membrane. Together, these data showed that a soluble factor(s) from late retina can promote rod fate in younger cells. In addition, very early mouse retinal progenitors (E11–E12) could form rods when cultured with an excess of older rat retinal cells; birthdating analyses indicate that rods are not normally generated by progenitors from this very early retina, which suggests that rod competence precedes rod genesis by at least 1 day. These experiments led to the idea that although progenitors may have an intrinsic bias in the types of cells they generate at any time in development, their fate can be influenced by factors in the microenvironment. Many subsequent studies have identified signaling factors in the developing retina that can influence the fate of the progenitors and, particularly, factors that can increase the percentage of these cells that differentiate into rods; however, since low-density cultures do not support robust rod differentiation (i.e., expression of


lineage trace (similar to above) in E16–E17 retinal explants (intact tissue) cultured the same amount of time. The clones in both cases were screened for rod, bipolar, amacrine, and Mu¨ller glial cell fates. The clone composition and size in both experimental systems were similar in isolation and in explants. This suggested that fate choice is largely cell autonomous and that any given cell type is not required to induce (specify) another. In sum, while many studies have shown a role for cell–cell interactions in the control of retinal cell fate, the relative importance of intrinsic and extrinsic regulation is still not resolved. These data have been used to assemble cell fate determination models (Figure 3). One model, progressive restriction, argues that early progenitors have competence to adopt all retinal cell fates and that this competence is gradually lost (restricted) over time. By this model, nonautonomous contributions are expected to specify cell fate in these multipotent progenitors over time. Early progenitors should be able to adopt late fates (if stimulated properly) and competence should extend beyond the normal genesis window to allow for feedback inhibition. Another model, serial competence, contends that progenitors have competence for only a few cell fates at any given time and that progenitors serially cycle through numerous restricted competence states. By this model, cell nonautonomous inputs are not required for fate specification. Presumably, the mechanism that controls competence dynamics would be reflected by the complement of transcription factors expressed in progenitors. In the next section, we discuss evidence that transcription factors regulate competence.

Competence

rhodopsin and other markers), it is possible that most of the factors identified to date have more of an effect on the expression of these identifiers, rather than the choice of the rod fate per se. Another concept that emerged from the early studies of retinal development was the idea that specific cell types use feedback regulation to control their density. Elimination of dopaminergic amacrine cells in the developing frog leads to an overproduction of these cells by progenitors at the ciliary marginal zone. This suggested that there is a nonautonomous feedback regulation to negatively control amacrine cell number. In vitro experiments found a similar effect in developing rat retina. When E16 retinal cells were co-cultured with an excess of P0 cells, fewer amacrines were generated. However, when the P0 cells were depleted of amacrine cells, there was an increase in the number of amacrine cells generated by the E16 cells. When the converse experiment was done, P0 cells co-cultured with an excess of E16 cells, an increase in amacrines and bipolars was seen along with a decrease in rods. This confirmed the presence of negative feedback on amacrine cells and implied feedback regulation of bipolar cell genesis. The concept of feedback regulation of retinal cell production was extended to ganglion cells by Waid and McLoon. They examined the influence of a late retinal environment on RGC fate determination using heterochronic co-cultures in chick. Early cells were inhibited from RGC fate when co-cultured with late retinal cells. This inhibition was not observed when RGCs were depleted from the challenging (late) cell population, which showed that RGCs can nonautonomously feed back to inhibit further RGC production. In a subsequent study from the McLoon lab, they blocked Notch signaling (an RGC inhibitor) at different times. Interestingly, when later time points were examined, they saw newborn RGCs in areas where RGC genesis had ceased in controls. This suggested that RGC competence extends beyond RGC genesis. Although the studies described above have supported a role for cell–cell interactions in the regulation of cell fate in the vertebrate retina, some types of studies have failed to find nonautonomous effects. Rapaport and colleagues conducted heterochronic transplants in frogs. When younger retinal tissue was transplanted into older hosts, the donor tissue did not adopt later fates or differentiate early. This result was also seen in co-cultured cells in vitro. Importantly, the donor cells directly adjacent to the older host cells were not fate shifted. This suggested that cell fate determination is not regulated by changing environmental stimuli, rather, that cell competence is limiting in frogs. Cayouette and colleagues combined lineage tracing and single cell culturing of rodent progenitor cells. In this experiment, E16–E17 rat progenitors were plated at single cell density and the resulting clones were examined 7–10 days later. In parallel, they conducted a retroviral

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RGC CON HOR AMA ROD

BIP

MUL

(a)

(b) Figure 3 Cell fate determination models. (a) Serial competence model. The progenitor changes over time in their potential to generate different types of retinal cells. These changes in competence would be mediated by changes in the complement of transcription factors present in the cells. (b) Progressive restriction model. The progenitor can initially generate all types of retinal neurons, but over time loses one or another of the transcription factors needed for a specific fate. This is shown in the figure as what is initially a rainbow-colored cell, progressing into a cell that is only red. In principle, the progressive restriction could be due to a loss in key transcription factors, or alternatively by the addition of new repressors.

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Transcription Factors and Competence How transcription factors regulate retinal progenitor competence is beginning to be characterized (Figure 4). Although most of these transcription factors are expressed in the postmitotic cells, and therefore are unlikely to regulate the competence of the progenitor cells per se, there are members of several different transcription factor families that are expressed in the mitotically active cells. One transcription factor expressed by progenitors that appears to convey competence is Pax6. This eye-field transcription factor is expressed in all progenitors and directly promotes the expression of other transcription factors (Ascl1, Ngn2, Atoh7, etc.). Specific deletion of Pax6 from progenitors leads to the apparent loss of competence to generate all retinal cell types except amacrine cells. A complementary result is obtained when FoxN4, a transcription factor expressed in a subpopulation of progenitors, is deleted in mice. In FoxN4 null mice, amacrine cells fail to develop. This suggests that the combination of FoxN4 and Pax6 can convey progenitors with competence for all retinal cell fates. Ikaros is a transcription factor that is expressed primarily by early-staged progenitors, and mice deficient in this gene have fewer early-born neurons. While these examples show that changes in transcription factors can affect the types of neurons produced by

bHLH

Homeodomain

Progenitor

Ascl1 Pax6 Rax Ngn2 Prox1 Six3/6 Olig2 Hes5 Hes1 Chx10 Lhx2

Precursor

Ath5 Ath3

Differentiation cell type RGC CON

NeuroD1

HOR

Brn3

Islet1

Crx

Otx2

Pax6

Prox1 Prox1

AMA

NeuroD1 Ath3 Ptf1

Pax6

BIP

bHLHb4

Chx10 Otx2 Islet1

ROD

NeuroD1

Crx

MUL

Hes1

Otx2

Other Sox9 Sox2 FoxN4

TRbeta2 RXRg RORbeta

Nrl

Nr2e3

Rax

Figure 4 Transcription factor code. Kageyama and others have proposed that a combination of bHLH and homeodomain transcription factors specify each retinal cell type. Although most of these factors are expressed primarily in postmitotic cells, and therefore might not be candidates for the changing competence models described above, several lines of evidence indicate that these factors are necessary for the full differentiation of these cell types.

progenitors, and hence their competence to generate specific neuronal types in the retina, it has also become clear that a more general level of competence, to generate neurons versus glia, is also conveyed by these factors. Another transcription factor expressed in a subset of progenitors is Ascl1 (Mash1), a member of the bHLH class. Prior to E15 in the rat retina, there is little Ascl1 expression in the retina, though progenitors by this age are producing RGCs, cones, rods, and horizontal and amacrine cells. Even after E15, Ascl1 is expressed in most, but not all, progenitors. Deletion of this gene in mice leads to an overproduction of Mu¨ller glia, apparently at the expense of rods and bipolar cells. It appears that Ascl1 imparts retinal progenitors with the competence for lategenerated retinal neurons, while also inhibiting Mu¨ller glial fate specification (Figure 5) in part by maintaining the expression of Hes6, but also by driving expression of key components of the Notch pathway, including Hes5 and Hes1, to maintain the progenitors in an undifferentiated state while they generate additional neurons. When Notch signaling is reduced, even for times as short as 6 hours, the retinal progenitors are irreversibly committed to exit the cell cycle and differentiate. Since the Notch effector genes, Hes1 and Hes5 are normally regulated during the cell cycle, such they are lowest during the G2 and M-phases, one model for the mechanism by which progenitors initiate differentiation is through a progressive slowing of the cell cycle as development proceeds (Figure 5). Immunolabeling for the active Notch intracellular domain in mouse retina has confirmed that Notch signaling is lowest in cells whose nuclei are at the apical surface. However, this is apparently not the case in the fish retina. Live imaging studies in cortex by Kageyama’s group confirm that Notch signaling oscillates through the cell cycle, with the lowest levels in the progenitors with nuclei located at the apical surface, those cells that are in G2- or M-phases of the cell cycle. Given the highly transient nature of Notch signaling and the very fast cell cycles in fish, further studies using highly destabilized reporters will be needed in the fish to determine whether this difference is real. Another member of the bHLH class of transcription factors, Atoh7 (Math5), is transiently expressed by a small subset of postmitotic progenitors. Lineage-tracing and genetic-deletion studies have shown that it is necessary for RGC competence. In addition to these transcription factors expressed in progenitors, there are many that are expressed in subpopulations of nascent neurons. For example, Ptf1a, NeuroD1, and Math3 are expressed in amacrine cells, and loss of one or more of these genes leads to defects in amacrine cell fate determination or survival. Moreover, overexpression of transcription factors such as NeuroD1 can drive progenitor differentiation into specific cell types, though the types appear to vary depending on the species and the method of


Notch inactivation (late progenitor)

Normal

Ascl1–/– SC

SC

SC GC

GC C

EP

GC C

EP

HC

751

C

EP

HC

HC

Ascl1 AM

AM

Hes6 DII1,3,4

R

Notch Hes1,5

LP

Neuron Progenitor (early and and Muller glia late)

LP

Hes6 DII1,3,4

BP

Notch Hes1.5

MG

AM R

Ascl1

BP

MG

Neuron Progenitor (late) muller glia

Ascl1

Hes1

R

Ascl1 Hes6DII1.3.4

LP

Notch Hes1,5 Neuron (late)

Notch-ICD

BP

MG

Muller glia

Hes1

Sox9

Active

S-phase

Off

DSM

Ascl-1 Notch Hes1.2

Figure 5 (Top row) Ascl1/Mash1 functions to maintain neuronal competence in the progenitors. Deletion of this gene in mice leads to an overproduction of Mu¨ller glia, apparently at the expense of rods and bipolar cells. It appears that Ascl1 imparts retinal progenitors with the competence for late-generated retinal neurons, while also inhibiting Mu¨ller glial fate specification by maintaining the expression of Hes6, and by driving expression of key components of the Notch pathway to maintain the progenitors in an undifferentiated state while they generate additional neurons. SC, stem cell; EP, early progenitor; LP, late progenitor; GC, ganglion cell; C, cone; HC, horizontal cell; AM, amacrine cell; R, rod; BP, bipolar cell; and MG, Mu¨ller glia. (Bottom row, left) Model of how Notch signaling changes with the cell cycle in mouse and chick retina. Notch signaling is high during the S-phase of the cell cycle and low to absent at the apical (ventricular) surface when cells are in G2 and M-phases of the cell cycle. This can be seen in the lower middle and right panels by the Notch ICD immunoreactivity (arrows) and the expression of Hes1, a downstream effector of Notch. By contrast, progenitor markers like Sox9 (red) and Ascl1-GFP (green), are expressed throughout the cell cycle in progenitor cells. Modified from Nelson, B. R., Hartman, B. H., Ray, C. A., et al. (2009). Acheate-scute like 1 (Ascl1) is required for normal delta-like (Dll) gene expression and notch signaling during retinal development. Development Dynamics 238: 2163–2178; and from Nelson, et al. (2007) Transient inactivation of notch signaling synchronizes differentiation of neural progenitor cells. Developmental Biology 304: 479–498.

overexpression. These studies show that few transcription factors fit cleanly in the simple models of progressive restriction or serial competence. Moreover, since few targets of these transcription factors have been identified, the nature of competence regulation remains unclear. Nevertheless, the transcription factor networks for two cell types, RGCs and rod photoreceptors, are beginning to be worked out.

Conclusions A great deal has been done over the past 50 years to understand the developmental mechanisms of vertebrate retinal histogenesis. Birthdating studies have revealed a characteristic genesis order of the retinal cell types. Lineage-tracing experiments have shown that retinal progenitors are multipotent and that fate choice is stochastic.

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Experiments that challenge the environment of retinal progenitors have revealed that both cell autonomous and cell nonautonomous factors contribute to fate determination. More recent techniques, such as expression fate mapping (lineage by gene expression) and live imaging of cell lineages, will further our understanding of retinal fate determination. In addition, the recent increase in early cell-type-specific markers will allow us to more precisely examine the effects of environmental challenges. While several factors have been identified, many more experiments are needed to elucidate the molecular mechanisms of retinal fate determination. See also: Coordinating Division and Differentiation in Retinal Development; Ganglion Cell Development: Early Steps/Fate.

Further Reading Cayouette, M., Barres, B. A., and Raff, M. (2003). Importance of intrinsic mechanisms in cell fate decisions in the developing rat retina. Neuron 40(5): 897–904. Elliott, J., Jolicoeur, C., Ramamurthy, V., and Cayouette, M. (2008). Ikaros confers early temporal competence to mouse retinal progenitor cells. Neuron 60(1): 26–39.

Li, S., Mo, Z., Yang, X., et al. (2004). Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors. Neuron 43(6): 795–807. Marquardt, T., Ashery-Padan, R., Andrejewski, N., et al. (2001). Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105(1): 43–55. Nelson, B. R., Hartman, B. H., Ray, C. A., Hayashi, T., BerminghamMcDonogh, O., and Reh, T. A. (2009). Acheate-scute like 1 (Ascl1) is required for normal delta-like (Dll) gene expression and notch signaling during retinal development. Development Dynamics 238: 2163–2178. Ohsawa, R. and Kageyama, R. (2008). Regulation of retinal cell fate specification by multiple transcription factors. Brain Research 1192: 90–98. Rapaport, D. H., Wong, L. L., Wood, E. D., Yasumura, D., and LaVail, M. M. (2004). Timing and topography of cell genesis in the rat retina. Journal of Comparative Neurology 474: 304–324. Reh, T. A. (1992). Cellular interactions determine neuronal phenotypes in rodent retinal cultures. Journal of Neurobiology 23: 1067–1083. Reh, T. A. and Kljavin, I. J. (1989). Age of differentiation determines rat retinal germinal cell phenotype: Induction of differentiation by dissociation. Journal of Neuroscience 9(12): 4179–4189. Shimojo, H., Ohtsuka, T., and Kageyama, R. (2008). Oscillations in notch signaling regulate maintenance of neural progenitors. Neuron 58: 52–64. Turner, D. L. and Cepko, C. L. (1987). A common progenitor for neurons and glia persists in rat retina late in development. Nature 328: 131–136. Turner, D. L., Snyder, E. Y., and Cepko, C. L. (1990). Lineageindependent determination of cell type in the embryonic mouse retina. Neuron 4: 833–845. Waid, D. K. and McLoon, S. C. (1998). Ganglion cells influence the fate of dividing retinal cells in culture. Development 125: 1059–1066. Watanabe, T. and Raff, M. C. (1992). Diffusible rod-promoting signals in the developing rat retina. Development 114: 899–906.

Retinal Pigment Epithelial–Choroid Interactions K Ford and P A D’Amore, Schepens Eye Research Institute, Boston, MA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Angiogenesis – The formation of new blood vessels from preexisting ones. Atrophy – To wither or deteriorate. Blood–retinal barrier – Specialized, nonfenestrated, tightly joined endothelial cells that form a transport barrier for certain substances between the retinal capillaries and the retinal tissue. Choroidal neovascularization – A condition whereby new blood vessels that originate from the choroid grow and break through Bruch’s membrane into the subretinal pigment epithelium (sub-RPE) or subretinal space. Dominant negative – A genetic mutation where the gene product adversely affects the normal, wild-type gene product within the same cell. Fenestration – A small pore (60–80 nm in diameter) within the endothelium wall that allows the passage of small molecules and a limited amount of proteins. Microphthalmia – An abnormal smallness of the eyes, occurring as the result of disease or of imperfect development. Phagocytosis – The process in which phagocytes engulf and digest microorganisms and cellular debris. Quiescence – The absence of proliferation. Trophic factor – A molecule that promotes cellular growth and/or survival.

Introduction Located at the back of the eye, the retinal pigment epithelium (RPE)–choroid complex is comprised of the RPE, a polarized, epithelial monolayer and the choroid, a highly fenestrated vascular bed. Separated by Bruch’s membrane (BrM), an elastic lamina, the RPE and choroid each play a vital role in normal eye physiology. Considerable evidence indicates that there is not only a great deal of interaction between the RPE and choroid, but that the integrity of this interaction is critical to normal eye function. Examination of the morphology of the choroidal microvasculature reveals that the vessels are ‘‘polarized,’’ with fenestrations preferentially localized to the capillary surface proximal to the RPE and BrM. Whereas the

cytoplasm of the endothelial cell is thinnest in this region, endothelial cell bodies and nuclei are more prominent distal to the RPE. These observations led to the speculation that the RPE exerts an inductive effect on the choriocapillaris by releasing a factor that diffuses across BrM to provide a trophic effect and to mediate the anatomic specializations in the capillary endothelial cells. Preservation of a normal interaction between the RPE and choroid is required for proper eye physiology. This review explores the intricacies of the RPE and choroid, and their interactions during development, in the adult and with aging.

RPE–Choroid Complex Development RPE Development The development of the RPE is dependent upon the coordination of transcription factor expression and inductive signals received from the tissues surrounding the developing eye. Eye development proceeds from two principal tissue components: the neural ectoderm, which buds from the wall of the forebrain to form the optic vesicle, and the surface ectoderm, which forms the lens (Figure 1). When the optic vesicle comes in contact with the surface ectoderm, it invaginates, forming the optic cup. The optic cup consists of an inner layer, which gives rise to the neural retina, and an outer layer, which forms the RPE. At the optic cup stage, the presumptive RPE and retina are separated by a thin remnant of lumen, which becomes filled by a material known as the interphotoreceptor matrix (IPM). Coincident with this is the onset of the expression of RPE65, which encodes a protein involved in the conversion of all-trans-retinal to 11-cisretinal. Prior to this stage, the RPE is a ciliated and pseudo-stratified epithelium; however, following IPM formation, RPE maturation commences. The onset of RPE maturation is marked by melanogenesis, which requires the activation of the tyrosinase promoter. As the RPE continues to differentiate, it displays a complete apical to basolateral polarity, with short apical microvilli and small basolateral membrane infoldings, and the formation of tight junctions between the RPE cells, which can be divided into three stages. The early stage of tight junction formation is characterized by the expression of key tight junction proteins, such as zona occludens 1 (ZO-1), occludin, and claudins. However, these tight junctional complexes are rudimentary so the RPE lacks complete barrier properties and is therefore leaky. As the

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Figure 1 Development of the RPE and choroid. (a) Eye development begins with the budding of the neural ectoderm from the wall of the forebrain to form the optic vesicle. (b) When the optic vesicle comes in contact with the overlying surface ectoderm, it invaginates, forming the optic cup, which consists of an inner layer and an outer layer. The inner layer gives rise to the neural retina, and the outer layer eventually forms the RPE. Choroidal development proceeds from two embryonic tissues: the mesoderm and cranial neural crest cells. The endothelial cells of the choroidal blood vessels are derived from the mesoderm, whereas neural crest cells give rise to the stromal cells, melanocytes, and pericytes. (c) Choroid development begins early in eye development, as the oxygenation of the retina is supplied solely by the choroid and the transient hyaloid vascular system. Initially, tubes and spaces form in the surrounding periocular region of the optic vesicle, and eventually expand to form a plexus. Primitive capillaries develop from this plexus adjacent to the RPE as the optic vesicle invaginates. In humans, the choroidal plexus fuses to form a singular vessel known as the annular vessel at the anterior region of the optic cup during the second and third months of gestation. This primitive plexus is then organized into a complex network, and a well-defined choriocapillaris layer also appears at this stage.

early phase ends, Na+ K+ ATPase (ATPase, adenosine triphosphatase) becomes concentrated at the apical surface, and the apical microvilli begin to elongate. In the second stage, the tight junctions become increasingly less permeable, presumably due to the alterations in the distribution of various tight-junction proteins. At this stage, the RPE now prevents the free diffusion of membrane proteins, and the basolateral membrane is remodeled. In the last stage of tight-junction formation, the composition of tight-junction protein isoforms stabilizes and the RPE begins to display barrier properties characteristic of a tight epithelium. Following completion of tight-junction formation, the RPE begins to express specialized proteins, such as the glucose transporter, that aid in the transport of essential nutrients from the RPE to the photoreceptors. As RPE maturation concludes, the RPE becomes fully functional and is able to interact with the photoreceptors. Choroid Development Blood vessels develop by vasculogenesis and angiogenesis. In vasculogenesis, primitive vascular cells assemble into a primitive capillary plexus, which is remodeled to define the pattern of the vascular architecture, whereas in angiogenesis, new vessels develop as sprouts from preexisting vessels. It is thought that the superficial vessels of the retina form by vasculogenesis at the optic nerve and expand along a gradient from the posterior to the anterior retina. However, new vessels then sprout via angiogenesis

and invade the retina to form intermediate and deep capillary beds. Nonetheless, the primitive vessels derived via vasculogenesis or angiogenesis must still be remodeled before they are considered mature. Remodeling involves the growth of new vessels and regression of others. In addition, alterations in lumen diameter and vessel wall thickness, which are dictated by the local needs of the tissue, must also occur. The choroid develops from two principle embryonic tissues: the mesoderm and cranial neural crest cells. The mesoderm gives rise to the endothelial cells of the choroidal blood vessels, whereas the stromal cells, melanocytes, and pericytes are all derived from the neural crest cells. Choroid development begins early in eye development, which is not surprising considering that the differentiation of the ocular tissues relies upon the oxygen and nutrients supplied by the primitive vascular system. In early eye development, the oxygenation of the retina is supplied solely by the choroid and the transient hyaloid vascular system. The vascularization of the retina itself is actually a late event. Initially, tubes and spaces form in the surrounding periocular region of the optic vesicle. These tubes, which are lined by the mesodermal endothelium, expand to form a plexus as eye development proceeds. Concomitant with optic vesicle invagination, the primitive capillaries develop from this plexus adjacent to the RPE. In humans, choroidal capillaries completely encircle the optic cup by the 13-mm stage of development and remain separated from the retina by the basement


membrane of the RPE. At the anterior region of the optic cup, the choroidal plexus fuses to form a singular vessel known as the annular vessel, and during the second and third months of gestation, this primitive plexus is organized into a complex network. A well-defined choriocapillaris layer also appears at this stage. Pigmentation does not appear in choroidal melanocytes until late gestation – between 6 and 7 months; however, it is complete at birth. BrM Development In mice, the formation of the choroidal vessels precedes the deposition of BrM, and maturation of the BrM layers is not complete until 6 weeks following birth. However, in humans and monkeys, BrM layers are completely formed in utero. BrM formation commences near the RPE, and is initially comprised of a single basement membrane derived from the RPE. The RPE is able to synthesize many of the extracellular matrix (ECM) components that comprise BrM; therefore, it is not surprising that BrM development begins near the RPE. On the 17th to 18th day of gestation in rats, collagenous fibrils gradually accumulate in the space between the RPE and choriocapillaris endothelium and begin to form a three-dimensional meshwork within BrM. The meshwork consists of three sublayers: the basement membranes of the RPE and choriocapillaris endothelium, and a collagenous layer. The final component of BrM, the central elastic layer, appears on the fifth postnatal day. Initially, dense elastic deposits appear within the collagenous layer, and subsequently accumulate to form a single continuous layer, which separates the collagenous layer into an inner and outer layer. Finally, an immature, but five-layered BrM is formed by the ninth day after birth.

RPE–Choroid Complex: Structure and Function

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The RPE serves many functions that are vital to normal eye function and physiology. These functions have been categorized based on the characteristics of three classes of cells: epithelium, macrophage, and glia. The major function of an epithelium is to control the passage of substances from one extracellular space to another; therefore, the epithelial aspect of the RPE is its role in actively transporting ions, water, and metabolic end products from the subretinal space to the blood. The RPE is also involved in the uptake of nutrients, such as glucose, retinol, and fatty acids from the blood to nourish the photoreceptors. One of its most critical functions is the exchange of retinal with the photoreceptors. The photoreceptors are unable to reisomerize all-trans-retinal; therefore, it is transported to the RPE where it is reisomerized to 11-cis-retinal, and then transported back to the photoreceptors, a process known as the visual cycle of retinal. The RPE also functions as a macrophage in that it phagocytoses the photoreceptor outer segments, which are constantly being renewed. As new membrane is added to the base of the outer segment, the older membrane is advanced toward the tip, and subsequently shed. The phagocytosis of the shed outer segments by the RPE is critical to normal photoreceptor function because it maintains the excitability of the photoreceptors. The vital nutrients, such as retinal, which are recycled and returned to the photoreceptors following outer segment digestion help rebuild the light-sensitive outer segments from the base of the photoreceptors. The final role that the RPE plays is that of a glial cell. RPE resembles glial cells in the manner in which they respond electrically to changes in the concentration of extracellular K+, which is determined by the photoreceptors. The RPE possesses tight junctions at both its basolateral and apical membranes, which generate transepithelial resistance (TER); furthermore, the tight junctions at the basolateral surface form the RPE portion of the blood–retinal barrier.

RPE Structure and Function It is apparent that the RPE performs a variety of complex functions that are essential for proper visual function. If any aspect of one of the many roles that the RPE serves is disrupted or fails, retinal degeneration, loss of visual function, and ultimately blindness may result. Therefore, the RPE is an indispensable component of the RPE–choroid complex. Anatomically, the RPE is juxtaposed to the outer segments of the photoreceptors at its apical surface, extending long apical microvilli that surround the outer segments. These microvilli provide a means for a complex structural interaction between the RPE and photoreceptors. BrM, which is at the basolateral side of the RPE, separates the RPE from the fenestrated endothelium of the choriocapillaris.

Choroid Structure and Function The choroid is a vascular bed that supplies nutrients and oxygen to the RPE and outer nuclear layer of the retina. It consists of larger vessels and a highly fenestrated choriocapillaris (Figure 2), whose endothelial basement membrane comprises one layer of BrM. Fenestrated endothelium is a characteristic of tissues that are involved in secretion and/or filtration. The photoreceptors are metabolically very active and the fenestrated choriocapillaris facilitates the transport of oxygen and nutrients to fulfill their metabolic needs. RPE tight junctions, in combination with sophisticated transport systems, determine which components of the choriocapillary secretions are transported to the photoreceptors.


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Figure 2 Corrosion cast of adult mouse choroidal vasculature. The choroid is a vascular bed that supplies nutrients and oxygen to the RPE and outer nuclear layer of the retina. It consists of larger vessels and a highly fenestrated capillary bed known as the choriocapillaris. (a) Scleral view of the entire adult choroid. Major vessels can be easily identified: posterior ciliary artery (black arrow), long posterior arteries (black arrowhead), and vortex vein (white arrowhead). (b, c) Scanning electron micrographs of vascular cast of the choroidal vasculature. (b) Posterior view showing one posterior ciliary artery (PCA) around the optic nerve. On each side, the artery divides regularly into smaller branches (arrows) and choriocapillaris (CC). (c) Anterior view showing the extremely dense choriocapillaris plexus.

RPE BL BL

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Figure 3 Ultrastructure of the RPE–BrM–choriocapillaris. The RPE and choriocapillaris are separated by a thick (1–4 mm) elastic lamina, BrM, which consists of five layers: the RPE basement membrane (BL), an inner collagenous layer (col), a central elastic layer (el), an outer collagenous layer (col), and the choriocapillaris (cc) endothelium basement membrane (BL), respectively, from top to bottom. BrM acts as a barrier for macromolecules and regulates the diffusion of small molecules between the RPE and the choriocapillaris.

BrM Structure and Function Located between the RPE and the choriocapillaris, BrM is a thick (1–4 mm) pentalaminar ECM, which consists of the RPE basement membrane, an inner collagenous layer, a central elastic layer, an outer collagenous layer, and the choriocapillaris endothelium basement membrane (Figure 3). BrM is comprised of several types of extracellular components, including many types of collagen, laminin, fibronectin, and proteoglycans. The collagenous layers of BrM are primarily composed of type I collagen and collagenous-associated proteins. It is believed that these components fortify the framework of BrM and aid in the resistance to the force that intraocular pressure exerts on the back of the eye. The basement membranes of the RPE and choriocapillaris endothelium are composed of type

IV collagen, which can indirectly interact with cells via laminin, and also binds to heparin and heparan sulfate proteoglycans. The central elastic layer consists of crosslinked elastin fibers that are associated with fibulin 5, an ECM protein that is thought to aid in elastin fiber assembly and serve as a link between the elastin fibers and cell surface receptors. BrM acts as a support and a barrier between the retina and the choroid, and is thought to support the many functions of the RPE. BrM is semipermeable, and therefore controls the transfer of molecules and cellular components between the RPE and the choroidal vasculature.

RPE–Choroid Interactions Interactions During Development The presence of the RPE is required for proper choroid development. Studies have shown that an intact, fully differentiated RPE is required for normal choroidal development; when the RPE is transdifferentiated into a neural retina by expressing fibroblast growth factor (FGF)-9 under the control of the tyrosine-related protein 2 (TRP-2) promoter, the choroid fails to develop. This is further illustrated in humans with colombas where RPE differentiation has failed and there are abnormalities in development of both the choroid and sclera. Basic fibroblast growth factor (bFGF) has also been implicated in choroidal development. Transgenic mice in which a dominant-negative bFGF receptor (FGFR1) was overexpressed in the RPE displayed choroidal abnormalities, such as incomplete and immature choroidal vessels. The RPE also secretes a variety of growth factors and an increasing body of evidence indicates that RPEderived vascular endothelial growth factor (VEGF) is essential to choroidal development. Studies in humans and rodents have illustrated that both VEGF and its


receptor, VEGFR2, are highly expressed by the RPE and the underlying mesenchyme, respectively, at the time of choriocapillaris formation. Furthermore, mice with an RPE-specific deletion of VEGF presented a variety of defects, including microphthalmia, loss of visual function, and the complete absence of the choriocapillaris. In addition, the RPE itself was discontinuous, suggesting that RPE-derived VEGF is not only important for choroidal development, but also for RPE survival. Whether the RPE abnormality is secondary to the defects in choroidal development or due to a direct effect of VEGF on RPE has not been elucidated. Interactions in the Adult Normal RPE–choroid interactions are not only important during development, but also in the adult. Several studies have revealed the impact of RPE loss on choroidal structure and function. The presence of an intact RPE is critical to proper choroidal function, as surgical RPE removal causes several changes throughout the choroid. Both large choroidal vessels and the choriocapillaris display a reduction in circulation, and depending upon the extent of RPE removal, choroidal nonperfusion can be permanent due to fibroblast infiltration. A landmark study in which the RPE was selectively destroyed by sodium iodate treatment illustrated that within 1 week following iodate injection, the choriocapillaris had reduced fenestrations and displayed signs of atrophy, such as degenerating endothelial cells, and pericapillary basal laminae that had begun to separate from the apparently shrunken endothelium. Together, these data illustrate the critical role the RPE plays in both survival and in the maintenance of the choriocapillaris. Growth factor secretion

RPE secretes a variety of growth factors, including VEGF, FGFs, transforming growth factor-b (TGF-b), and ciliary neutrophic factor (CNTF). The RPE also secretes pigment-epithelium-derived factor (PEDF), which functions not only to maintain the retina by acting as a neuroprotective factor, but also to provide antiangiogenic activity to inhibit endothelial cell proliferation, thereby stabilizing the choriocapillaris endothelium. VEGF, a well-characterized angiogenic factor, also plays a role modulating blood vessel permeability. Differential splicing of VEGF premessenger RNA (mRNA) gives rise to multiple isoforms, with the most notable being VEGF120, VEGF164, and VEGF188 in mice, and VEGF121, VEGF165, and VEGF189 in humans. VEGF120 does not bind heparin sulfate proteoglycans (HSPGs) and is readily diffusible, whereas VEGF164 is partially sequestered on the cell surface and in the ECM. VEGF188, with high affinity for heparan sulfate, is therefore primarily cell-surfaceand matrix-associated. The RPE primarily expresses the

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diffusible isoforms, VEGF164 and VEGF120, while VEGF188 is virtually undetectable. RPE-derived VEGF is essential for both survival and maintenance of the underlying choriocapillaris endothelium. Interestingly, PEDF and VEGF are secreted by the RPE in a polarized fashion in opposing directions. PEDF is secreted to the apical surface of the RPE to support the neurons and photoreceptors of the retina, whereas VEGF is primarily secreted to the basolateral surface where it acts on the choroidal endothelium (Figure 4). Despite the fact that a small fraction of total VEGF, a potent angiogenic factor, is secreted to the apical surface by the RPE, the outer nuclear layer of retina remains completely avascular, presumably due to the balance between antiangiogenic (PEDF) and angiogenic (VEGF) factors. Receptor expression In parallel with the many factors secreted by the RPE, the choroid expresses a number of corresponding receptors, including FGF receptors, type I and II TGF-b receptors, and VEGFR2. Of particular interest is the fact that VEGFR2 is observed primarily in the choriocapillaris adjacent to the RPE, with substantially less VEGFR2 expression observed in the major vessels of the choroid. Furthermore, VEGFR2 is localized to the apical surface of the choriocapillaris endothelium and to the photoreceptors where it is constitutively activated, which is surprising given that adult choroidal vasculature is mature and quiescent. Isoform-specific VEGF mouse model In support of a critical role for RPE-derived VEGF in the maintenance of the adult choriocapillaris, we have shown that the absence of soluble VEGF isoforms in the RPE leads to changes that recapitulate the classical features of dry age-related macular degeneration (AMD). Mice expressing only VEGF188 (i.e., lacking the diffusible isoforms that they normally express) display signs of RPE dysfunction, such as increased autofluorescence, loss of barrier proprieties, and accumulation of basal deposits that are similar to drusen, extracellular deposits that build up beneath the basement membrane of the RPE within BrM. These changes occur prior to the formation of both focal choroidal atrophy and RPE attenuation, which progress to large areas of RPE loss. The abnormalities are age dependent and increase in severity over time. Choroidal change impact on RPE The RPE–choroid interactions do not merely function to serve the choroid; changes in the choroid have also been shown to impact the RPE. Choroidal ischemia has been reported to lead to opaque RPE lesions and subsequent serous retinal detachment. Furthermore, very early studies on the effects of choroidal congestion on RPE–retina function revealed that increased choroidal pressure may

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Figure 4 Polarized secretion by the RPE and localization of VEGFR2. (a) The RPE secretes both VEGF and PEDF in polarized fashion in opposing directions. PEDF is secreted to the apical surface of the RPE to support the neurons and photoreceptors of the retina, whereas VEGF is primarily secreted to the basolateral surface where it acts on the choroidal endothelium. RPE-derived VEGF is essential for both survival and maintenance of the underlying choriocapillaris endothelium, and PEDF functions not only as a neuroprotective factor, but also as an antiangiogenic factor to maintain the avascularity of the outer nuclear layer of the retina. (b) Higher magnification of selected area in (a) showing fenestrations in choriocapillaris and localization of the VEGFR2 receptor. In parallel with the VEGF secreted by the RPE, the choroid expresses the corresponding VEGF receptor, VEGFR2. VEGFR2 is observed primarily in the choriocapillaris adjacent to the RPE, with substantially less VEGFR2 expression observed in the major vessels of the choroid.

cause RPE–retina dysfunction by altering normal fluid movement across the RPE, modifying the integrity of the subretinal space.

RPE–Choroid Changes with Age Various changes occur in the RPE–choroid complex with aging. In light of the close association among the RPE, BrM, and choroid, it is not surprising that an alteration in a single component of this complex compromises the normal RPE–choroid interaction and ultimately leads to disease. Studies have shown that the density and diameter of the choriocapillaris and medium-sized choroidal vessels substantially decline with age, resulting in decreased choroidal blood volume and blood flow. The aging RPE exhibit changes such as a reduction in cell density and a loss of RPE melanin. Melanin pigmentation is believed to play a protective role, acting to protect cells against oxidative stress. Oxidative changes in RPE melanin may be attributed to complexing of melanin with lipofuscin, pigment granules composed of lipid-containing residues of lysosomal digestion, which generate reactive oxygen species upon excitation with blue light, thereby making the aged RPE more susceptible to oxidative damage. BrM Changes Changes in BrM include increased thickness, accumulation of lipids, and subsequent alterations of the BrM

permeability. Collectively, these changes can limit the diffusion of water-soluble proteins, leading to a range of problems. Furthermore, drusen are known to accumulate in the aging eye (Figure 5). There is a strong correlation between the presence of drusen and ocular pathology, such as AMD. It has been speculated that drusen lead to a gradual reduction in the diffusion of RPE-derived factors, such as VEGF, which may be causal in the atrophy of segments of the RPE and underlying choriocapillaris, known as geographic AMD. Drusen accumulation and coalescence may also lead to breaks in BrM that are believed to be the initiating event in the formation of choroidal neovascularization (CNV), in which new blood vessels sprout from preexisting choroidal vessels and invade the overlying RPE and retina. The development of CNV associated with wet AMD leads to visual loss due to the fact that the neovessels are leaky and cause damage to the surrounding tissues.

Gene Expression Gene expression of the RPE–choroid also changes with age. There is considerable upregulation of the expression of genes and proteins involved in leukocyte extravasation, and the accumulation of leukocytes at the RPE–BrM interface suggests that leukocytes are possibly recruited to aid in the removal of cellular waste. Furthermore, there have been reports of increased macrophages in the aged RPE–choroid; mice harboring mutations that render them


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Figure 5 The accumulation of drusen beneath the RPE within BrM. In the aging eye, it is common for drusen to accumulate at the interface between the inner collagenous layer of BrM and the basal lamina of the RPE. Drusen are extracellular deposits that are strongly correlated with ocular pathology, such as age-related macular degeneration (AMD). It is proposed that drusen deposits between the RPE and the BrM as well as within BrM, and causes a gradual reduction in the diffusion of RPE-derived factors, such as VEGF, which may lead to the atrophy of segments of the RPE and underlying choriocapillaris, also known as geographic AMD. Drusen accumulation and coalescence may also cause breaks in BrM that are believed to be the initiating event in the formation of choroidal neovascularization (CNV), in which new blood vessels sprout from preexisting choroidal vessels and invade the overlying RPE and retina. Adapted from Johnson, L. V. and Anderson, D. H. Age-related macular degeneration and the extracellular matrix. New England Journal of Medicine 351(4): 320–322. Copyright ã 2004 Massachusetts Medical Society. All rights reserved.

deficient in macrophage recruitment display hallmarks of AMD. It is also thought that the aged RPE–choroid synthesizes proteins that not only attract leukocytes, but that also activate the complement pathway, which is a part of the immune response and can lead to inflammation. Recent associations between polymorphisms in a member of the complement pathway reinforce the role of inflammation in the development of AMD.

Conclusions Despite the fact that the RPE and choroid are separated by BrM, there is a great deal of interaction between the tissues. The presence of an intact, fully differentiated RPE is not only required for proper choroidal development, but is also essential for survival and maintenance of adult choriocapillaris endothelium specializations

(fenestrations) and integrity. The RPE secretes a variety of factors, one of the most notable being VEGF. The secretion of VEGF by the RPE is somewhat of a double-edged sword. Although VEGF is vital to choroidal homeostasis during both development and in adult, breakdown of the RPE barrier upon direct contact with choroidal endothelial cells is thought to involve a VEGF-mediated mechanism, for VEGF has been shown to mediate the vessel growth and permeability associated with wet AMD. Thus, maintenance of proper RPE–choroid interaction is vital to normal function, and any perturbation to this system can ultimately lead to disease. See also: Breakdown of the RPE Blood–Retinal Barrier; Choroidal Neovascularization; Developmental Anatomy of the Retinal and Choroidal Vasculature; Immunobiology of Age-Related Macular Degeneration; RPE Barrier.

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Further Reading Gogat, K., Le Gat, L., Van Den Berghe, L., et al. (2004). VEGF and KDR gene expression during human embryonic and fetal eye development. Investigative Ophthalmology and Visual Science 45(1): 7–14. Hartnett, M. E., Lappas, A., Darland, D., et al. (2003). Retinal pigment epithelium and endothelial cell interaction causes retinal pigment epithelial barrier dysfunction via a soluble VEGF-dependent mechanism. Experimental Eye Research 77(5): 593–599. Ivert, L., Kong, J., and Gouras, P. (2003). Changes in the choroidal circulation of rabbit following RPE removal. Graefe’s Archive for Clinical and Experimental Ophthalmology 241(8): 656–666. Korte, G. E., Reppucci, V., and Henkind, P. (1984). RPE destruction causes choriocapillary atrophy. Investigative Ophthalmology and Visual Science 25(10): 1135–1145. Mancini, M. A., Frank, R. N., Keirn, R. J., Kennedy, A., and Khoury, J. K. (1986). Does the retinal pigment epithelium polarize the choriocapillaris? Investigative Ophthalmology and Visual Science 27(3): 336–345. Marneros, A. G., Fan, J., Yokoyama, Y., et al. (2005). Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function. American Journal of Pathology 167(5): 1451–1459. Ramrattan, R. S., van der Schaft, T. L., Mooy, C. M., et al. (1994). Morphometric analysis of Bruch’s membrane, the choriocapillaris,

and the choroid in aging. Investigative Ophthalmology and Visual Science 35(6): 2857–2864. Rousseau, B., Larrieu-Lahargue, F., Bikfalvi, A., and Javerzat, S. (2003). Involvement of fibroblast growth factors in choroidal angiogenesis and retinal vascularization. Experimental Eye Research 77(2): 147–156. Saint-Geniez, M. and D’Amore, P. A. (2004). Development and pathology of the hyaloid, choroidal and retinal vasculature. International Journal of Developmental Biology 48: 1045–1058. Saint-Geniez, M., Maldonado, A. E., and D’Amore, P. A. (2006). VEGF expression and receptor activation in the choroid during development and in the adult. Investigative Ophthalmology and Visual Science 47(7): 3135–3142. Saint-Geniez, M., Maharaj, A. S., Walshe, T. E., et al. (2008). Endogenous VEGF is required for visual function: Evidence for a survival role on Mu¨ller cells and photoreceptors. PLoS ONE 3(11): e3554. Steinberg, R. H. (1985). Interactions between the retinal pigment epithelium and the neural retina. Documenta Ophthalmologica 60: 327–346. Strauss, O. (2005). The retinal pigment epithelium in visual function. Physiological Reviews 85(3): 845–881. Zhao, S. and Overbeek, P. A. (2001). Regulation of choroid development by the retinal pigment epithelium. Molecular Vision 2(7): 277–282.

Retinal Pigment Epithelium: Cytokine Modulation of Epithelial Physiology S S Miller, A Maminishkis, R Li, and J Adijanto, National Eye Institute, Bethesda, MD, USA Published by Elsevier Ltd., 2010.

Glossary Cytokines – A large and diverse family of polypeptide regulators that are used in cell regulation. ELISA – The enzyme-linked immunosorbent assay is a biochemical technique that uses an enzymelinked antibody to detect its corresponding ligand within a sample. It can be used to assay for chemokines and cytokines. Subretinal space (SRS) – The extracellular space between the retinal photoreceptors and the apical surface of the retinal pigment epithelium. Tight junctions – A structure of specialized proteins that form a seal between neighboring cells and regulate ion and small molecule selectivity and resistance in the paracellular pathway between epithelial cells. Tight junction proteins include occluding, claudins, and junction adhesion molecules (JAMs).

Introduction In the back of the vertebrate eye, the apical membrane of the retinal pigment epithelium (RPE) and the photoreceptor outer segments form a very tight anatomical relationship (Figure 1). This structural feature supports a whole host of mechanical, electrical, and metabolic interactions that maintain the health and integrity of the neural retina throughout the life of the organism. Like all epithelia, the RPE plasma membrane contains a wide variety of proteins, enzymes, and small molecules that are specifically segregated to the apical or basolateral sides of the epithelium, which face the neural retina and choroidal blood supply, respectively (Figure 2). The asymmetrical distribution of these functionally distinct molecules is maintained by junctional complexes that surround each cell and by the continuous synthesis and regulated traffic of these molecules to each membrane. Epithelial polarity is defined by the steady-state maintenance of this asymmetric distribution and is critical for the ongoing vectorial transport of ions, metabolites, fluid, and waste products across the RPE. Epithelial polarity is also fundamentally important for controlling

changes in the volume and chemical compositions of the extracellular spaces on either side of the RPE, following transitions between light and dark. In the distal retina, the extracellular or subretinal space (SRS) separates the photoreceptor outer segments and the RPE apical processes. The chemical composition of this space is tightly buffered by the cells which surround it (Mu¨ller cells, photoreceptors, and RPE). On the opposite side of the RPE, an extracellular space is formed between its basolateral membrane and Bruch’s membrane, which is adjacent to the choriocapillaris. The physiological and pathophysiological states of the RPE/distal retina complex are significantly affected by changes in the chemical composition of these extracellular spaces as evidenced in disease processes such as age-related macular degeneration (AMD) or uveitis. AMD develops within the RPE/distal retina complex and eventually leads to RPE impairment and loss of photoreceptor function. The RPE’s ability to control and respond to varying levels of oxidative insult from light quanta, outer segment phagocytosis, vitamin A uptake and delivery, and oxygen consumption diminishes with age. These changes significantly affect the chemical composition of the surrounding extracellular spaces, SRS and choroid, and are a major factor in disease pathogenesis. In recent years, significant advances have been made in identifying the role of the immune system in neurodegenerative disease in general and in AMD, in particular (summarized by Hageman and colleagues and by Nussenblatt and Ferris). This article summarizes recent experiments from our lab and others, which show that inflammation induced changes in the environment surrounding human RPE can significantly alter intracellular signaling and physiology. This study provides a basis for understanding disease progression and regression. This article is divided into three main parts. We begin with a description of our development of a robust and welldefined primary cell culture model of human fetal retinal pigment epithelium (hf RPE). We use this model to analyze how metabolic waste products, produced in the retina following light/dark transitions, can be disposed of by CO2/ HCO3 and lactate transporters located in the apical and basolateral cell membranes. In the second part, we use this cell culture model to analyze RPE antioxidant mechanisms that are protective against disease processes, such as AMD or uveitis. In the third part, we describe a series of experiments that use this model to define the impact of cytokines on human RPE function. Finally, we briefly focus on the role of interferon gamma (INFg) in controlling RPE physiology.

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Retina Rods and cones Subretinal space Pigment epithelium Choroid Sclera

A

B

C

Ciliary body

Cornea

A B

Lens

C

Iris

Figure 1 Schematic diagram of the eye. The retinal pigment epithelium is located in the back of the eye between neural retina and choroid.

Human RPE: Morphology, Polarity, and Function The availability of native human tissue, fetal or adult, is limited and extant models of cultured human RPE have been, in varying degrees, less than adequately characterized or understood, not reproducible or available in large quantities, and lacking expression of melanin pigment and key functional proteins such as bestrophin (Best1), and RPE-specific protein 65 (RPE65). Therefore, we developed a set of standard procedures for producing confluent monolayers of hf RPE cells and demonstrated that they have the morphology, polarity, and function of the native tissue from which they were derived (Figure 3). Light and electron microscopy (EM) studies confirmed the presence of apical processes (microvilli) and basal infoldings that increased the elaboration of the apical and basolateral membranes, respective, in cultured hfRPE cells. We carried out immunoblot and immunofluorescence experiments for a variety of proteins to help define the polarity of this model. In the course of these experiments, we discovered that human RPE tight junctions contain a variety of

membrane proteins (claudins) that are important for regulating the selectivity and conductance of the paracellular pathway, and we also confirmed the presence of several visual cycle and cytoskeleton proteins. Intracellular recordings confirmed many membrane physiological properties and demonstrated the polarity of purinergic and adrenergic receptors at the apical membrane, which serve to regulate cell calcium and transepithelial fluid absorption (Figure 2). By enzyme-linked immunosorbent assay (ELISA), we showed that these monolayers constitutively secrete pigment epithelium-derived factor (PEDF) to the apical bath and vascular endothelial growth factor (VEGF) to the basal bath; the former provides neuroprotection for the retina and the latter would allow active regulation of endothelial cell fenestration, a structural feature critical for choroidal circulation. pHi – Induced Changes in Fluid Absorption In previous experiments we showed that acetazolamide, a carbonic anhydrase (CA) inhibitor, reduced net 36Cl flux across frog RPE. Subsequent animal models and clinical


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Choroid basal Na

K ADP

Cl (Lac)

nHCO3

HCO3 (Lac)

Cl (CFTR)

Ca2+

↑cAMP

PLB ER

↑HCO3

Ca2+

sAC

−

Na Lac

cAMP

ATP PKA

Ca2+

Cl

Cl

ATP

[K] = 90 mM [Cl] = 85 mM [HCO3] = 23 mM [Na] = 3–10 mM

[Ca2+] = 100 nM pHI = 7.4

IP3 K G Na Anion

P2Y

ATP/UTP

H

3Na↑ cAMP

Na

α1 Ep

β

Na 2HCO3

Lac

H Na 2Cl K

2K

Ep/Iso

ATP

tmAC

+ Forskolin

SSTR2

SST14

JV

Subretinal space (apical) Figure 2 Schematic diagram of retinal pigment epithelium (RPE) summarizing some membrane proteins, channels, and receptors that are responsible for a variety of RPE functions such as fluid transport, pH maintenance, or cell-volume regulation. The arrows highlighted in yellow indicate a main pathway for solute-driven fluid transport across the RPE, consisting of a sodium, potassium, 2 chloride co-transporter (Na/K/2Cl co-transporter) at the apical membrane, and a cyclic AMP-activated chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), and Ca2+-activated Cl-channels at the basolateral membrane. The apical membrane contains a variety of receptors, for example, purinergic (P2Y), adrenergic (a-1 and b), and somatostatin (SSTR2), which when bound to their specific ligands, activate Ca2+ and cAMP second-messenger signaling systems. The plasma membrane localization of previously described ion transporters such as the Na/2HCO3 co-transporter, Na/H exchanger, 3Na/2K ATPase, H/Lac co-transporter, Cl/HCO3 exchanger, and potassium channels are also shown. From Maminishkis, A., et al. (2002). Investigative Ophthalmology and Visual Science 43(11): 3555–3566. ã Association for Research in Vision and Ophthalmology.

trials have utilized CA inhibitors to reduce diseaseinduced abnormal accumulation of retinal fluid. CAs catalyze the reversible hydration of CO2 to HCO3 and protons, which are transported across the plasma membrane (e.g., Na/HCO3 or H/lactate co-transporters) to regulate cell pH. As a first step, we have identified and localized several highly expressed CAs in human RPE and begun study of their physiology. A total of 16 CAs have been identified in human tissues. In human fetal RPE cell cultures, 14 of the 16 known isozymes have been confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Immunocytochemical studies indicate that CA II is localized intracellularly, as in many other cell types. CA IV, XII, and XIV are localized to the apical surface, while CA IX, the most abundantly expressed isozyme in hfRPE cultures, is expressed apically and laterally (Figure 4). However, it should be noted that CA IX messenger RNA (mRNA) and proteins are not expressed in native adult or fetal human RPE. CA inhibitors have had limited success in alleviating the effects of retinal disease, partly because of systemic side effects, but mainly because of their lack of specificity. The positive clinical outcomes for some patients with retinal edema suggest that nonspecific CA inhibitors, such as acetazolamide, may be affecting multiple CAs or other transportrelated mechanisms that can either increase or decrease

net fluid absorption across the RPE in varying degrees in different patients. This is supported by in vivo animal studies, in which intravenous administration of acetazolamide to rabbits increased fluid clearance from the SRS. The regulatory role of CAs is potentially important in human RPE, which critically depends on HCO3 transport to maintain fluid absorption ( JV) out of the SRS (Figure 5).

Modulation of SRS Metabolic Load and Chemical Composition In the intact eye (cat/monkey), the transition from light to dark causes significant alterations in SRS pH, Ca2+, and K. In addition, the transition from light to dark increases photoreceptor O2 consumption by 2-fold as measured in situ in cat and nonhuman primate retina. The rates of retinal O2 consumption in light and in dark were used by Linsenmeier and Winkler and their colleagues to estimate the associated changes in glucose metabolism that leads to the concomitant release of carbon dioxide, lactic acid, and water from the photoreceptor inner segments into the SRS. This metabolic acid load is potentially damaging to all of the cells that surround the SRS (i.e., photoreceptors, Mu¨ller cells, and RPE). It raises the


764

Native

P0-flask

(a)

P1-insert

(b)

RPE65 NaKATPase Ezrin

(c)

Cytokeratin rbCRALBP

220 kd 120 kd 100 kd 80 kd 60 kd 50 kd 40 kd 30 kd 20 kd

(d) P1-insert (e) Figure 3 Photomicrographs showing native (a) and cultured human fetal retinal pigment epithelium (hfRPE) ((b) PO cultured on flask; (c) P1 cultured on insert). (d) Westerns blots for five hfRPE specific proteins. (e) Transmission-electron micrograph of hfRPE cells grown on inserts. From Maminishkis, A., et al. (2006). Investigative Ophthalmology and Visual Science 47 (8): 3612–3624. ã Association for Research in Vision and Ophthalmology.

question of how the RPE could help prevent this accumulation of metabolic acid and water in the SRS. In in vivo studies of rabbit eye, it was estimated that 70% of fluid absorption across the RPE is linked to metabolite transport to the choroidal blood supply. In addition, in vitro studies of frog RPE showed that steady-state fluid absorption decreased by 70%, following the removal of HCO3 from both bathing solutions, implicating HCO3 transport in a regulatory role on fluid transport. The RPE functionally expresses several different HCO3 transport proteins at the apical and basolateral membranes as illustrated in Figure 5. In an earlier study, a 4,40 -diisothiocyano-2,20 -stillbene-disulfonic acid (DIDS)sensitive electrogenic Na/2HCO3 co-transporter was localized to the apical membrane of frog and bovine RPE; DIDS is a bicarbonate transport inhibitor. At the basolateral membrane, HCO3 is transported out of the RPE through a pH-sensitive Cl/HCO3 exchanger with a possible contribution from a Na/HCO3 co-transporter. These HCO3 transporters in the RPE are linked to Na and Cl transport, which are major driving forces for

fluid transport. Recently, the identities of some of these HCO3 transporters have been characterized in our laboratory and by other groups. NBC1 (Na/2HCO3 cotransporter) and NBC3 (NBCn1; electroneutral Na/ HCO3 co-transporter) were localized to the apical membrane. AE2 (Cl/HCO3 exchanger) mRNA transcripts were detected, but protein expression in the RPE remains to be determined. The identity of the basolateral membrane Na/nHCO3 co-transporter (NBC) is still unknown. In vitro, we mimic the increased retinal CO2 production, following the transition from light to dark by increasing apical bath CO2 level from 5% to 13%. This maneuver increased NaCl uptake at the apical membrane and can enhance CA-mediated Na/HCO3 co-transport across the RPE, thus increasing net NaHCO3 absorption. This increase in solute transport would drive additional fluid across the RPE as observed in in vitro experiments. The transport of metabolic waste products from the SRS to the choroidal blood supply by the RPE helps maintain ionic and pH homeostasis of the SRS. The RPE handles the increased metabolic load by transporting

Retinal Pigment Epithelium: Cytokine Modulation of Epithelial Physiology Carbonic anhydrase mediated HCO3-transport

+ H+

HCO3

CO2 + H2O

HCO3 + H+

Cl AE2

NBC

Na

H+ + HCO3

Choroid

MCT3

nHCO3 + H+

765

CA IX

Lac

CA ll

CO2 + H2O

CO2 + H2O

CO2 + H2O

CA ll

HCO3 + H+ NBC1

CA IV, IX, XII, XIV

H+ MCT1

NHE

Apical

Basal

Na 2HCO3

CA XIV

CO2 + H2O

H+

Lac

Na Subretinal space

HCO3

+ H+

Immunostain and western qRT-PCR

CA II

CA IV

CA IX CA XII CA XIV

CA II

CA III

CA IV CA Vα CA Vβ

CA VII CA VIII CA IX CA X CA XI CA XIIα CA XIIβ CA XIII CA XIV

Figure 4 Carbonic anhydrase (CA)-mediated CO2/HCO3 transport in retinal pigment epithelium (RPE). Membrane-bound CA IV, XII, and XIV are expressed exclusively at the apical membrane (immunostaining of CA XIV shown in insert on lower left). CA IX is expressed at both the apical and basolateral membranes of cultured human fetal RPE. CA II is expressed in the cytosol and can be recruited to the inner leaflet of the membrane. Experimental data (immunostaining, Western blots, and RT-PCR) that support the localization of the various CAs are listed in the table (lower right). Membrane-bound CAs hydrate CO2 into HCO3 and H+, which are substrates for NBC1 (sodium bicarbonate co-transporter) and MCT1 (proton lactate co-transporter) at the apical membrane. Cytosolic CA II regulates CO2 and HCO3 equilibrium in the RPE. The anion exchanger isoform 2 (AE2) mediates Cl/HCO3 exchange at the basolateral membrane, while a sodium proton exchanger (NHE) at the apical membrane helps regulate intracellular pH.

CO2 across the RPE in the form of HCO3 through HCO3transporters, and this process is mediated by the catalytic activity of CAs. This increase in Na and HCO3 absorption provides the driving force for increased net fluid absorption across the RPE, which dehydrates the SRS and creates retinal adhesion, thus allowing the RPE to maintain proper anatomical relationship with the photoreceptors. In the retina, 95% of glucose consumption is metabolized through glycolysis into lactic acid, which is subsequently deposited into the SRS. In addition to the high glycolytic activity of the retina, several other mechanisms cause additional lactic acid to be released by the retina following light–dark transition: (1) increased glucose metabolism at the outer retina; (2) reduced retinal oxygen level in the dark-adapted eye, leading to an increased anaerobic lactate production; and (3) glutamate-induced lactate release from Mu¨ller cells. The RPE disposes of this metabolic load by transporting lactic acid to the choroid

through monocarboxylate transporters (MCTs) of the MCT family. We previously demonstrated that the RPE is extremely resistant to pH change compared to other epithelia and that part of this regulation comes from H/Lac cotransporters at the apical and basolateral membranes. In human RPE, Philp and colleagues showed that monocarboxylate transporter 1 (MCT1), a H/Lac cotransporter, is immunolabeled at the apical membrane (Figure 5). They also showed that MCT3, a H/Lac cotransporter expressed exclusively in the RPE and choroid plexus basolateral membranes, meditates lactate efflux from the RPE into the choroidal blood supply (Figure 5). In addition, a Cl/Lac exchanger, possibly anion exchanger 2 (AE2), has been shown to transport lactate at the basolateral membrane. The importance of lactate transport in the mammalian eye has also been demonstrated in mice lacking MCT1, MCT3, and MCT4 expression – the mutant mice gradually lose photoreceptor function and

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Choroid

NBC

CFTR

Na nHCO3

Cl

Anion

+ + cAMP Cl Ca2+ Cl

AE2

VB (− 47 mV)

HCO3 H+ Lac (Lac)

Na nLac

Na

Na

MCT3

Cl HCO3

HCO3

H2O

CA II

Na 2HCO3 CO2

NKCC1

NBC1

Na 2HCO3

K 2Cl Na

CO2

H+

3Na ATP

K 2Cl Na 2k Subretinal space

NHE

Na

H+ Lac

VA (− 50 mV)

MCT1

H+ Lac

TEP ≈ 3 mv

Figure 5 Na, Cl, HCO3, and lactate transport mechanisms in retinal pigment epithelium (RPE). CO2 enters the apical membrane via diffusion and HCO3 is transported into the cell by NBC1 (sodium bicarbonate co-transporter). As CO2 enters the cell, it can be hydrated into HCO3 in a reversible reaction catalyzed by carbonic anhydrase II. Cl enters the apical membrane via NKCC1 (sodium potassium chloride co-transporter – Na/K/2Cl) and exits the basolateral membrane via CFTR or Ca2+-activated Cl channels. Lactic acid is transported across the apical membrane by MCT1, and out of the basolateral membrane through MCT3 (H/Lac co-transporter) and AE2 (Cl/Lac exchanger). These ion-transport mechanisms at the apical and basolateral membranes mediate net solute transport across the RPE, which drives fluid absorption. The apical and basolateral membrane potentials are given by VA and VB, respectively, and their difference is the transepithelial potential (TEP).

were completely blind after 41 weeks. Further, altered visual function in MCT3-null mice demonstrates the importance of lactate transport specifically in the RPE.

Oxidative Stress The RPE encounters significant levels of oxidative stress on a daily basis and this onslaught promotes mitochondrial (mt) damage and decreases in mt potential and respiration, which may contribute to inflammation and the onset of age-related diseases such as AMD (summarized by Jarrett and colleagues). In opposition to these oxidative stresses, there exist mt protective mechanisms that provide direct antioxidant protection and those that enhance glutathione (GSH) production; furthermore, there is evidence that all of these protective mechanisms weaken with age. Different cell types can exert different levels of protection; for example, it has been shown that hfRPE monolayers are significantly more resistant to oxidative stress than ARPE19 cells. Voloboueva and colleagues used the hfRPE primary cultures to examine mt and other pathways that are putative targets for therapeutic intervention against oxidative stress. In one set of experiments they studied the protective effects of a-lipoic acid (R-form), a potent intracellular antioxidant that has been shown in other systems, to induce all three cellular protective mechanisms. The R form of lipoic acid is a coenzyme in mt that has

been shown to reverse the age-related decrease in mt function. Measurements of cell viability, mt potential, cell death, oxidative stress, apoptosis, and GSH/GSSH show that lipoic acid can protect hfRPE cells in three ways: (1) directly scavenge reactive oxidative species; (2) repair and protect mt enzymes; and (3) activate antioxidant defenses through phase 2 enzymes. Our results suggest that (R)-a-lipoic acid can be used as an all-purpose therapeutic intervention against the slow accumulation of oxidative damage that can occur in AMD. Cigarette smoke is an important risk factor for AMD and causes significant oxidative damage in RPE that also can be mitigated by (R)-a-lipoic acid. RPE mitochondria are themselves a main generation site of oxidants and a critical and sensitive target of specific cigarette-smoke components. Acrolein is present in the gas phase of cigarettes (25–140 mg per cigarette), and it is estimated that the gas phase of one cigarette reaches a concentration of 80 mM in the airway surface fluid. Acrolein has a high hazard risk in cigarette smoke and causes oxidative stress in cells by reacting with sulfhydryl groups. In the physiological range (0.1–100 mM), it causes significant mt damage in hfRPE that can be ameliorated by (R)-a-lipoic acid, for example, by inducing GSH and other phase-2 antioxidant protective enzymes. Pretreatment by (R)-alpha-lipoid acid has a protective effect against peroxide induced mt oxidative stress in several ways: (1) by lowering cell calcium; (2) by increasing mt electron chain complexes I, II, and III


activity levels; (3) by increasing mt membrane potential; and (4) by increasing total antioxidant power as well as GSH peroxidase/GSH/superoxide dismutase levels. Collectively, these data indicate that lipoic acid may be an effective therapeutic strategy against age-related, oxidantinduced RPE degeneration. As summarized recently by Dunaief, AMD patients have elevated levels of iron within the RPE that also can lead to oxidative damage to mitochondria. Based on that observation, Voloboueva and colleagues showed that ferric ammonium citrate increased intracellular iron and oxidant production and decreased GSH and mt complex IV activity in human fetal cultured RPE. They also showed that N-tert-butyl hydroxylamine (Nt-BHA), a known mt antioxidant, reduced oxidative stress, mt damage, and age-related iron accumulation. These data show that the application of Nt-BHA may be an effective therapeutic strategy against AMD.

RPE–Immune System Interactions in and around the SRS The integrated effect of proinflammatory molecules on RPE function depends on the polarized location of the cognate receptors and the access of their ligands (cytokines and chemokines) to the apical and basolateral membranes, and the interactions of downstream signaling pathways. For the experiments summarized in Table 1, we used a mixture of three proinflammatory cytokines, interleukin 1 beta (IL-1b), interferon gamma (IFNg), and tumor necrosis factor-alpha (TNF-a) to stimulate confluent monolayers of hfRPE. These proinflammatory cytokines are elevated in patients with uveitis and are detected in the vitreous and blood of patients with proliferative diabetic retinopathy (PDR) and AMD with choroidal neovascularization (CNV). As a first step in understanding how the RPE in vivo can actively control the inflammatory environment in the SRS and choroid, Shi and colleagues used confluent monolayers of human fetal RPE primary cultures to (1) measure the constitutive and polarized secretion of angiogenic/angiostatic cytokines by the RPE; (2) determine how this pattern of polarized secretion changes in the inflammatory state; and (3) demonstrate that the inflammatory state alters RPE physiology. Constitutively, the human RPE secretes massive amounts of monocyte chemoattractant protein 1 (MCP-1) to the SRS and lesser amounts of IL-6 and IL-8 (Table 1), all of which contribute to the ongoing downregulation of the immune environment of the retina. RPE activation was achieved using a cocktail of IL-1b, TNF-a, and INFg with similar concentrations as that detected in the diseased eye. We showed that IL-1b receptors are mainly localized to the apical membrane and TNF-a and INFg (subunit 1)

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receptors are mainly localized at the basolateral membrane. This cocktail significantly increased the secretion of various cytokines/chemokines to both baths, but significantly more to the apical bath. The increase in angiogenic cytokine secretion exceeds the increase in angiostatic cytokine secretion. However, two chemokines generally thought to be angiostatic, interferon-inducible T-cell a-chemoattractant (I-Tac) and monokine induced by g interferon (MIG), were secreted to the apical bath in significant quantities. The mechanisms by which these chemokines exert their effects and their role in eye physiology are not yet known. Similarly intriguing and not understood are the secretions into the apical bath of interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein 3 (MCP-3), and the Rantes chemokine. In animal model experiments from Charlotte Reme’s group, blue-light-induced oxidative damage induces the invasion of blood-borne monocytes and activation of retinal microglia, thus stimulating the secretion of cytokines to induce an inflammatory response. Our experiments strongly suggest that the RPE is a significant source of cytokines and chemokines. Thus both retinal microglia and RPE can contribute to the inflammatory response in a diseased eye. Our further demonstration that basolateral addition of the cocktail acutely increases fluid absorption across the RPE (Figure 6), from the apical to basal baths (retina to choroidal side of tissue), and significantly decreases transepithelial resistance after a 24-h treatment is important because of the possibility that with age or accumulated oxidative stress these changes can alter chemokine/cytokine gradients across the RPE. These gradients regulate the attraction of monocytes to the RPE basement membrane and, thus, play a role in the accumulation of drusen with age. We believe that this concept is important for understanding early events that underlie chronic disease processes, such as AMD, a notion revisited below.

Modulation of RPE Proliferation and Migration by Cytokines and Growth Factors Breakdown of the inner or outer blood–retinal barrier can lead to significant alterations in the chemical composition of the SRS, including cytokines and growth factors, which trigger the activation of normally quiescent RPE cells. In proliferative vitreoretinopathy (PVR), RPE cells proliferate and migrate to the vitreous cavity along with other types of cells (e.g., glial cells, fibroblasts, and macrophages) and form fibrocellular membranes on the retinal surface or in the vitreous. These newly formed membranes, if left untreated, eventually contract, resulting in retinal detachment and eventual vision loss. Several isoforms of platelet-derived growth factor (PDGF) are present in retinal membrane from patients

Inflammatory cytokine mixture alters polarized secretion of chemokines and cytokines by cultured human fetal retinal pigment epithelium (hfRPE)

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Table 1



Jv (μl ·cm–2 ·h–1)

30

ICM (Ba)

769

ICM (Ba)

20 10 0 Probe out 4

1000

3

800 TEP (mV)

2 1

600

0 400

RT (Ω·cm2) Δ

10 min

–1 200

–2 (a) ICM (Ap + Ba) Jv (μl · cm−2 · hr−1)

20

10

0 4

300

3

275

2

250

1

225 200

0 (b)

2 RT (Ω ·cm ) Δ

TEP (mV)

Probe out

10 min

Figure 6 Inflammatory cytokine mixture (ICM) induced changes in hfRPE fluid transport (JV). In all panels, the top trace is JV, which is plotted as a function of time; net fluid absorption is indicated by positive values and TEP and total tissue resistance (RT) are plotted in the lower traces. (a) Addition of ICM to the basal bath increased JV by 13 ml cm–2 hr–1 with no significant changes in TEP and RT. (b) Concomitant addition of ICM to apical and basal baths increased JV by 10 ml cm–2 hr–1 with no change in TEP and a slight increase in resistance that is not statistically significant. From Shi, G., et al. (2008). Investigative Ophthalmology and Visual Science 49(10): 4620–4630. ã Association for Research in Vision and Ophthalmology.

with PVR and PDR and are elevated in the vitreous of PVR eyes. Recently we showed that PDGF-C, -D are highly expressed in human fetal and adult RPE and that the mRNA levels of these two isoforms are up to 100-fold higher than PDGF-A and -B. PDGF-C and -D have been implicated in PVR and lens epithelial cell proliferation, but relatively little is yet known about their function in RPE. In other systems, they play an important role in angiogenesis and wound healing.

PDGFR-a and PDGFR-b, the receptors for PDGF-C and -D, respectively, are mainly localized to the apical membrane of human fetal RPE as shown in Figure 7. PDGF-CC, -DD, and -BB significantly stimulated hfRPE cell proliferation, while PDGF-DD, -BB, and AB significantly stimulated cell migration. Furthermore, the stimulatory effects of PDGF were abrogated by a proinflammatory cytokine cocktail composed of TNF-a, IL-1b, and IFNg. Comparison of the component effects


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Apical

Basal

(a)

(b)

Figure 7 Immunofluorescence localization of PDGF receptors on hfRPE. The main part of each panel is an en face view of a cell culture monolayer shown as a maximum intensity projection through the z-axis. The top and right side of each panel is a cross section through the Z-plane of multiple optical slices obtained using the Apotome. In all experiments shown, the nucleus was stained with DAPI (blue), and the tight junction protein (ZO-1) was immunolabeled in red. The platelet-derived growth factor receptor PDGFR-a (green) is shown in (a), and while the beta subunit, PDGFR-b (green) is shown in (b). Both were detected on the apical membrane of hfRPE. From Li, R., et al. (2007). Investigative Ophthalmology and Visual Science 48(12): 5722–5732. ã Association for Research in Vision and Ophthalmology.

showed that IFNg was more effective in suppression than the entire cocktail or any subset of the cocktail, indicating that the downstream cytokine signaling pathways are interactive. Identifying the elements of this putative network and the specific nature of these interactions could provide targets for therapeutic intervention. For example, the proinflammatory cocktail may activate PDGF secretion by the RPE. In preliminary experiments, we showed that IFNg increased the polarized secretion of PDGF-AA to the apical bath, providing a possible autocrine signal mediating RPE proliferation/migration. We have shown that the cytokine cocktail induces cell apoptosis, alters cytoskeleton distribution, and significantly decreases transepithelial resistance, which can help mediate leukocyte traffic to the SRS. The cytokine cocktail-induced in hibition of RPE proliferation/migration indicates a potential therapeutic role against proliferative responses at the retina/RPE/choroid interfaces. Since IFNg has a strong inhibitory effect on RPE proliferation and migration, it is natural to ask how this signaling pathway might provide the basis for inhibition. Native human adult RPE and hfRPE cells constitutively express two transcription factors, interferon regulatory factors 1 and 2 (IRF-1 and IRF-2), which are well-characterized members of the IFN regulatory family and key factors in the regulation of cell growth through their effects on cell cycle. In hfRPE, we showed that stimulation by IFNg significantly increased IRF-1 protein levels with no effect on IRF-2. If these two transcription factors are mutually antagonistic, as shown in other systems, this may explain the strong inhibitory effect of IFNg on RPE proliferation

and migration, which we hypothesize is caused by an increase in the ratio of IRF-1/IRF-2.

IFNg Regulation of RPE Fluid Transport Immunoblots, immunofluorescence, intracellular recordings, pharmacology, and fluid transport data indicate a basolateral location of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel, in native adult and fetal human RPE. As a first step in unraveling the network of cytokine interactions, we focused on INFg since it is a main determinant of several key effects produced by the inflammatory cocktail. IFNg has been implicated in the pathogenesis of a number of inflammatory diseases of infectious or presumed autoimmune origin and it has been detected in vitreous aspirates of patients with uveitis, PVR, and other inflammatory ocular diseases. In human RPE, IFNg activates several intracellular signaling pathways, including the canonical janus-activated kinase and signal transducers and activators of transcription protein (JAK/STAT) pathway and P38 mitogenactivated protein kinase (MAPK), leading to the elevation of cyclic adenosine monophosphate (cAMP) and the subsequent activation of protein kinase A-dependent chloride channels – CFTRs. This results in a significant increase in net fluid absorption across the epithelium (Figure 8). These data and the data summarized below provide a possible basis for the etiology of chronic inflammatory diseases, such as posterior uveitis and AMD. In the diseased eye, the IFNg-induced dehydration of the SRS


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Figure 8 IFNg-stimulated fluid transport (JV) increase is inhibited by 5 mM CFTRinh-172, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), added to basal bath. JV is plotted as a function of time in the top trace and net fluid absorption (apical to basal bath) is indicated by positive values; TEP () and RT (△) are plotted as function of time in the two lower traces.

ns Transmigrated granulocytes % of control

120

*

100 80 60 40 20 0

Upper well

-

Lower well

-

IL-8

-

could increase the concentration of the already-accumulating chemokines and thereby help draw monocytes and neutrophils to the RPE basement membrane or across the RPE to the SRS. This helps control the continuing accumulation of debris from incompletely digested photoreceptors, oxidative stress, and accumulation of drusen that normally occur in and around the RPE over the first five decades of life. Based on a variety of risk factors that activate the immune system (e.g., monocytes), these protective gradients may dissipate with age, aided perhaps by the loss of RPE barrier function and the steady buildup of an immunologically hostile environment.

Fc-JAM-C Fc-JAM-A IL-8

Leukocyte Migration across the RPE: A Model of Disease Progression

(a) ns

Transmigrated monocytes % of control

160

The integrity of the RPE monolayer depends on the inter-epithelial junctions that include tight and adherens junctions and desmosomes. The main constituents of tight junctions are three families of transmembrane proteins: occludins, claudins, and junctional adhesion molecules

ns

140 120 100 80 60 40 20

0 Upper well Lower well

MCP-1

Fc-JAM-C Fc-JAM-A MCP-1

(b) Figure 9 IL-8 and MCP-1 regulated transmigration of leukocytes. (a) Basolateral to apical transepithelial migration of

granulocytes. The number of transmigrating granulocytes is shown as % of control. Exogenous addition of modified junctional adhesion molecule Fc-JAM-C competes with JAM-C/ JAM-C interactions between adjacent cells, significantly reducing IL-8 induced transmigration of granulocytes. (b) In contrast, Fc-JAM-C had no significant effect on the basolateral to apical transepithelial migration of monocytes toward MCP-1. Addition of another RPE JAM isoform Fc-JAM-A, did not alter transmigration of granulocytes or monocytes. From Economopoulou, M., et al. (2009). Investigative Ophthalmology and Visual Science 50(3): 1454–1463. ã Association for Research in Vision and Ophthalmology.

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( JAMs). The third member of the JAM family, JAM-C, has been identified in various cell types and implicated in inflammatory processes and shown to participate in the transmigration of leukocytes through endothelial and gut epithelial cells. Economopoulou and colleagues found that JAM-C is localized at the tight junctions of intact monolayers of adult and fetal human RPE, it is found at the initial cell–cell contacts of newly forming junctions, and that it helps initiate hfRPE junction formation and polarization. JAM-C also promotes the transepithelial migration of granulocytes through intact monolayers of cultured hfRPE driven by physiological gradients of interleukin 8 (IL-8). Thus, in the intact eye, JAM-C may be an important determinant of RPE initial junction formation, cell polarization, and immune-system-mediated pathophysiology at the retina–RPE interface (Figure 9). Recent animal model studies have implicated monocyte chemoattractant protein 1 (MCP-1) and fractalkine receptor in retinal microglia as critical regulators of drusen accumulation, local inflammation, and the development of AMD. As demonstrated by Shi and colleagues, the RPE secretes significant amounts of MCP-1 and IL-8 to the apical side in a polarized manner (Table 1). Both chemokines could form gradients across the RPE that coordinate monocyte and neutrophil movement to the RPE basement membrane. This could provide local surveillance/protection against the accumulation of immunologically active debris (drusen). The transformation over time of monocytes into macrophages would slowly degrade the RPE’s ability to maintain protective chemokine gradients for the removal of immunologically active debris and eventually lead to degeneration/disease. See also: Injury and Repair: Light Damage; Phototransduction: The Visual Cycle; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration.

Further Reading Adijanto, J., Banzon, T., Jalickee, S., and Miller, S. S. (2009). CO2-induced ion and fluid transport in human retinal pigment epithelium. Journal of General Physiology 133(6): 603–622. Blaug, S., Quinn, R., Quong, J., Jalickee, S., and Miller, S. S. (2003). Retinal pigment epithelial function: A role for CFTR? Documenta Ophthalmologica 106: 43–50. Bryant, D. M. and Mostov, K. E. (2008). From cells to organs: Building polarized tissue. Nature Reviews. Molecular Cell Biology 9(11): 887–901. Daniele, L. L., Sauer, B., Gallagher, S. M., Pugh, E. N., Jr., and Philp, N. J. (2008). Altered visual function in monocarboxylate transporter 3 (Slc16a8) knockout mice. American Journal of Physiology Cell Physiology 295: C451–C457.

Donoso, L. A., Kim, D., Frost, A., Callahan, A., and Hageman, G. (2006). The role of inflammation in the pathogenesis of age-related macular degeneration. Surveys of Ophthalmology 51: 137–152. Dunaief, J. L. (2006). Iron induced oxidative damage as a potential factor in age-related macular degeneration: The Cogan lecture. Investigative Ophthalmology and Visual Science 47: 4660–4664. Economopoulou, M., Hammer, J., Wang, F., et al. (2009). Expression, localization, and function of junctional adhesion molecule-C (JAM-C) in human retinal pigment epithelium. Investigative Ophthalmology and Visual Science. 50: 1454–1463. Fisher, S. K., Lewis, G. P., Linberg, K. A., and Verardo, M. R. (2005). Cellular remodeling in mammalian retina: Results from studies of experimental retinal detachment. Progress in Retinal and Eye Research 24: 395–431. Gehrs, K. M., Anderson, D. H., Johnson, L. V., and Hageman, G. S. (2006). Age-related macular degeneration – emerging pathogenetic and therapeutic concepts. Annals of Medicine 38: 450–471. Illek, B., Fu, Z., Schwarzer, C., et al. (2008). Flagellin activates inflammatory response and cystic fibrosis transmembrane conductance regulator-dependent Cl secretion: Role for p38. American Journal of Physiology Lung Cell Molecular Physiology. 295: L531–L542. Jarrett, S. G., Lin, H., Godley, B. F., and Boulton, M. E. (2008). Mitochondrial DNA damage and its potential role in retinal degeneration. Progress Retinal Eye Research 6: 596–607. Jia, L., Liu, Z., Sun, L., et al. (2007). Acrolein, a toxicant in cigarette smoke, causes oxidative damage and mitochondrial dysfunction in RPE cells: Protection by (R)-alpha-lipoic acid. Investigative Ophthalmology and Visual Science 48: 339–348. Li, R., Maminishkis, A., Wang, F. E., and Miller, S. S. (2007). PDGF-C and-D induced proliferation/migration of human RPE is abolished by inflammatory cytokines. Investigative Ophthalmology and Visual Science 48: 5722–5732. Maminishkis, A., Chen, S., Jalickee, S., et al. (2006). Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Investigative Ophthalmology and Visual Science 47: 3612–3624. Nussenblatt, R. B. and Ferris, F., 3rd (2007). Age-related macular degeneration and the immune response: Implications for therapy. American Journal of Ophthalmology 144: 618–626. Philp, N. J., Ochrietor, J. D., Rudoy, C., et al. (2003). Loss of MCT1, MCT3, and MCT4 expression in the retinal pigment epithelium and neural retina of the 5A11/basigin-null mouse. Investigative Ophthalmology and Visual Science 44: 1305–1311. Shi, G., Maminishkis, A., Banzon, T., et al. (2008). Control of chemokine gradients by the retinal pigment epithelium. Investigative Ophthalmology and Visual Science 49: 4620–4630. Strauss, O. (2005). The retinal pigment epithelium in visual function. Physiological Reviews 85: 845–881. Voloboueva, L. A., Killilea, D. W., Atamna, H., and Ames, B. N. (2007). N-tert-butyl hydroxylamine, a mitochondrial antioxidant, protects human retinal pigment epithelial cells from iron overload: Relevance to macular degeneration. FASEB Journal 21: 4077–4086. Voloboueva, L. A., Liu, J., Suh, J. H., Ames, B. N., and Miller, S. S. (2005). (R)-alpha-lipoic acid protects retinal pigment epithelial cells from oxidative damage. Investigative Ophthalmology and Visual Science 46: 4302–4310. Wangsa-Wirawan, N. D. and Linsenmeier, R. A. (2003). Retinal oxygen: Fundamental and clinical aspects. Archives of Ophthalmology 121: 547–557. Winkler, B. S., Starnes, C. A., Twardy, B. S., Brault, D., and Taylor, R. C. (2008). Nuclear magnetic resonance and biochemical measurements of glucose utilization in the cone-dominant ground squirrel retina. Investigative Ophthalmology and Visual Science 49: 4613–4619.

RPE Barrier L J Rizzolo, Yale University School of Medicine, New Haven, CT, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Adherens junctions – A component of the apical junctional complex that provides mechanical strength to cell–cell adhesions and, together with the tight junction, regulates cell size, shape, and proliferation. Apical junctional complex – An assembly of tight, adherens, and gap junctions that join neighboring cells of an epithelial monolayer together. The junctions form a belt that completely encircles each cell at the apical end of the lateral membranes. Apical membrane – The region of the plasma membrane that interdigitates with the photoreceptors of the neural retina. It is separated from the basolateral membranes by the apical junctional complex. Basolateral membrane – The region of the plasma membrane that rests on Bruch’s membrane and faces the choroid. It is separated from the apical membrane by the apical junctional complex. Claudins – A family of proteins that forms tight junctional strands and determines the selectivity and permeability of the tight junctions. Paracellular space – The space between the neighboring cells of an epithelial monolayer. Subretinal space – The thin space that lies between the apical membrane of the retinal pigment epithelium and the photoreceptors. It becomes a wide space with retinal edema and detachment. Tight junctions – A component of the apical junctional complex that regulates transepithelial diffusion through the paracellular space, retards diffusion of lipids and membrane proteins between the apical and basolateral membranes, and, together with the adherens junction, regulates cell size, shape, and proliferation. Transepithelial electrical resistance (TER) – An amalgam of the electrical resistances of the apical membrane, basolateral membrane, and paracellular space. It is commonly used as a reflection of the electrical resistance of tight junctions. When the sum of the membrane resistances greatly exceeds the paracellular (shunt) resistance, the TER approximates the electrical resistance of the tight junctions.

Introduction Blood–tissue barriers were first revealed by the inability of injected proteins, or protein-bound dyes, to move from the blood into certain tissues. Only the brain, testes, and placenta shared this property. The cells that formed the barrier exhibited reduced transcytosis and were bound together by seemingly impermeable tight junctions. Transcytosis is one mechanism to move serum solutes across the cells of the barrier, whereas tight junctions partially occlude the paracellular spaces to reduce transepithelial diffusion between the cells. This initial conception has since been expanded to include all mechanisms of transcellular transport and the metabolic and catabolic pathways that alter solutes during transport. The blood–retinal barrier has two divisions. The inner layers of the retina are supplied by a vascular bed, whose endothelia form the inner blood–retinal barrier. This endothelial barrier typifies most of the blood–brain barrier. This article focuses on the outer blood–retinal barrier, which is more similar to the choroid plexus and testes blood–tissue barriers. These barriers are a collaboration of a fenestrated capillary bed with an epithelium. In the outer retina, the collaboration is between the choriocapillaris and the retina pigment epithelium (RPE). As fenestrae make the capillaries porous, the RPE forms the barrier to serum components (Figure 1). Certainly, the Bruch’s membrane that separates the capillaries and RPE serves as a filter; however, this aspect of the outer blood–retinal barrier is discussed elsewhere in the encyclopedia. A good example of a metabolic pathway that participates in barrier function is the visual cycle. Vitamin A, transported by the serum, is endocytosed and transformed into cis-retinal as it is transported across the cell and exported to the photoreceptors. Ultimately, the paracellular and transcellular transport of ions and small organic solutes should be considered as a unit. Because explorations of how these pathways interact remain in their infancy, the two topics are discussed separately in this encyclopedia. This article focuses on the paracellular pathway, but includes the transcellular pathway whenever a connection between the two can be made. We discuss the assembly of RPE tight junctions, their retina-specific properties, and how the RPE and its tight junctions are regulated by the surrounding tissues.

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Structure and Function Tissue Level The distinctive structure of the outer blood–retinal barrier leads to unique functions. Unlike other epithelia and endothelia, the RPE separates two solid tissues – the choroid and the neural retina. Early in embryogenesis, the apical surface of the RPE borders a fluid-filled lumen, the lumen of the optic vesicle. As development proceeds, this lumen is reduced to a potential space known as the subretinal space (Figure 2). In this space, the microvilli of the RPE’s apical pole interdigitate with the outer segments of the photoreceptor cells. The intimate contact of the RPE and photoreceptors allows retinoids of the visual cycle to readily shuttle back and forth across the subretinal space, and allows disk membranes shed by the photoreceptors to be phagocytosed

by the RPE. The ionic composition of the subretinal space is carefully regulated to support the functions of the photoreceptors and the RPE. To this end, the RPE absorbs water. Water continuously enters the retina from the inner vascular bed and vitreous and is transported by the RPE into the choroid for removal by the choroidal circulation. Failure of this process results in retinal edema and even retinal detachment. Contrast this with another region of the blood–brain barrier, the epithelium of the choroid plexus. The epithelium of the choroid plexus is also derived from the neuroepithelium, but in this case, the lumen of the neural tube expands to form the ventricular system. Rather than absorb fluid, the choroid plexus secretes copious volumes of cerebral spinal fluid. To understand the functional differences between the RPE and the epithelium of the choroid plexus, we need to look more closely at the structure of the barrier.

Retina Subretinal space

Lateral membranes enlarged Cross-section Enface view view

Rotate plane

RPE

90⬚

Bruch’s membrane Paracellular path Non-vectorial diffusion along electrochemical gradients Tight junctions selectively retard certain ions and organic solutes

Transcellular path Active transport along unidirectional vectors Membrane pumps and transporters Modification of solutes during transport

Tight junctions appear as membrane kisses

Tight junctions appear as a belt of anastomosing strands

Figure 1 Mechanisms that regulate transport across the outer blood–retinal barrier.

Early development

Late development

Lumen of optic vesicle

Connector to: - Neural tube - 3rd ventricle (with optic nerve)

RPE Retina Subretinal space

Figure 2 The lumen of the embryonic optic vesicle becomes the subretinal space.

RPE Barrier

Cellular Level The RPE is a monolayer of cells that are joined by a complex of junctions known as the apical junctional complex. The apical junctional complex encircles each cell to bind the monolayer together much like the plastic rings that hold together a six-pack of canned beverages. The complex consists of three junctions (tight, adherens, and gap) whose functions are intertwined. Adherens junctions bind neighboring cells together. Tight junctions form a partially occluding seal that semiselectively retards diffusion through the paracellular spaces of the monolayer (Figure 1). Both junctions regulate proliferation, cell size, and the polarized distribution of plasma membrane proteins. The junctions of the complex work in concert, but we focus on how tight junctions contribute to barrier function. Tight junctions form a barrier between the apical and basolateral poles of the cell. They work in conjunction with intracellular trafficking pathways to create and maintain an apicobasal polarity that is essential for the blood–retinal barrier to function. Unlike most epithelia, the Na,K-ATPase (ATP, adenosine triphosphate) is enriched in the apical membrane rather than localized to the basolateral membrane. Although this initial discovery suggested that the RPE is an upside-down epithelium, it is now known that only a few RPE proteins have an atypical distribution. What is crucial for RPE function is the distribution of membrane channels and transporters. The RPE and the epithelium of the choroid plexus both have an apical Na,K-ATPase that provides the energy for vectorial transport. It is the distribution of the various ion channels and transporters that determines whether the RPE absorbs water or the epithelium of the choroid plexus secretes it. Briefly, the polarized distribution of transporters in the RPE results in the active transport of chloride from the apical to basal side of the cell. As a result, the apical side of the monolayer has a positive charge relative to the basal side. Sodium and potassium are transported down this electrical gradient to balance the chloride transport. The osmotic gradient that results pulls water in the apical to basal direction. The polarized distribution of the various channels and transporters differs in the epithelium of the choroid plexus to support the opposite, basal to apical, transport of water. There are general housekeeping mechanisms that recognize the targeting signals encoded in a protein’s structure to deliver it to the correct membrane. In some cases, different tissuespecific isoforms encode different targeting signals; in others, tissue-specific variations in the targeting machinery give each epithelium its unique character. The vectorial transport mechanism outlined above would have little effect, if transepithelial ion gradients were dissipated by the paracellular pathway. Early microscopists believed that a zonular band of junctions occluded the paracellular space by completely encircling each cell. Their

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name for this junction, zonula occuldens or tight junction, is misleading because the junction is selectively leaky. The degree of leakiness, and selectivity for certain ions, not only varies among epithelia, but is essential for epithelial function as well. The permeability and semiselectivity of the junctions are matched to transcellular transport mechanism. For example, RPE tight junctions need to be leakier to sodium than to chloride, because RPE pumps chloride across the cell and needs a leak for cations to passively follow the chloride flux. The inadequate capacity for sodium and potassium to cross the cell is ameliorated by the sodiumselective leak through the tight junction. A compelling example of how transcellular transport is matched to tight junction selectivity is provided by the kidney. The diuretic hormone, aldosterone, changes the flux of water by acting simultaneously on a membrane sodium channel and a tight junction protein that regulates sodium selectivity. In the retina, the volume and composition of the subretinal space vary with the day/night cycle. It remains to be investigated how the properties of tight junctions and membrane transporters are coordinated to manage the subretinal space during this cycle. Molecular Level Proteins of the apical junctional complex fall into transmembrane, adaptor, and signaling/regulatory categories. Adaptors link the transmembrane proteins to signaling proteins and a cortical band of actin filaments. Besides known members of the tight junction (Figure 3), proteomics suggest that over 912 proteins are associated with the tight junction. The adaptor proteins express multiple copies of PDZ domains. This protein-binding domain of approximately 100 amino acids takes its name from the three proteins that defined this class of protein-binding domain: postsynaptic density protein 95 (PSD-95), disks large, and zonula occludins 1 (ZO-1). PDZs are the largest family of protein-binding domains and form the basis of many protein complexes. Each adaptor protein expresses Apical membranes Transmembrane proteins 24 claudins, 3 JAMs, occludin, CAR, CRB3 Adaptor proteins ZO-1, -2, -3; MAGI-1, -3; MUPP1, Par 3,6; AF-6, Pals 1, PATJ A fila ctin Signalling proteins and regulators me aPKC, GEF-H1, CDK4, Rab13, Gα12’ nts ZONAB, symplekin, AP-1, cingulin, paracingulin, angiomotin, JEAP, ZAK, MASCOT, PTEN, WNK4, c-YES, PP2A Lateral

membranes Figure 3 Composition of the tight junctions.

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multiple homologs of PDZ domains that have distinct, but sometimes overlapping binding specificities. Together with other protein-binding motifs, for example, SRC homology 3 domain, guanylate kinase, bi-tryptophan domain that binds proline-rich peptides, Dilute domain, and Phox and Bem1P domain, the known adaptor proteins have the capacity to bind the large number of regulatory proteins that proteomics suggests. A junction of such complexity would be inconsistent with the original view of the tight junction as a static barrier. The tight junction is a highly dynamic structure with the potential to rapidly respond to environmental stimuli. Photobleaching studies demonstrate that ZO-1 and occludin, a regulator of permeability, rapidly associate and dissociate from the junction. Occludin has a very short half-life and its degradation is regulated by endocytic

and ubiquitin pathways. Therefore, the cell exerts a fine control over the properties of the tight junction that can rapidly respond to changes in the environment. This flexibility extends to the claudin family of transmembrane proteins. Membrane proteins typically have a half-life on the order of days, but the claudins that have been studied have a half-life of 4–12 h. Claudins form the anastomosing network of strands that are observed by freeze-fracture electron microscopy (Figure 4) and schematized in Figure 1. There are 24 or more claudins. Each epithelium expresses a subset of claudins. The subset of claudins, expressed and localized to the tight junction, determines the selectivity and permeability of the junction. In the kidney example given above, aldosterone decreases the expression of claudin 4 to increase the sodium leak through the tight junctions.

m m m E7 g

(a)

g

(b)

(c)

m E10

PF EF PF

EF (e)

(d)

m

m

E14

(f) Figure 4 Strands of the tight junction gradually coalesce during development. Freeze-fracture replicas show how sparse, disconnected strands on E7 become a necklace of strands with discontinuities by E10 and a continuous, uninterrupted network by E14. Microvilli (m) at the top of each panel indicate the apical end of the lateral membrane. Arrows, tight junctional strands; Arrowheads, discontinuities. EF, E-face; PF, P-face; Bar ¼ 0.25 mm.

RPE Barrier

Although the basic structure of the outer blood–retinal barrier is conserved among species, there are speciesspecific variations in the composition of the subretinal space, the properties of transmembrane transport, and the composition of the tight junctions. In human RPE, the principal claudins appear to be claudins 3, 10, and 19. Claudin 19 has been linked to kidney disease and visual impairment. By contrast, chick RPE expresses primarily claudins 1 and 20 with lesser amounts of claudins 2, 4L2, 5, and 12. Claudins 1 and 3 are fairly ubiquitous with most epithelia expressing one or the other. Claudin 2 increases sodium permeability in some contexts. However, the study of how claudins affect permeability is in its infancy particularly in regard to the effects of cellular context on function.

Regulation of RPE Tight Junctions Clues from Embryonic Maturation What is a mature, differentiated RPE monolayer? Which markers and how many should we use to render this judgment? Might enhancing the expression of some markers in a culture experiment lessen the expression of others? If cellular pathways form an integrated web, would over- or underexpression of a protein have deleterious effects? A default path for human embryonic stem cells appears to be an RPE-like cell, but those cells lack some RPE proteins and express non-RPE proteins. The RPE is the first retinal cell to form during development, but despite its undeniable RPE character, the early RPE cell is only partially differentiated. It will undergo many transformations, as the neural retina and choroid differentiate on either side of it. The RPE is very plastic. Depending upon pathology or culture conditions, one can observe many partially differentiated states, or even transform RPE to other phenotypes. This may be the reason why RPE transplants fail when retinal degeneration is advanced. Dedifferentiation would be a normal response of healthy RPE to this abnormal environment. In support of this hypothesis, RPE transplants were most successful when the RPE and neural retina were cotransplanted. In some ways, culture is like a disease state where a degenerate neural retina and choroid no longer send RPE the signals that maintain key functions. In the chick culture model described, we found that retinal secretions promote RPE differentiation over the course of days rather than the months required without the retina. More work will be needed to determine whether these findings apply to mammalian eyes. A study of chick embryonic development illustrates the maturation process. We can describe early, intermediate, and late phases of development. The early phase extends from the time that the RPE forms on embryonic day 3

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(E3) until the inner segments of photoreceptors protrude the outer limiting membrane on E9. The intermediate phase extends from E9 to E15, when photoreceptors begin to elaborate outer segments. The late phase extends from E15 until hatching on E21. During the intermediate phase, the layers of Bruch’s membrane gradually form. Fenestrations in the walls of the choroidal capillaries begin to form in the intermediate phase, but are not fully elaborated until the middle of the late phase. In parallel with the formation of fenestrations in the capillaries, infoldings of the basolateral membrane begin to form in the intermediate phase, but are not completely elaborated until the middle of the late phase. As the basolateral membranes elaborate infoldings, apical membrane microvilli elongate in coordination with the elongation of photoreceptor inner and outer segments. Some plasma membrane proteins have a distribution that is polarized between the apical and basolateral membranes as early as E7. Nevertheless, some proteins become polarized later in development in parallel with the morphological changes of the apical and basolateral membranes. Examples include basigin, monocarboxylate transporters, and the Na,K-ATPase. This coordination of the development of photoreceptors, RPE plasma membranes, Bruch’s membrane, and the choriocapillaris is conserved between chickens and mammals. It is in the context of this maturing environment that the RPE completes its differentiation. The RPE is the first retinal layer to overtly differentiate. Despite an epithelial morphology and the expression of RPE markers, early embryonic RPE is still immature. Between E7 and E18 of chick development, 40% of the transcriptome changes with substantial effects on the extracellular matrix, junctional complexes, cell surface receptors, signal transduction pathways, cytoskeleton, regulators of gene expression, and transmembrane transport proteins. Some genes turned on and others off, but most changed their level of expression relative to one another. These data suggest why RPE cultures that express the same tissue-specific markers can function so differently. Without direction from the neural retina or choroid to establish balanced gene expression, cultured RPE can adopt the range of behaviors it displays during development in vivo. Assembly of Tight Junctions during Differentiation Most studies of the assembly of the apical junctional complex were performed in mouse blastocysts or in culture using kidney or intestinal cell lines. The rapid kinetics of assembly in these models makes it difficult to parse the role of the putative assembly proteins. The consensus is that a primordial adherens junction forms first, followed by the segregation of tight junction components into a nascent tight junction. Nectin, E-cadherin, and junctional

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RPE Barrier

adhesion molecule-A (JAM-A) trigger the formation of the primordial adherens junction. These transmembrane proteins form homodimers with their counterparts on the neighboring cell. Their cytoplasmic domains crystallize a complex of proteins by binding an adaptor protein. For example, JAM-A binds the adaptors, AF-6 (Afadin), PAR3 (Partitioning defective-3 homologue), and ZO-1. Their multiple protein-binding domains enable these adaptors to assemble a complex. For example, PAR-3 binds PAR-6 (Partitioning defective-6 homologue) and the atypical protein kinase C, which contributes to cell polarity. JAM-A also localizes ZO-1 to the apical side of the complex to initiate the formation of a tight junction. The tight junction transmembrane proteins, occludin and claudin, bind this nascent complex. Anti-JAM-A antibodies block the formation of tight junctions, as evidenced by the mislocalization of occludin and a low transepithelial electrical resistance (TER). Nevertheless, the adherens junction did form, as evidenced by the localization of E-cadherin and ZO-1. Taken together, these data suggest that JAM-A may play a role in assembling adherens junctions, but plays a more critical role in assembling tight junctions. As the apical junctional complex assembles slowly during normal RPE development, the process may be studied in greater detail. In chick, primordial adherens junctions are already present in the neuroepithelium that forms the RPE on E3, days before rudimentary tight junctions begin to form on E7. Many of the proteins described above in the assembly of the apical junctional complex are present at this time, but the adherens junction will remodel throughout development both morphologically and molecularly. In each phase of development, different cadherins will appear and disappear. The early phase includes many tight-junctional proteins: ZO-1, occludin, and the assembly proteins AF-6, JAM-A, PAR3, and PAR6. Nonetheless, tight junctions are absent until claudins expression begins on E7 and short, sparse tight-junctional strands begin to appear (Figure 4). During the intermediate phase, tightjunctional strands grow in number and length to gradually coalesce into a complete network that encircles each cell. The tight junction first becomes functional between E10 and E12 (defined by the ability to block the transepithelial diffusion of horseradish peroxidase). During the late phase, structural modifications of the tight junctions continue. Like the adherens junctions, these morphological changes are accompanied by molecular changes. Some claudin messenger RNAs (mRNAs) appeared early, but others appeared during the intermediate or late phases. The expression of some of the early-appearing claudins decreased during the late phase. During the intermediate phase, there was a switch in the expression of ZO-1 isoforms, an event also observed during tight-junction formation in pre-implantation embryos. ZO-3 did not appear until the late phase of development. Although changes in protein expression parallel gene expression to some extent, it appears that the

claudins and ZO proteins are also regulated by effects on protein stability and subcellular localization. The molecular and morphological changes in the apical junctional complex imply the function of the outer blood–retinal barrier changes during this long maturation process. The changes in claudin expression imply changes in the selectivity and permeability of the tight junctions. It would be reasonable to expect that this would be coupled with changes in transepithelial transport. Among the changes in the transcriptome, many involve membrane transporters. Several should be mentioned, because there is also physiological and cell biological data to corroborate the changes in gene expression. Changes in the expression and polarized distribution of the monocarboxylate transporters and Na,K-ATPase have already been mentioned. Early in development, several facilitated glucose transporters are expressed, but more are expressed later in development, including a sodium-coupled glucose transporter. The appearance of the latter transporters corresponds to the time that the tight junctions become relatively impermeable to glucose. These changes are essential because the retina has a high demand for glucose. It appears that housekeeping transporters that are sufficient for the RPE’s individual needs are replaced by a transcellular, active transport mechanism at the time the blood–retinal barrier forms. These conclusions are based on studies of a primary cell culture model of RPE maturation.

Culture Models to Study Regulation of the Outer Blood–Retinal Barrier RPE can be cultured on matrix-coated filters that are suspended in a culture dish (Figure 5). This architecture separates the media compartment into apical and basal chambers. The cultures spontaneously polarize with the basal membrane against the filter substrate and the apical microvilli projecting into the upper chamber. The culture architecture allows the cells to feed from the basolateral membranes, as in vivo. By contrast, plastic-grown cells need to feed from the apical membranes. Another advantage is that it is easy to measure barrier function by placing tracers in one medium chamber and measuring their flux

Apical chamber

RPE monolayer

Ohm meter

Basolateral chamber

Figure 5 RPE cell culture.

Filter

Higher resistance = lower permeability

RPE Barrier

across the monolayer into the opposite chamber. Further, electrodes may be placed to measure a TER. This flexibility is important because selectivity and permeability of tight junctions are regulated semi-independently. The TER is commonly used to assess tight junctions, but TER is an amalgam of transcellular and paracellular resistances to a current that is carried by all the ions of the extracellular space. The TER often approximates the resistance of the tight junctions, because the transjunctional current is often much greater than the transcellular current. Junctions of epithelia with a similar TER can differ in their ion selectivity. Therefore, it is valuable to measure ion fluxes directly. Further, the permeation of mannitol (a small organic tracer) can be modulated independently of TER, and vice versa. Accordingly, multiple assays of barrier function are required for a full assessment. Although it is relatively easy to culture RPE from many species, including human adults, it is difficult to establish cultures that form an effective barrier. A good example of the problems encountered is the human-derived ARPE19 cell line. This spontaneously transformed cell line has many RPE-specific properties, but its tight junctions are immature. Many claudins were expressed that are undetected in native tissue, and some of the claudins expressed in vivo were undetected in ARPE19. By modifying culture conditions, barrier function, morphology, and melanin expression could be improved. Although claudin expression was affected somewhat, large differences between native RPE and ARPE19 remained. Like the chick studies described above, genomic analyses of native and cultured human RPE have become or are becoming available, which will allow a molecular definition that can compare native to cultured RPE. Several culture systems have been devised that allow RPE to form a barrier that resembles native RPE. The most highly differentiated are primary or secondary cultures that were isolated from the intermediate phase of RPE development. This stage corresponds to E14 in the chick, postnatal day 5 in rat, and 18–22 weeks gestation in the human. Notably, human fetal RPE appears to have a broader window, as cultures isolated from 13-week fetuses also have excellent properties. Each culture relies on highly specialized medium that includes low amounts, or no, serum. In some cases, the medium appears to remove the need for choroidal or retinal stimulation, but these cultures require 1–2 months in culture to fully mature. Rat and chick cultures that were maintained in serum-free medium were very sensitive to the addition of serum to the apical medium chamber, as might be seen in pathology. For each species, apical serum decreased barrier function, as measured by the TER. In contrast to the other models, the chick model was designed to study tissue interactions. It used primary cultures that formed incomplete tight junctions with a low TER in a serum-free medium. However, the cultures were

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very sensitive to retinal secretions. A medium conditioned by the organ culture of neural retinas induced the formation of complete tight junctions with a TER that was similar to native RPE. Reconstitution experiments showed that contact with the neural retina was required for the proper polarized distribution of the Na,K-ATPase and certain integrins. A central finding was that retinal interactions promoted differentiation on a timescale of days rather than months. Besides cellular junctions, the retinal conditioned medium affected the expression of genes related to the visual cycle, phagocytosis, cytoskeleton, and transmembrane transport. Besides gene expression, retinal conditioned medium affects the half-life and subcellular localization of claudins and ZO proteins. By regulating membrane transporters and tight junctions, the retina and RPE appear to collaborate in regulating the subretinal space. This is an area of research that needs to be explored in greater detail.

RPE in the Larger Context of Ocular Biology and Disease Many diseases are coming to be viewed as a low-grade inflammatory process, including age-related macular degeneration. Investigations of inflammatory diseases of the intestine (Crohn’s) and central nervous system (multiple sclerosis) demonstrate that disease can affect the tight junctions in part through the action of inflammatory cytokines. Certainly, disease also affects the membrane transporters of epithelia and endothelia. In the renal field, there has been progress in understanding the interrelationships of tight junctions and membrane transporters and how they are coordinately regulated. The retina field lags behind, but the availability of good culture models and the advances in genomics and systems biology hold great promise for the future. See also: Breakdown of the RPE Blood–Retinal Barrier; Phototransduction: The Visual Cycle; Retinal Pigment Epithelium: Cytokine Modulation of Epithelial Physiology.

Further Reading Bradbury, M. W. B. (1979). The Concept of a Blood–Brain Barrier. New York: Wiley. Burke, J. M. (2008). Epithelial phenotype and the RPE: Is the answer blowing in the Wnt? Progress in Retinal and Eye Research 27(6): 579–595. Cereijido, M. and Anderson, J. M. (eds.) (2001). Tight Junctions. Boca Raton, FL: CRC Press. Cereijido, M., Contreras, R. G., Shoshani, L., Flores-Benitez, D., and Larre, I. (2008). Tight junction and polarity interaction in the transporting epithelial phenotype. Biochimica et Biophysica Acta 1778: 770–793.

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Grunwald, G. B. (1996). Cadherin cell adhesion molecules in retinal development and Pathology. Progress in Retinal Eye Research 15: 363–392. Guillemot, L., Paschoud, S., Pulimeno, P., Foglia, A., and Citi, S. (2008). The cytoplasmic plaque of tight junctions: A scaffolding and signalling center. Biochimica et Biophysica Acta 1778: 601–613. Klimanskaya, I., Hipp, J., Rezai, K. A., et al. (2004). Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning and Stem Cells 6: 217–245. Le Moellic, C., Boulkroun, S., Gonzalez-Nunez, D., et al. (2005). Aldosterone and tight junctions: Modulation of claudin-4 phosphorylation in renal collecting duct cells. American Journal of Physiology – Cell Physiology 289: C1513–C1521. Radtke, N. D., Aramant, R. B., Petry, H. M., et al. (2008). Vision improvement in retinal degeneration patients by implantation of retina

together with retinal pigment epithelium. American Journal of Ophthalmology 146: 172–182. Rajasekaran, S. A., Beyenbach, K. W., and Rajasekaran, A. K. (2008). Interactions of tight junctions with membrane channels and transporters. Biochimica et Biophysica Acta 1778: 757–769. Rizzolo, L. J. (2007). Development and role of tight junctions in the retinal pigment epithelium. International Review of Cytology 258: 195–234. Strauss, O. (2005). The retinal pigment epithelium in visual function. Physiological Reviews 85: 845–881. Van Itallie, C. M. and Anderson, J. M. (2006). Claudins and epithelial paracellular transport. Annual Review of Physiology 68: 403–429. Wilt, S. D. and Rizzolo, L. J. (2001). Unique aspects of the blood–brain barrier. In: Anderson, J. M. and Cereijido, M. (eds.) Tight Junctions, pp. 415–443. Boca Raton, FL: CRC Press.

Retinal Vasculopathies: Diabetic Retinopathy N C Steinle and J Ambati, University of Kentucky, Lexington, KY, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Cotton-wool spots (CWSs) – Also known as soft exudates, these may be found in nonproliferative diabetic retinopathy (NPDR). They are composed of accumulations of neuronal debris within the retinal nerve fiber layer, and result from disruption and stasis of axoplasmic flow. Diabetes control and complications trial (DCCT) – A study that contributed to our understanding that intensive glycemic control is associated with a reduced risk of newly diagnosed retinopathy and a reduced progression of existing retinopathy in people with diabetes. Diabetes mellitus (DM) – A metabolic disorder characterized by sustained hyperglycemia secondary to lack or diminished efficacy of endogenous insulin. Diabetic retinopathy (DR) – A retinal disease consequent to development of DM. Insulin-dependent diabetes mellitus (IDDM) – A term sometimes used to refer to type I diabetes. Intraretinal microvascular abnormalities (IRMAs) – The tortuous, hypercellular micro vessels that develop in NPDR. New vessels elsewhere (NVE) – The neovascularization of the retina found greater than one disk diameter from the optic nerve head. New vessels on disk (NVD) – The neovascularization on or within one disk diameter of the optic nerve head. Non-insulin-dependent diabetes mellitus (NIDDM) – An older term for type II diabetes or adultonset diabetes. Nonproliferative diabetic retinopathy (NPDR) – DR characterized by intraretinal microvascular changes which precede the proliferative phase. Optical coherence tomography (OCT) – A noninvasive imaging technique that permits analysis of retinal structure in the living eye. Proliferative diabetic retinopathy (PDR) – DR characterized by the presence of retinal neovascularization. The early treatment diabetic retinopathy study (ETDRS) – A large study of progression and treatment of DR. United Kingdom Prospective Diabetes Study (UKPDS) – A study that contributed to our

understanding that intensive glycemic control is associated with a reduced risk of newly diagnosed retinopathy and a reduced progression of existing retinopathy in people with diabetes. Vascular endothelial growth factor (VEGF) – A growth factor that promotes development of endothelial cells.

Background Diabetes mellitus (DM) is a metabolic disorder characterized by sustained hyperglycemia secondary to lack or diminished efficacy of endogenous insulin. The terminology used for classification of different types of diabetes is evolving and can be a source of confusion. Traditionally, there have been two types of DM: type I diabetes and type II diabetes (these terminologies are used throughout the remainder of this article). Immune-mediated diabetes is the latest, and perhaps most descriptive, terminology applied to type I diabetes. Autoimmune destruction of insulin-producing pancreatic islet cells is postulated as instrumental in the pathogenesis of type I diabetes. Previous terminology used for type I diabetes included insulin-dependent diabetes mellitus (IDDM), and juvenileonset diabetes. Type II diabetes is characterized by relative deficiencies of insulin and/or peripheral insulin resistance. It was previously known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes. DM is a common medical problem and is a major global source of morbidity and mortality. The incidence of DM is thought to be increasing throughout the world in part due to an increasing incidence of obesity and sedentary lifestyles. In the United States, it is estimated that 7.8% of the total population has DM, and it causes a vast array of long-term systemic complications which have a significant impact on both quality and quantity of life. Patients with DM have heart disease, death rates, and stroke rates that are 2–4 times higher than adults without diabetes, and DM is the leading cause of end-stage renal disease in the United States. Further, people with diabetes are more susceptible to many other illnesses and, once acquired, often have worse prognoses (e.g., pneumonia). From an ophthalmic standpoint, DM causes numerous complications. Chief among these complications are diabetic retinopathy (DR), unstable refractions, accelerated

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cataracts, rubeosis iridis, which can lead to neovascular glaucoma, cranial nerve palsies, reduced corneal sensitivity, papillopathy, and poor wound healing. The incidence of blindness is 25 times higher in patients with diabetes than in the general population. Furthermore, DR is the most common cause of blindness in patients aged 20–74 years, accounting for 12 000–24 000 new cases of blindness in the United States each year.

Risk Factors for DR The prevalence of DR in the diabetic population increases with the duration of diabetes and patient age. Studies have shown that after 20 years of diabetes, nearly 99% of patients with type I DM and approximately 60% of patients with type II DM have some degree of DR (Figure 1). DR rarely develops in children younger than 10 years of age, regardless of the duration of diabetes. The risk of DR increases after puberty. Approximately 5% of type II diabetics have DR at presentation; this observation is a reflection of the typically insidious onset of hyperglycemia in type II diabetes many years before the diagnosis is firmly established. In addition to duration of DM, other risk factors for the development of DR include poor glycemic control, the type of diabetes (type 1 more than type 2), and the presence or absence of associated conditions such as hypertension, smoking, dyslipidemia, nephropathy, and pregnancy. The Diabetes Control and Complications Trial (DCCT) and

Pathogenesis The pathogenesis of DR is the current subject of intense research. It is theorized that exposure to chronic hyperglycemia results in a number of biochemical and physiologic alterations that ultimately produce retinal vascular changes and subsequent retinal injury and ischemia. The list of hematologic and biochemical abnormalities theorized to play a role in the development of DR

Insulin, 30

60 40 20 0 0

12 24 Duration of diabetes

36

Figure 1 Incidence of diabetic retinopathy (DR) increases over time. Duration of DM is directly associated with an increased prevalence of DR in people with both type I and type II DM. The figure represents the percent of diabetic patients with retinopathy according to duration of disease in patients under the age of 30 years who were treated with insulin (primarily type I diabetics) and patients over the age of 30 years who were not treated with insulin (primarily type II diabetics). Retinopathy increased over time in both groups, affecting virtually all patients with type I diabetes by 20 years. The increased incidence in type II diabetes at 3 years is likely secondary to the difficulty in determining the exact time of onset of type II DM. Data from Klein, R., Klein, B. E., Moss, S. E., Davis, M. D. and DeMets, D. L. (1984). The Wisconsin Epidemiologic Study of Diabetic Retinopathy: III. Prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. Archives of Ophthalmology 102: 527.

Percentage of patients

Percent with retinopathy

100

the United Kingdom Prospective Diabetes Study (UKPDS) demonstrated that intensive glycemic control is associated with a reduced risk of newly diagnosed retinopathy and a reduced progression of existing retinopathy in people with DM (type I in DCCT (Figure 2) and type II in UKPDS). According to the DCCT, intensive insulin therapy reduced the incidence of new cases of DR by as much as 76% compared with conventional therapy. The UKPDS found similar results in type II diabetics; each 1% point reduction in glycosylated hemoglobin was associated with a 37% reduction in development of retinopathy. Further, the UKPDS showed that control of hypertension was also beneficial in reducing progression of DR. Pregnancy is occasionally associated with rapid progression of DR; thus, women with diabetes who become pregnant require more frequent evaluation of the retina. Pregnant women without any DR are at a 10% risk of developing nonproliferative diabetic retinopathy (NPDR) during their pregnancy. Of those with preexisting NPDR, 4% progress to proliferative retinopathy (Figure 3).

50 Conventional

40 30

P < 0.001

20 Intensive

10 0 0

1

2

3

4 5 6 Year of study

7

8

9

Figure 2 Intensive glycemic control slows progression of retinopathy. Cumulative incidence of progressive retinopathy in patients with type 1 diabetes and early nonproliferative retinopathy who were treated with either conventional or intensive insulin therapy for 9 years. Intensive glycemic control reduced the risk of DR progression over time by 54%, although intensive therapy was associated with transient worsening in the first year ( p < 0.001). Data from Diabetes Control and Complications Trial Research Group (1993). The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The New England Journal of Medicine 329: 977.


Rate of progression of retinopathy (per 100 patient-years)

16 14 12 10 8 6 4 2 0 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 Glycosylated hemoglobin (%) Figure 3 Progression of DR in relation to glycemic control. The figure shows the rate of progression of retinopathy in patients with type 1 diabetes according to mean glycosylated hemoglobin values (solid line). Better glycemic control was associated with slower rates of DR progression. The dashed lines represent the 95% confidence intervals. Data from Diabetes Control and Complications Trial Research Group (1993). The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The New England Journal of Medicine 329: 977.

includes the following: impairment of retinal blood vessel autoregulation, the occurrence of retinal microthrombosis and subsequent ischemia, accumulation of advanced glycosylation end products, and damage caused by reactive oxygen species. The role that growth factors (e.g., vascular endothelial growth factor (VEGF)) play in the formation of DR is discussed later. There have also been recent considerations of categorizing DR as an inflammatory disease. Trials investigating anti-inflammatory agents for prevention or treatment of DR in humans are ongoing. Specific retinal vascular changes theorized to be instrumental in DR include the loss of pericytes, basement membrane thickening, and impaired endothelial cell function. The walls of retinal capillaries consist of endothelial cells and pericytes and are devoid of smooth muscle and elastic tissue. Endothelial cells form a single layer on a basement membrane and are linked by tight junctions that form the inner blood–retinal barrier. Pericytes are found external to the endothelial cells and have pseudopodial processes that envelop the capillary. It is believed that pericytes have contractile properties and are thought to participate in autoregulation of the microvascular capillary circulation (analogous in function to the smooth muscle found in larger arteries). The classic histologic finding of early DR in the human retina is the loss of microvascular pericytes; however, the exact mechanism by which pericytes are preferentially lost early in DR is

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unknown. Thickening of the retinal capillary basement membrane is another well-known lesion found in DR. In addition to basement membrane thickening, patients with DR are also found to have vacuolization and deposition of fibrillar collagen in their basement membranes. Similar to the loss of pericytes, the exact biochemical events that lead to basement membrane alterations in DR are not fully evident. Several studies implicate the sorbitol pathway in this process. The sorbitol pathway is the name given to the sequence of reactions that convert glucose to fructose involving the enzymes aldose reductase and sorbitol dehydrogenase. In this pathway, glucose is reduced first to sorbitol, which is then oxidized to fructose. However, since the latter reaction occurs slowly in many cells, sorbitol may build to high, and possibly, toxic concentrations. The toxicity of sorbitol is theorized to perhaps lead to basement membrane alterations. The final vascular change that appears to be instrumental in DR is the loss of endothelial cell function and the subsequent breakdown of the blood–retinal barrier. One possible cause of the blood–retinal barrier breakdown is opening of the tight junctions (zonulae occludentes) between adjacent microvascular endothelial cell processes. Several proteins are known to be involved with tight junction function, namely ZO-1, occluding and claudin. Studies have shown that high glucose levels appear to inhibit ZO-1 expression. Further experiments have shown reduced expression and anatomical distribution of occludin in experimental diabetes. Finally, studies have shown that intravitreal injections of the growth factor VEGF in rats increased production of nitric oxide (NO) and increased phosphorylation of ZO-1 and occludin, changes that result in increased breakdown of the blood–retinal barrier. Advanced stages of DR are marked by the proliferation of new blood vessels. Retinal neovascularization is a devastating process that can lead to blindness in DR. Neovascularization develops through angiogenesis, in which capillaries develop from preexisting blood vessels. In DR, the regulatory mechanisms of angiogenesis can become compromised, which leads to uncontrolled endothelial cell division. On a molecular level, angiogenesis is a complicated pathway involving interplay between a number of angiogenic messengers, proteolytic enzymes, and the preexisting vessels themselves. The common final product in angiogenesis is activation of vascular endothelial cells. Several endothelial cell mitogens have been isolated and studied, including VEGF, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), protein kinase C (PKC), antiopoietins, integrins, and ephrins. VEGF is commonly considered the most potent angiogenic factor, and some of the other molecules may act indirectly through VEGF. As the activated endothelial cells proliferate, they secrete proteolytic enzymes that degrade the parent vessel’s basement

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membrane as well as the extracellular matrix. Once the endothelial cells gain access to the extravascular space, they migrate and form new capillary sprouts. Mesenchymal cells are then recruited to form smooth muscle cells in arterioles and a new basement membrane is deposited to complete the process of angiogenesis. The resulting abnormal vessels lack structural integrity and are prone to leak fluid, leading to retinal edema. Further, the abnormal vessels often are associated with a fibrovascular membrane. This membrane can become adherent to both the retina and the posterior hyaloid face. As the vitreous contracts, the fibrovascular membrane can cause tractional forces on the retina, leading to retina edema, vitreous hemorrhages, retinal heterotropia, retinal tears, and tractional retinal detachments. Fortunately, the majority of DR follows a fairly predictable course; thus, screening examinations and prophylactic interventions can be implemented to reduce the devastating and potentially blinding consequences of advanced DR.

Classifications The classification of DR is classically based on the severity of intraretinal microvascular changes and the presence or absence of retinal neovascularization. Thus, DR is divided into two main forms: nonproliferative and proliferative. NPDR is characterized by intraretinal microvascular changes which precede the proliferative phase. Proliferative diabetic retinopathy (PDR) is characterized by the presence of retinal neovascularization. These two classifications of DR (NPDR and PDR) have been useful for the analysis of treatment efficacy in the literature and serve as general indicators for treatment strategies. However, it should be noted that an individualized approach to the treatment of DR is prudent, as every patient with DR has a unique combination of findings, symptoms, and rate of progression.

Nonproliferative Diabetic Retinopathy

Figure 4 (a) Severe non-proliferative diabetic retinopathy. Color fundus photograph and a red-free fundus photograph of the left eye of a patient with severe NPDR. The photographs demonstrate numerous diffusely scattered microaneurysms, dot-blot hemorrhages, and hard exudates. (b) Microaneurysms and retinal hemorrhages. Arteriovenous phase fluorescein angiogram image of the same left eye (a). The microaneurysms demonstrate marked early hyperfluorescence, whereas the retinal hemorrhages block fluorescein and thus appear hypofluorescent.

The retinal microvascular changes found in NPDR are, by definition, limited to the confines of the retina and do not extend beyond the innermost retinal layer – the internal limiting membrane. Characteristic findings in NPDR include microaneurysms, retinal hemorrhages, retinal edema, hard exudates, cotton-wool spots (CWSs), areas of capillary nonperfusion, intraretinal microvascular abnormalities (IRMAs), and venous beading. NPDR primarily causes visual decline through either capillary nonperfusion leading to macular ischemia, or through increased vascular permeability, resulting in macular edema. The first visible sign of NPDR is the retinal capillary microaneurysm. Clinically, microaneurysms are identified as red dots from 15 to 60 mm in diameter (Figure 4).

On histologic examination, microaneurysms are hypercellular saccular outpouchings of the capillary wall. They are often found in relation to areas of capillary nonperfusion. Postulated mechanisms behind microaneurysm formation include the release of vasoproliferative factors (e.g., VEGF) with endothelial cell proliferation, weakness of the capillary wall secondary to the loss of pericytes, abnormalities of the adjacent retina, and increased intracapillary pressure. Microaneurysms can be differentiated from punctate retinal hemorrhages, which are also seen in DR, through fluorescein angiography. Microaneurysms will demonstrate marked early hyperfluorescence against the darker choroidal background; whereas, retinal hemorrhages


will block fluorescein and thus appear hypofluorescent (Figure 4(b)). Late fluorescein angiography frames often demonstrate leakage emanating from microaneurysms as a result of the breakdown in the blood–retinal barrier. Individual microaneurysms typically appear and disappear over time. Microaneurysms often foreshadow progression of DR as an increase in microaneurysms often is associated with progression of DR. The retinal hemorrhages most commonly seen in NPDR include both dot-blot hemorrhages and retinal nerve fiber layer hemorrhages. Dot-blot hemorrhages are punctate, intraretinal hemorrhages that arise from the venous end of retinal capillaries and are located within the compact middle layers of the retina. These compact, vertically aligned, middle retinal layers confer upon the retinal hemorrhages their characteristic red, dot-blot appearance (Figure 4). Retinal nerve fiber layer hemorrhages arise from the more superficially located precapillary arterioles. The horizontal alignment of the retinal nerve fiber layer gives these hemorrhages their classic flame shape. As discussed previously, another consequence of DR is excessive vascular permeability, which can result in retinal edema, usually in the macular region. Retinal edema is often accompanied by macular hard exudates, which are lipid deposits that accumulate in association with lipoprotein leakage from decompensated endothelial tight junctions. On clinical examination, hard exudates are yellowish intraretinal deposits often found at the border of edematous and nonedematous retinal tissue (Figure 4). Macular edema initially accumulates between the outer plexiform and inner nuclear layers. With chronic edema, the entire thickness of the retina becomes edematous and can assume a cystoid appearance. Retinal thickening secondary to macular edema is best detected by indirect slit-lamp biomicroscopy; in addition, optical coherence tomography (OCT), can be used to detect thickening and may be used to assess response to therapy (Figure 5). CWSs, also known as soft exudates, may often be found in NPDR. CWSs are composed of accumulations of neuronal debris within the retinal nerve fiber layer. These result from disruption and stasis of axoplasmic flow. As CWSs heal, debris is removed from the nerve fiber layer by autolysis and phagocytosis. Clinically, CWSs are seen as yellowish, fluffy superficial lesions which obscure the underlying blood vessels. Interestingly, CWSs are only found in the postequatorial retina where the nerve fiber layer is of sufficient thickness to allow visualization of the CWSs. As NPDR progresses, it can lead to the obliteration of retinal capillaries. These areas of capillary nonprofusion are seen on fluorescein angiography as patches of hypofluorescence. Adjacent to areas of nonperfusion, tortuous, hypercellular vessels often develop. It is difficult to determine whether these vessels are actually dilated preexisting capillaries or whether they represent new vessels forming within the retina. These vessels have been

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Figure 5 Macular edema secondary to diabetic retinopathy. OCT (two images) demonstrating retinal thickening and cystoid intraretinal spaces created by extensive capillary leakage and secondary macular edema in a patient with severe NPDR.

referred to as IRMAs, a term which encompasses both possibilities. The main distinguishing features of IRMAs are their intraretinal location, failure to cross major retinal blood vessels, and absence of leakage on flouroscein angiography. As areas of capillary nonperfusion become extensive, it is common to see an increase in intraretinal hemorrhages or dilated segments of retinal veins (referred to as venous beading). The degree of retinal capillary nonperfusion is directly associated with the severity of IRMAs, intraretinal hemorrhages, and venous beading. NPDR is further categorized into four levels of severity: mild, moderate, severe, and very severe (Table 1). The clinical extent of microaneurysms, retinal hemorrhages, venous beading, and IRMA determine the level of severity of nonproliferative disease. Mild and moderate NPDR are characterized by relatively few microaneurysms and intraretinal hemorrhages and only minimal venous changes or IRMA. Severe NPDR is characterized by diffuse intraretinal hemorrhages, two quadrants of venous beading, or moderate IRMA in at least one quadrant. If any two of these features are present, the retinopathy is considered to be very severe NPDR. The Early Treatment Diabetic Retinopathy Study (ETDRS) found that severe NPDR had a 15% chance of progression to high-risk PDR within 1 year. Very severe NPDR had a 45% chance of progression to high-risk PDR within 1 year.

Macular Edema Macular edema is the most common cause of visual impairment in patients with NPDR. Due to the

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Retinal Vasculopathies: Diabetic Retinopathy Classification of diabetic retinopathy

Nonproliferative diabetic retinopathy (NPDR) Mild NPDR: At least one microaneurysm Criteria not met for other levels of DR Moderate NPDR: Hemorrhage/microaneurysm standard photograph #2A or Soft exudates (cotton-wool spots), venous beading, and intraretinal microvascular abnormalities definitely present Criteria not met for severe NPDR, very severe NPDR, or PDR Severe NPDR: Hemorrhage/microaneurysm standard photograph #2A in all four quadrants or Venous beading in at least two quadrants or Intraretinal microvascular abnormalities standard photograph #8A in at least one quadrant Very servere NPDR: Any two or more of criteria for severe NPDR Criteria not met for PDR Proliferative diabetic retinopathy (PDR) Early PDR: New vessels Criteria not met for high-risk PDR High-risk PDR: Neovascularization of the disk 1/4 to 1/3 disk area or Neovascularization of the disk and vitreous or preretinal hemorrhage or Neovascularization elsewhere 1/2 disk area and vitreous or preretinal hemorrhage Advanced PDR: Posterior fundus obscured by preretinal or vitreous hemorrhage or Center of macula detached

breakdown of the blood–retinal barrier, leakage of fluid and plasma constituents leads to retinal edema (Figure 6). If the retinal edema threatens the center of the fovea, there is a higher risk of visual loss. In the ETDRS, the 3-year risk of moderate visual loss was 32% (moderate visual loss was defined as a doubling of the initial visual angle or a decrease of three lines or more on a logarithmic visual acuity chart). The ETDRS investigators classified macular edema by its severity. More specifically, macular edema was defined as clinically significant macular edema (CSME) if any of the following features were present: (1) thickening of the retina at or within 500 mm of the center of the macula; (2) hard exudates at or within 500 mm of the center of the macula, if associated with thickening of the adjacent retina; or (3) a zone of thickening larger than one disk area if located within one disk diameter of the center of the macula (Table 2). Many of the current treatment paradigms for the management of diabetic

macular edema are derived from the ETDRS. The ETDRS demonstrated that eyes with CSME benefited from focal argon laser photocoagulation treatment when compared to untreated eyes in a control group. Furthermore, focal argon laser photocoagulation treatment for CSME reduced the risk of moderate visual loss, increased the chance of visual improvement, and was associated with only minor losses of visual field. Specifically, in patients with CSME involving the center of the macula, focal treatment reduced moderate visual loss by 60% after 3 years of follow-up. In patients with less than CSME, little difference was noted between the untreated and treated groups during the first 2 years of follow-up, after which there was a trend toward less frequent visual loss in the treated group. Treatment patterns regarding CSME continue to evolve. In patients with refractory CSME, intravitreal administrations of corticosteroids have been shown to be beneficial. Intravitreal anti-VEGF agents have also been shown to improve CSME. Currently, several trials investigating corticosteroid use as well as anti-VEGF agents in the treatment of CSME are underway. Pars plana vitrectomy and detachment of the posterior hyaloid may also be useful for treating CSME. Surgical intervention may prove particularly beneficial when there is evidence of posterior hyaloidal traction and diffuse macular edema.

Proliferative Diabetic Retinopathy As the degree of retinal ischemia increases, extensive hypofluorescent areas representing retinal nonperfusion are seen on fluorescein angiography. Eventually neovascularization may develop in an attempt to revascularize hypoxic retinal tissue. This neovascularization is the hallmark of PDR. It has been estimated that over one-quarter of the retina has to be nonperfused before PDR develops. PDR affects 5–10% of the diabetic population (Figure 7). Type I diabetics are at particular risk for PDR with an incidence of about 60% after 30 years. Although new vessels may arise from anywhere in the retina, neovascularization is particularly common on the optic disk itself (this location is termed new vessels on disk, or NVD). NVD is defined as neovascularization on or within one disk diameter of the optic nerve head. It can be differentiated from normal vessels by utilizing fluorescein angiography, which demonstrates profuse leakage in NVD but not in normal vasculature (Figure 8). Neovascularization of the retina found greater than one disk diameter from the optic nerve head is termed new vessels elsewhere, or NVE. NVE typically is found along the course of the major retinal vessels. The rate of growth of NVD or NVE is extremely variable. In some patients, neovascularization may show little change over many months, while in others definite changes in neovascularization may be seen in as little as 1–2 weeks. As neovascularization


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Stratus oct Retinal thickness analysis report-4.0.1(0056) Scan type: Fast macular thickness map Scan date: Scan length: 6.0 mm

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Figure 6 OCT demonstrating CSME. OCT of a diabetic patient with CSME. The patient has marked thickening of the central macula secondary to CSME. The OCT retinal cross section reveals cystic intraretinal and subretinal changes. Table 2 ETDRS

Clinically significant macular edema as defined by

Thickening of the retina 500mm from the center of the macula or Hard exudates and adjacent retinal thickening 500 mm from macular center or Zone of retinal thickening at least 1 disk area in size located 25% of cone OFF bipolar cells may see direct contacts from rods, the upper end suggesting that this pathway may play an integral role in visual processing. Despite attempts by various groups to characterize the physiological response properties of the rod pathways, the exact sensitivities and operating ranges of each pathway remain unclear. The lack of physiological evidence elucidating the role of these pathways in rod vision arises from common signaling mechanisms used by each. Thus, genetic or pharmacological manipulation of each rod-signaling pathway will also influence other pathways, making it impossible to unambiguously identify the properties of each. Nevertheless, the evidence now points to the rod bipolar pathway as the primary carrier of singlephoton responses near absolute visual threshold, with an 5–10-fold reduction in the sensitivity of the rod–cone and rod–OFF pathways that may carry signals at higher light levels where cone function is merged. Cone Pathways Our understanding of the cone pathways is far less complete than our understanding of rod pathways in the mammalian retina. The physiological properties of the cones and cone pathways remain as one of the frontiers in retinal neurobiology, especially as cone function dominates our visual experience. To date, as many as nine types of cone bipolar cells have been identified in mammalian retinas, which may connect to as many as 10–15 types of retinal ganglion cells. Other than the specific pathway for S-cones, there is presently little understanding of specific circuits that carry cone responses to ganglion cells.

Adaptation to Mean Background Light A common feature of all sensory systems is the ability to adapt to increases in the mean level of a stimulus by reducing the gain of the system. Such sensitivity adjustments allow the sensory system to remain maximally responsive as the stimulus intensity changes. Both rods and cones in the retina, as well as their circuitry,

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exhibit adaptive mechanisms that are designed to increase the dynamic range of the receptor. However, in the context of the dynamic range of the visual system, the influences of adaptation on the lower limit of scotopic and upper limit of photopic vision are opposite. To retain maximal sensitivity near absolute visual threshold, the retina must maximize its gain for the single-photon response and any adaptive mechanism would allow the rod circuitry to aid in the transition from scotopic to mesopic vision. Conversely, the upper light limits of our visual experience are ultimately defined by adaptation in the cone photoreceptors, which continue to operate even when a majority of the photopigment is bleached. Adaptation: Rod Pathways Adaptation within a neural circuit with considerable convergence will begin centrally and move peripherally. Under these circumstances, downstream cells will be the first to detect sufficient signal to adapt for the weakest stimuli, and the rod pathways are no exception. In the rod bipolar pathway, weak background light begins to reduce the gain of ganglion cells and AII amacrine cells before adaptation is detectable in rod bipolar cells or rod photoreceptors. Some mechanisms that provide this gain reduction have been identified, particularly at the rod-to-rod bipolar, and the rod bipolar-to-AII amacrine synapses. For instance, at the rod-to-rod bipolar synapse, the influx of Ca2þ through mGluR6 transduction channels reduces the bipolar cell gain for subsequent stimulation. In addition, at the rod bipolar-to-AII amacrine synapse depression mediated by depletion of the available pool of vesicles can be evoked at individual synapses by single-photon responses. These adaptive mechanisms allow the rod bipolar pathway to extend its range to higher light levels where they merge with the other rod pathways. However, the extension of the dynamic range of vision to lower light levels requires that the retina remains maximally responsive to single photons, and thus these adaptive mechanisms would impair absolute threshold. Adaptation: Cone Pathways It has been well documented that adaptation of ganglion cell responses in the cone pathways occurs at lower light levels than where adaptive features of cone pathways have been documented. Cone and horizontal cell recordings have demonstrated that cone adaptation is observed at light levels that exceed those required for the adaptation of cone-driven signals in ganglion cells. This adaptation of the postsynaptic cone circuitry, in turn, prevents these pathways from saturating, thereby allowing the extension of the cone operating range through mesopic to photopic vision. Adaptation, both in the cones and in the postreceptoral circuitry, have been found to be mutually

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exclusive. Ultimately, the upper limits of cone vision are directly imposed by receptoral adaptation of the cones themselves and not by the postsynaptic circuitry.

Conclusions The vertebrate retina has developed many strategies to maximize the dynamic range of vision. At the initial stages of light detection the evolution of two photoreceptor types, the rods and cones, allows the visual system to signal a wide range of light intensities. By dividing the output of these receptors across many retinal pathways, each of which is subject to its own optimization and adaptation, the human eye is capable of providing the brain with information that extends the range of vision to encompass approximately 12 orders of magnitude of light intensity. See also: Anatomically Separate Rod and Cone Signaling Pathways; Information Processing: Bipolar Cells; Information Processing: Ganglion Cells; Morphology of Interneurons: Bipolar Cells; Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Rod Photoreceptor Cells: Soma and Synapse.

Further Reading Barlow, H. B. (1956). Retinal noise and absolute threshold. Journal of the Optical Society of America 46: 634–639.

Barlow, H. B., Levick, W. R., and Yoon, M. (1971). Responses to single quanta of light in the retinal ganglion cells of the cat. Vision Research Supplement 3: 87–101. Baylor, D. A., Lamb, T. D., and Yau, K. W. (1979). Responses of retinal rods to single photons. Journal of Physiology 288: 613–634. Dacheux, R. F. and Raviola, E. (1986). The rod pathway in the rabbit retina: A depolarizing bipolar and amacrine cell. Journal of Neuroscience 6: 331–345. Dunn, F. A. and Rieke, F. (2008). Single photon absorptions evoke synaptic depression in the retina to extend the operational range of rod vision. Neuron 57: 894–904. Dunn, F. A., Lankheet, M. J., and Rieke, F. (2007). Light adaptation in cone vision involves switching between receptor and post-receptor sites. Nature 449: 603–606. Field, G. D. and Rieke, F. (2002). Nonlinear signal transfer from mouse rods to bipolar cells and implications for visual sensitivity. Neuron 34: 773–785. Hecht, S., Schlaer, S., and Pirenne, M. H. (1942). Energy, quanta, and vision. Journal of General Physiology 25: 819–840. Hornstein, E. P., Verweij, J., Li, P. H., and Schnapf, J. L. (2005). Gap-junctional coupling and absolute sensitivity of photoreceptors in macaque retina. Journal of Neuroscience 25: 11201–11209. Okawa, H. and Sampath, A. P. (2007). Optimization of single-photon response transmission at the rod-to-rod bipolar synapse. Physiology 22: 279–286. Sampath, A. P. and Rieke, F. (2004). Selective transmission of single photon responses by saturation at the rod-to-rod bipolar synapse. Neuron 41: 431–443. Singer, J. H., Lassova, L., Vardi, N., and Diamond, J. S. (2004). Coordinated multivesicular release at a mammalian rod synapse. Nature Neuroscience 7: 826–833. Smith, R. G., Freed, M. A., and Sterling, P. (1986). Microcircuitry of the dark-adapted cat retina: Functional architecture of the rod–cone network. Journal of Neuroscience 6: 3505–3517. Sterling, P., Freed, M. A., and Smith, R. G. (1988). Architecture of rod and cone circuits to the on-beta ganglion cell. Journal of Neuroscience 8: 623–642. van Rossum, M. C. and Smith, R. G. (1998). Noise removal at the rod synapse of mammalian retina. Visual Neuroscience 15: 809–821.

Xenopus laevis as a Model for Understanding Retinal Diseases O L Moritz and D C Lee, University of British Columbia, Vancouver, BC, Canada ã 2010 Elsevier Ltd. All rights reserved.

Glossary AP20187 – A small molecule modeled on a dimer of the immunosuppressive drug FK506. One molecule of AP20187 can bind with high affinity to two FK506binding protein (Fv) domains. Thus, Fv domains can be used to produce fusion proteins that will dimerize in the presence of AP20187. This in turn can be used to control the activity of proteins or enzymes whose activities are influenced by dimerization, such as caspases. Bardet–Biedl syndrome – An autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, hypogonadism, developmental delay, and mental retardation. Genes implicated in this syndrome are involved in ciliary transport processes. Caspase-9 – An initiator caspase in the apoptotic cascade. Caspases are a family of cysteine proteases, which play essential roles in programmed cell death. Once activated, caspase-9 triggers activation of other caspases, precipitating the apoptotic process. Optical coherence tomography (OCT) – A technique that permits three-dimensional imaging within tissues that scatter light. The technique is frequently used in ophthalmology to noninvasively image the cell layers of the retina. Peripherin/RDS – A transmembrane glycoprotein found in the outer segment of both rod and cone photoreceptor cells. It is thought to be a structural protein important for disk morphogenesis. Mutations in the gene encoding peripherin/RDS are associated with a variety of autosomal dominant retinal dystrophies; also referred to in publications as rds, peripherin, or peripherin-2. Rab proteins and Arf4 – The members of the Ras superfamily of monomeric guanine-nucleotide-binding proteins (G proteins), which are involved in the regulation of membrane trafficking. Retinal degeneration – A phenotype associated with many different retinal disorders, involving progressive death of retinal cells, usually of a specific cell type. Retinitis pigmentosa – A hereditary retinal dystrophy characterized by defective dark adaptation, progressive loss of peripheral vision that may eventually extend to loss of central vision, and the appearance of black pigment in the fundus.

RNA helicase Ddx39 – A member of the DEAD box protein family of putative RNA helicases. Family members are characterized by the conserved motif Asp-Glu-Ala-Asp (D-E-A-D), and are involved in RNA metabolism. Stargardt’s disease – An autosomal recessive juvenile-onset form of macular dystrophy arising from mutations in the ABCA4 gene. The ABCA4 gene product is expressed in photoreceptor cells and is thought to be an ATP-dependent transporter for N-retinylidene-PE.

Introduction The amphibian retina has many unique properties that have intrigued visual scientists for decades. Many studies have been conducted on the retina of Xenopus laevis, a frog species commonly used as a laboratory animal. These frogs were introduced to the research community in the 1930s and soon became widely available, after the discovery that they can be induced to lay eggs by injection of human chorionic gonadotropin (known as the Hogben test for pregnancy). As a research subject, X. laevis have advantages over other amphibians in that they have low maintenance requirements (they are entirely aquatic and do not require live food), and they can easily be induced to lay eggs. This made them a desirable model organism for developmental biologists studying fertilization and embryonic development; however, they have also been adopted by vision researchers as a model amphibian retina. The large size of the principle rod photoreceptors makes them highly amenable to biochemical, morphological, and electrophysiological studies (Figure 1). Early studies on the properties of the X. laevis visual system laid the groundwork for the use of this animal as a model organism for the study of visual disorders, while more recently developed techniques for genetic manipulation have resulted in X. laevis models of inherited retinal disease that are particularly amenable to certain forms of analysis.

Early Work on X. laevis – Biochemistry, Electrophysiology, and Microscopy Modern research on the amphibian retina dates back to the extraordinary anatomical studies of Cajal and

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Figure 1 Expression of a bovine rhodopsin P23H mutant transgene causes retinal degeneration in Xenopus laevis. Combined confocal/DIC (upper panels) or confocal micrographs (lower panels) of developmental stage 47/48 wild-type retinas (left panels) or transgenic retinas expressing bovine-rhoP23H (right panels). Animals were raised in bright cyclic light for 2 weeks, beginning at fertilization. The 12-mm retinal cyrosections were stained with wheat germ agglutinin (red), B630N anti-rhodopsin (green), and Hoechst 33342 nuclear stain (blue). The central retinas of animals expressing bovine P23H rhodopsin (right panels) have almost no remaining rods, with those that remain having short and irregular outer segments. Note the relatively large diameter (7 mm) of the rod photoreceptors relative to those of the mammalian retina. Due to the relatively large diameter of the photoreceptors, only a single row of nuclei is required in the ONL. RPE, retinal pigment epithelium; OS, outer segment; INL, inner nuclear layer; ONL, outer nuclear layer. Scale = 10 mm.

others in the 1800s that defined many of the principal cell types of the retina. Most early studies dealt with Rana and Bufo species. However, X. laevis have been used as a subject of retinal research dating back more than 50 years. In the 1950s, Wald and co-workers used X. laevis for investigations of visual pigment biochemistry. From the most abundant rod photoreceptors, it was found that the principal pigment exhibited maximal absorbance at 523 nm. The amphibian retina is also of interest to electrophysiologists. Although the first electroretinogram (ERG) was recorded by Holmgren in the 1860s from a frog eye, X. laevis frogs were not used extensively for electrophysiological studies until the 1970s, with initial studies of X. laevis retinal physiology performed by Ripps and coworkers, correlating visual pigment content and photoreceptor threshold.

In the 1950s and 1960s, detailed analysis of the ultrastructure of photoreceptors was obtained by electron microscopy, leading to the current understanding of photoreceptor disk membrane structure, and the mechanisms of disk membrane synthesis and renewal. Many of these studies utilized amphibian retina, again typically Rana species, although Lanzavecchia examined the ultrastructure of X. laevis rods and cones in 1960. Further studies by Kinney and Fisher further characterized the morphogenesis and ultrastucture of the principal rod photoreceptor in X. laevis. In the 1940s and 1950s, work by Sperry and co-workers demonstrated regeneration of the optic nerve after transection in various amphibians. Beginning in the late 1960s, Jacobson and co-workers conducted investigations of retinal development in X. laevis, with particular emphasis on the development of retinotectal projections. These studies employed embryonic surgery (e.g., inversion of the eye) to identify the origin of signals for optic nerve axon guidance, and demonstrated that the location of synapses of ganglion cell axons in the optic tectum are specified by the location (dorsal, ventral, nasal, or temporal) of the cell bodies in the retina. These early investigations established the X. laevis retina as a viable subject for retinal research that is still in use, including ongoing studies of X. laevis disk shedding and renewal, electrophysiology, biochemistry, and retinal development, that are further improving our understanding of retinal function. Additionally, several studies discussed below have directly modeled human retinal disorders in X. laevis, in order to better understand retinal dysfunction.

X. laevis as a Model for Vitamin A Deprivation One of the first instances of modeling retinal disease in X. laevis was a study by Witkovsky and co-workers on vitamin A deprivation in tadpoles. This model reproduced features of night blindness (i.e., decreased rod sensitivity) seen in vitamin-A-deprived patients. Among other novel findings, the authors demonstrated that bleaching of photopigment causes a greater reduction in photoreceptor sensitivity than can be accounted for by reduction in pigment quantity alone. This was accounted for in later studies, which demonstrated that opsin has a greater tendency to activate the visual transduction cascade than rhodopsin. More recently, studies on vitamin A deprivation in X. laevis have been continued by Solessio and co-workers, who have also pioneered the use of psychophysical measurements of X. laevis visual sensitivity.

X. laevis as a Model for Glaucoma Early studies by Jacobson and Keating involving optic nerve transection in X. laevis are similar to paradigms


currently used in research of optic nerve injury or glaucoma, although more typically the research subject is a mammal. However, unlike a mammalian optic nerve, a severed X. laevis optic nerve regenerates (a property that would be highly desirable in glaucoma patients). Research focused on X. laevis models of retinal regeneration of this type is currently being continued by Belecky-Adams and co-workers, who recently identified a possible role of the RNA helicase Ddx39 in the regulation of stem cells in the retina.

X. laevis and Studies of the Transport of Rhodopsin The first investigations of the mechanism of outer segment renewal utilized amphibians injected with radioactive amino acids in a pulse–chase paradigm that allowed newly synthesized membranes to be visualized by autoradiography. The original experiments by Young and coworkers demonstrated that new disks were formed at the base of the rod outer segment, and subsequently became discontinuous with the outer segment plasma membrane. These studies were further extended by Besharse and Hollyfield, who examined the influence of light on disk synthesis in X. laevis photoreceptors, demonstrating diurnal regulation of disk membrane synthesis. Collectively, these studies demonstrated the extremely high rate of rod photoreceptor disk membrane synthesis in X. laevis retina, estimated at roughly 50-fold higher than in mammalian photoreceptors. These findings suggested that, due to the enormous rate of outer segment membrane synthesis, the amphibian retina would be an excellent choice for studying the biosynthesis of rhodopsin. This work was pioneered by Papermaster and Dreyer, and subsequently continued by Papermaster and co-workers. These studies largely used Rana and Xenopus species, and resulted in identification of molecular and ultrastructural details of the rhodopsin transport pathway. Components of the biosynthetic machinery and transport mechanisms first identified in amphibia included associated features of the connecting cilium (the pericilliary ridge complex), and vesicles transporting rhodopsin (RTCs or rhodopsin transport carriers). Deretic was able to reconstitute rhodopsin transport in amphibian retinal extracts, and using this assay demonstrated that the rhodopsin transport signal was located in the cytoplasmic C-terminal domain. This in vitro assay and associated methodologies subsequently led to identification of a number of molecules associated with rhodopsin transport, including the small G proteins, rab6, rab8, rab11, and arf4. However, due to the lack of an effective system for culturing photoreceptors, it was not possible to incorporate molecular biology approaches into the study of

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amphibian rhodopsin transport pathways until the 1990s, when techniques for the production of transgenic X. laevis developed by Kroll and Amaya, and cloning of promoters suitable for driving expression in X. laevis rods, allowed the study of rhodopsin transport in a genetically manipulated amphibian retina. Tam and colleagues found that rhodopsin-green fluorescent protein (GFP) fusion proteins expressed in X. laevis retina were transported correctly to rod outer segments. This allowed further identification and in-vivo demonstration of the function of the outer segment localization signal QVAPA, located at the extreme C-terminus of rhodopsin. Several mutations affecting this region cause retinitis pigmentosa (RP). These studies also demonstrated the extraordinary utility of transgenic X. laevis for conducting comparisons between transgenic animals, as numerous primary transgenic animals carrying different transgenes can be generated in a relatively short amount of time. For example, the rhodopsin outer segment localization signal was identified and refined using 15 distinct transgene constructs. Using the same transgenic X. laevis system, the function of rab8 in rhodopsin transport was explored using dominant-negative and constitutively active mutants. Expression of these mutant rab8-GFP fusion proteins in X. laevis rods generated the first X. laevis models with an inherited retinal degeneration (RD) phenotype, as these fusion proteins proved to be quite toxic to retinal rods, and the phenotype was passed to F1 offspring. The expression of dominant-negative GFP-rab8T22N caused a particularly rapid death of photoreceptors associated with accumulation of rhodopsin-containing intracellular vesicles in the vicinity of the base of the connecting cilium. Although it was not clear at the time, subsequent studies indicate that the resulting phenotype may be closely related to the RD associated with Bardet–Biedel syndrome (BBS). Similar investigations of the small G-protein Arf4, which binds the rhodopsin outer segment localization signal, are ongoing. The RD observed in this study demonstrated a unique central-to-peripheral distribution subsequently seen in all other X. laevis models of RD. This is associated with the rapid growth of the eye in young X. laevis, which results in continuous addition of new photoreceptors to the peripheral retina. Thus, a single cryosection can demonstrate all stages of photoreceptor degeneration moving from central retina to periphery.

Modeling RP in Transgenic X. laevis Subsequently, genetically modified transgenic X. laevis was used in a number of studies that directly examined mutations associated with the human disorder RP, an inherited form of RD. The pioneering study involved

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transgenic expression of mutant forms of peripherin/rds, a protein found at the periphery of rod outer segment disks. Peripherin/rds and its mutants were expressed as GFP fusion proteins using the rod opsin promoter to drive expression in retinal rods, at levels sufficiently high to cause RD in some cases. Confocal microscopy of the intrinsic GFP fluorescence showed that several fusion proteins had unique localization patterns distinct from wild type that respectively suggested either specific disruptions in normal function, or misfolding and endoplasmic reticulum (ER) retention. Furthermore, electron microscopy revealed unique abnormalities in disk organization, possibly associated with disruption of the normal functions of the peripherin/rds C-terminus. Previous attempts to use rhodopsin-GFP fusion proteins to develop similar models of RP were unsuccessful, most likely due to low expression levels. In order to adapt the system for the study of RD induced by rhodopsin mutants, a system was devised for detection of nonfluorescent transgene products based on nonconserved rhodopsin antibody epitopes, such that epitope tags involving minimal (or no) sequence changes could be introduced. Initially, this system was applied to the study of an RP-causing mutation (Q348ter) that disrupts the previously identified rhodopsin outer segment localization signal. In these studies, the power of the X. laevis system for drawing comparisons between transgenes was further expanded. Rhodopsin mutants defective in signal transduction properties were combined with rhodopsin mutants responsible for RP to dissect the role of rhodopsin signal transduction in rod cell death pathways. The results demonstrated rhodopsin mislocalization was associated with axonal sprouting and cell death, regardless of whether rhodopsin signal transduction properties were inhibited. The same system was subsequently applied to the study of the rhodopsin mutation P23H, the most common cause of autosomal dominant RP in North America. Despite numerous studies of this rhodopsin mutant in cultured cells and transgenic rodents, there was no clear consensus as to the effects of this mutation on rhodopsin function; in cultured cells, it was classified as a mutant defective in folding and ER exit, while transgenic animal studies suggested it was transported correctly to rod outer segments, where it caused RD that was exacerbated by light. Studies in transgenic X. laevis compared several different forms of P23H rhodopsin, including P23H rhodopsins based on different species, and P23H rhodopsins defective in signal transduction and chromophore binding. Interestingly, all forms of P23H rhodopsin caused RD, but varied in terms of ER retention, expression level, and light sensitivity. P23H rhodopsins that exhibited dramatic ER retention (X. laevis P23H rhodopsin) caused RD under all circumstances, while P23H rhodopsins that were

transported in small quantities to the OS (bovine P23H rhodopsin) caused RD only on light exposure (Figure 1). Furthermore, for bovine P23H rhodopsin, disruption of the chromophore-binding site was associated with reduced expression levels and RD, regardless of light exposure. This result reconciles the differences seen between previous studies, suggesting that in some forms of P23Hinduced RD, chromophore binding promotes ER exit of newly synthesized rhodopsin. The dramatic sensitivity of these phenotypes to the underlying rhodopsin sequence was confirmed by Zhang and colleagues, who demonstrated light-sensitive RD in an X. laevis rhodopsin that differed from that used previously only in the sequences of the epitope tags. In addition to providing insight into the mechanisms underlying RD, these studies dramatically emphasize the difficulties in extrapolating results reported from a single disease model to human disease states. A unique finding in this system was the presence of considerable quantities of truncated P23H rhodopsin, in which a significant portion of the N-terminal domain (including the mutated H23 residue) was removed; in fact, this was the dominant species observed in retinas expressing bovine P23H rhodopsin. This truncated species was also previously observed in cultured cells, although in smaller quantities. Identification of this species in other transgenic models would be difficult due to lack of a suitable reagent for detection, but was readily achieved in X. laevis due to the availability of both N- and C-terminal specific antibodies that did not cross-react with endogenous rhodopsin. Subsequent studies of the same X. laevis models of P23H-rhodopsin-induced RD probed the association of chromophore binding and ER exit. In order to address the question of whether the causative factor in light-induced RD was a reduction in the supply of free 11-cis retinal, or isomerization of 11-cis retinal bound to P23H rhodopsin as chromophore, the sensitivity of RD to different wavelengths of light was examined. It was determined that the profile of light sensitivity was consistent with photoisomerization of rhodopsin (which maximally absorbs green light) rather than free chromophore (with maximal absorbance in the UV). This also brings to mind similar studies of the constitutively active rhodopsin mutant K296E, classified as misfolding by some studies in cultured cells, suggesting that the active conformation of rhodopsin and/or loss of chromophore can be associated with altered kinetics of ER exit. Studies of additional RP-causing rhodopsin mutations in transgenic X. laevis are ongoing, and have been reported at international meetings, including K296E rhodopsin and the glycosylation-defective mutants T4K and T17M. Glycosylation-defective rhodopsin mutants are also reported to be associated with light-exacerbated RD in X. laevis.


Inducible RD In an alternate approach to modeling RP in the transgenic X. laevis retina, Hamm and co-workers designed a druginducible model of RD driven by a modified form of caspase-9. Dimerization and subsequent activation of this caspase-9 transgene is driven by AP20187, a small molecule based on a dimer of FK506. Administration of AP20187 to these transgenic X. laevis induces rapid RD that is not dependent on any particular environmental condition (such as special lighting). The system is designed to examine the effects of rod degeneration on other cell types, including cone photoreceptors and cells of the inner nuclear layer, and to examine the capacity for regeneration of rods in the X. laevis retina. This study was also the first to provide functional (i.e., electrophysiological) data for an X. laevis model of RD. Interestingly, this model demonstrated a dramatic reduction in electrophysiological responses to stimuli designed to isolate cone function (e.g., a rapid flicker stimulus), despite the fact that no associated cone death was detected. This reduction in cone sensitivity may be associated with the cones themselves, or with other cells associated with the cone pathway and the ERG B-wave (e.g., cone bipolar cells and/or Mu¨ller glia). The restoration of responses to flicker stimuli was associated with a thickening of the inner nuclear layer, and a similar thickening can be observed by optical coherence tomography (OCT) in human RP patients.

Other Transgenic X. laevis Models of Retinal Disease In addition to rhodopsin and peripherin/rds mutations responsible for RP, gene products related to Stargardt’s disease have been expressed in X. laevis retina as GFP fusion proteins in order to examine their localization properties, although this has not yet resulted in a replication of a RD phenotype. In studies by Kefalov and colleagues, cone opsins were expressed in X. laevis rods. As cone photoreceptors are considerably noisier than rod photoreceptors, this allowed a determination of the proportion of cone dark noise (activation of transduction in the absence of photons) that is purely due to the cone pigment sequence, and not other aspects of the transduction cascade or photoreceptor environment. Although generating a model of disease was not a goal of this study, the resulting animals could be considered a model for congenital stationary night blindness, which is due to abnormally high activity of the visual transduction cascade in the absence of photons.

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X. laevis as a Model for Eye Development/ Developmental Disorders The rapid development of the X. laevis embryos makes these animals of particular interest to developmental biologists, including those concerned with eye development. The first studies of the development of the X. laevis eye were conducted by Hollyfield through radioactive monitoring of the growth of the developing retina. Chung and colleagues histologically monitored the structural changes in the developing larval X. laevis retina and correlated these changes with electrophysiological changes, notably that while the receptive field of a ganglion cell remains constant in the developing larvae through metamorphosis, the inhibitory peripheral region expands to the entire retina of the adult X. laevis. The development of the X. laevis eye has been manipulated by both the overexpression and by the knockdown of transcription factors. El-Hodiri and colleagues have extensively studied the transcriptional regulation of photoreceptor development. More recently, they identified a retinal homeobox gene family member, Rx-L, which regulates photoreceptor-specific gene expression. Expressed in developing embryos, the knockdown of Rx-L expression adversely affected photoreceptor development, causing subtle phenotypes of altered photoreceptor morphology. Using a similar embryonic transfection paradigm, Knox and colleagues found that overexpression of the transcription factors, Nrl and Nr2e3, in X. laevis retina resulted in an increase in numbers of rods, with concomitant reduction in cone photoreceptors, indicative of the roles of these factors in determining the developing photoreceptor cell fate. This system may prove extremely useful in modeling developmental disorders of the retina with similar underlying mechanisms.

X. laevis Models of Retinal Regeneration Some recent studies have investigated the fascinating capacity of the X. laevis retina to repair itself after severe traumatic injury. In these studies, the entire retina is excised from an X. laevis tadpole eye. The retina subsequently demonstrates a dramatic capacity to completely regenerate by transdifferentiation of the remaining cells of the retinal pigment epithelium (RPE). Certain aspects of this transdifferentiation can be reproduced in culture, and it appears to be dependent on diffusible factors (possibly fibroblast growth factor 2 (FGF2)) released from the choroid. These results could have implications for traumatic eye injuries such as retinal detachment, retinal degenerative disorders, and glaucoma.

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Summary As an unconventional system for modeling retinal disease, X. laevis presents a number of advantages. As it is quite easy to generate transgenic X. laevis, they are an excellent system for comparing the effects of multiple transgenes. Other advantages include the relative ease of microscopic and electrophysiological studies due to the large size of the photoreceptor cells, regenerative capacity of the retina, and non-cross-reactivity of mammalian antibodies. However, there are also significant disadvantages, such as the current lack of knock-out or gene-replacement capabilities, long generation time (1 year), pseudotetraploid genome, and relatively small eyes, such that it is clearly not an ideal system appropriate for all experiments. Rather, X. laevis models of retinal disease are a very useful addition to the library of systems and models available to vision researchers. See also: The Photoreceptor Outer Segment as a Sensory Cilium; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Retinal Degeneration through the Eye of the Fly; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration; Secondary Photoreceptor Degenerations; Zebra Fish as a Model for Understanding Retinal Diseases; Zebra Fish–Retinal Development and Regeneration.

Further Reading Araki, M. (2007). Regeneration of the amphibian retina: Role of tissue interaction and related signaling molecules on RPE transdifferentiation. Development, Growth and Differentiation 49: 109–120.

Besharse, J. C., Hollyfield, J. G., and Rayborn, M. E. (1977). Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light. Journal of Cell Biology 75: 507–527. Deretic, D., Williams, A. H., Ransom, N., et al. (2005). Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4). Proceedings of the National Academy of Sciences of the United States of America 102: 3301–3306. Hamm, L. M., Tam, B. M., and Moritz, O. L. (2009). Controlled rod cell ablation in transgenic Xenopus laevis. Investigative Ophthalmology and Visual Science 50(2): 885–892. Hollyfield, J. G. (1971). Differential growth of the neural retina in Xenopus laevis larvae. Developmental Biology 24: 264–286. Pan, Y., Nekkalapudi, S., Kelly, L. E., and El-Hodiri, H. M. (2006). The Rx-like homeobox gene (Rx-L) is necessary for normal photoreceptor development. Investigative Ophthalmology and Visual Science 47: 4245–4253. Papermaster, D. S., Schneider, B. G., Zorn, M. A., and Kraehenbuhl, J. P. (1978). Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas. Journal of Cell Biology 77: 196–210. Sperry, R. W. (1944). Optic nerve regeneration with return of vision in Anurans. Journal of Neurophysiology 7: 57–69. Tam, B. M. and Moritz, O. L. (2007). Dark rearing rescues P23H rhodopsin-induced retinal degeneration in a transgenic Xenopus laevis model of retinitis pigmentosa: A chromophore-dependent mechanism characterized by production of N-terminally truncated mutant rhodopsin. Journal of Neuroscience 27: 9043–9053. Tam, B. M., Moritz, O. L., Hurd, L. B., and Papermaster, D. S. (2000). Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis. Journal of Cell Biology 151: 1369–1380. Tam, B. M., Xie, G., Oprian, D. D., and Moritz, O. L. (2006). Mislocalized rhodopsin does not require activation to cause retinal degeneration and neurite outgrowth in Xenopus laevis. Journal of Neuroscience 26: 203–209. Witkovsky, P., Gallin, E., Hollyfield, J. G., Ripps, H., and Bridges, C. D. (1976). Photoreceptor thresholds and visual pigment levels in normal and vitamin A-deprived Xenopus tadpoles. Journal of Neurophysiology 39: 1272–1287. Young, R. W. and Droz, B. (1968). The renewal of protein in retinal rods and cones. Journal of Cell Biology 39: 169–184. Zhang, R., Oglesby, E., and Marsh-Armstrong, N. (2008). Xenopus laevis P23H rhodopsin transgene causes rod photoreceptor degeneration that is more severe in the ventral retina and is modulated by light. Experimental Eye Research 86: 612–621.

Zebra Fish as a Model for Understanding Retinal Diseases A A Lewis, C C Heikaus, and S E Brockerhoff, University of Washington, Seattle, WA, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary Achromatopsia – A disease characterized by defects in the cone photoreceptors resulting in extreme light sensitivity and color blindness or rod monochromacy. Apoptosis or programmed cell death – A form of cell death characterized by a series of biochemical and morphological changes resulting in the formation of apoptotic bodies and removal by the immune system. Bystander effect – This describes the transmission of death from mutant or injured cells to healthy neighboring cells. Cyclic guanosine monophosphate (cGMP) – A cyclic nucleotide derived from guanosine triphosphate (GTP). cGMP acts as a regulator of the cyclic-nucleotide-gated ion channels in photoreceptors. Cyclic nucleotide phosphodiesterases (Pde) – A family of enzymes that hydrolyze the phosphodiester bond in the second-messenger molecules cAMP and cGMP. They regulate the localization, duration, and amplitude of cyclic nucleotide signaling. Electroretinography (ERG) – A method for evaluating visual response. Electrodes are placed against the cornea and used to measure the electrical responses of various cell types in the retina to a light flash of varying intensity. GAF domains – A large group of protein domains that bind small molecules; in the Pde proteins these domains bind cyclic nucleotides. The GAF acronym comes from the names of the first three different classes of proteins identified to contain them: cGMP-specific and-regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA. Gap junctions – The intercellular connections that occur between some types of cells. These junctions allow the movement of various molecules and ions between cells. Optokinetic response (OKR) – A method for evaluating zebrafish vision. Fish are placed in a small dish in the center of a rotating drum decorated with vertical stripes. If the fish can see, its eyes will follow the rotating stripes with regular reflexive saccades.

Retinitus pigmentosa (RP) – A group of diseases characterized by defects in the rod photoreceptors resulting in night blindness. Progressive RP often results in cone loss and tunnel vision in some cases progressing to total blindness. Scotopic vision – The low light vision that is produced exclusively by rod function. Zebrafish or Danio rerio – A tropical freshwater fish of the minnow family that has gained prominence as a scientific animal model. For further information see the Zebrafish Information Network (ZFIN), an online database of zebrafish genetic, genomic, and developmental information.

Introduction Inherited photoreceptor degenerations are a major cause of incurable blindness. Degenerations can affect rods, causing night blindness, cones, causing color and daylight blindness, or both cell types, leading to complete blindness. Although there are many models of retinal degeneration caused by variety of mutations in different genes, it is still not possible to completely describe the molecular cascade causing cell death in any of these disorders and therefore it is equally difficult to prevent the degenerative process. Fundamental new information about the biochemistry of photoreceptor cell death is required to enhance our understanding of retinal degeneration and to develop new successful therapies. Zebrafish have gained prominence as a model organism for studies of retinal development, disease, and vision because they offer some distinct advantages over other genetically tractable systems. Zebrafish develop rapidly ex utero and can be maintained transparent allowing cells in the retina to be visualized in live larvae in real time using confocal and multiphoton imaging techniques. This provides the opportunity to visualize morphological and biochemical changes occurring in diseased photoreceptors with cellular and subcellular resolution. Here, we describe two mutations in the zebrafish cone phosphodiesterase (pde6c) gene that result in retinal degeneration. These mutants provide a unique opportunity to learn more about the biochemical triggers and inhibitors of cell death within the retina.

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Retinal Disease Photoreceptors are the primary sensory cells within the visual system. There are two main types of photoreceptors within the vertebrate eye: rods, which are monochromatic and respond in low light levels, and cones, which respond to higher light levels and specific wavelengths within the visual spectrum. The system of light absorption, ion fluctuation, and neuronal transmission are processes that require significant energy and, thus, the retina is one of the highest-energy-consuming tissues in the body. The high level of oxygen consumption by photoreceptors makes them particularly susceptible to injury and perturbation often resulting in cell death. Retinal degeneration is a leading cause of blindness in the developed world. The most common form of degeneration is age-related macular degeneration, which first affects the cones within the central retina (macula) and then progresses to the periphery. Retinal degeneration can also occur as a result of genetic mutations. Achromatopsia and Retinitis pigmentosa (RP) together define a large class of heritable diseases that affect vision in humans. Both of these diseases are caused by a wide variety of mutations that disrupt visual transduction and photoreceptor maintenance. Achromatopsia is characterized by defects in the cone photoreceptors while rods remain functional, resulting in extreme light sensitivity and color blindness or rod monochromacy. Achromatopsia symptoms are generally seen at birth and the vision loss is only rarely progressive. RP, in contrast, develops during childhood and in later stages of life starting with degeneration of the rod photoreceptors, and progressing in some cases to total blindness. RP is estimated to affect 1 in 10 000 people. Due to the diversity of genes associated with these disease states, most therapeutics have focused on the general prevention of cell death as a method for limiting progressive vision loss.

Animal Models of Vision Many animal models are used to study the visual system, and each possesses various strengths and weaknesses. By using a variety of systems scientists are able to capitalize on the strengths of all of them. The two most common systems for retinal modeling have been the fruit fly, Drosophila melanogaster, and mice, with a variety of work occurring in related species such as rabbit or ferret. Drosophilae have mainly been used to study ocular development and patterning. Surprisingly, despite the obvious structural differences between the insect compound eye and our own, many of the same signaling cascades are used to establish the structure and patterning of the Drosophila eye. Drosophilae have an extremely short

gestation period and well-established methods for rapid genetic manipulation, and these features have been used to understand the roles of multiple genes in eye development. However, there are still many limitations in the use of this organism, particularly in the modeling of retinal disease. Several differences exist between the insect and mammalian phototransduction cascade, and the structural differences of the compound eye also limit the applicability of this organism for disease studies. The most commonly used mammalian animal model of the retina is the mouse. Mice have a number of advantages as a model system. There is an extensive literature on a variety of mutants that have been studied for many years. There are also multiple techniques for genetic manipulation, including the ability to modify genes and genomic loci and several techniques for retinal explantation for in vivo imaging. However, mice are nocturnal creatures and depend primarily on their olfactory system for foraging and predator identification. As a result, unlike the human retina, the mouse retina is dominated by rod photoreceptors and contains only 3% cone photoreceptors. While this makes mice ideal for studying rod photoreceptor disease and function, the study of cone physiology and function is less straightforward in this system. Additionally, mouse eye development occurs in utero, making it difficult to image and understand the early stages of eye development and retinal perturbation. The Advantages and Techniques of the Zebrafish Model System The zebrafish system has a number of benefits for studying visual development and disease. First, zebrafish vision, similar to humans, is cone dominated. They rely upon their vision for food acquisition and have four different cone types: red, green, blue, and ultraviolet. In the zebrafish, ocular development occurs externally over the first 5 days postfertilization (dpf). During zebrafish development, the cone photoreceptors mature first between 3–5 dpf followed by the rods, which mature between 15–20 dpf. Thus, early zebrafish vision is dependent solely on cone-mediated vision. The rapid development of the visual system and early cone dominance have facilitated several genetic screens using young embryos to select specifically for cone related defects (see below). Zebrafish were originally developed as a model system because they are inexpensive to keep, and take only 3 months to reach sexual maturity after which a mating pair will produce 200–300 eggs per week for at least a year. Further, development of many different genetic tools has added greatly to the versatility of this model for scientific study. In particular, mutagenesis protocols were optimized in the mid-1990s to introduce high frequencies of mutations. This made it possible to conduct


large-scale forward genetic screens. Hundreds of different mutants affecting many aspects of vertebrate development and function have been identified. The constantly improving annotation of the nearly completed genome makes identifying mutated genes straightforward, and an impressive collection of vision mutants is available. Another advantage is that cellular behavior can be observed in the intact, living organism. During the first 2 weeks of development, zebrafish larvae can be maintained in a translucent state and visualized live using either exogenous or genetically encoded fluorescent markers (see examples in Figure 1). Zebrafish can be maintained alive and healthy in agar on the microscope stage for days. Thus, cells can be imaged over the course of development or degeneration without perturbing the extracellular environment. At this point, transgenic lines containing fluorescent markers in various cell types are simple to generate and maintain. Transient injection of plasmid DNA at the one-cell stage will also produce a mosaic expression of genes throughout the fish allowing for the study of isolated cells within the intact animal. Multiple promoters have been developed to drive expression in various types of cells within the eye. While no procedures exist to manipulate specific genes within the genome, several methods have been developed which allow scientists to circumvent this limitation. Morpholinos are synthetic RNA-like oligonucleotides that can be injected at the one-cell stage or electroporated into the fish at later stages. These morpholinos bind to corresponding RNA sequences within the cell, resulting in the degradation of the RNA and loss of protein expression. The loss-of-protein function is not as complete as is seen in knockouts and is only transient, but it has allowed

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researchers to study the effects of specific knockdowns during development. Additionally, several labs have developed a method known as targeting-induced local lesions in genomes (TILLING) in which high-throughput polymerase chain reaction (PCR) methods are used to identify specific gene mutations from libraries of randomly mutagenized fish. Very recently, another method has been established in which nucleases are used to produce double-strand breaks at specific loci within the genome. These double-strand breaks are repaired by nonhomologous end joining, often resulting in deletions or insertions at the break site that can lead to frame-shift mutations in the target gene. The targeting specificity of these double-strand breaks is established by fusion to an array of zinc finger domains that bind to specific DNA sequences. Each zinc finger recognizes a 3-bp sequence and three zinc fingers are fused together to create a 9-bp recognition domain. Further, these zinc finger nuclease arrays must dimerize to activate the nuclease such that a total 18-bp recognition sequence is required. Currently, the technology for generating the zinc finger arrays is cumbersome, but soon this will be a rapid and convenient way to generate targeted zebrafish mutants. Methods have also been developed to create chimeric fish allowing for the rapid evaluation of the cell autonomous nature of genetic phenotypes. To produce these fish, eggs are grown to the blastula stage and then cells from one egg are removed and inserted into another egg. This procedure does not affect fish development and wild-type chimeras grow normally. The production of chimeric fish can be used to determine the critical cell population for the phenotypic changes associated with a mutation. It can also be useful for the evaluation of neighboring and surrounding effects.

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Figure 1 The zebrafish eye. These images illustrate the translucence of the eye and the ability to image the eye in vivo. For these experiments, zebrafish are anesthetized and embedded in a 0.5% agar solution during imaging. The zebrafish can survive under these conditions for up to 2 days. (a) A fluorescent image of the eye with the cone photoreceptors expressing the transgene for green fluorescent protein (GFP) under the control of a cone-specific promoter (TaCP), shown over the differential interference contrast (DIC) image of the eye. (b) An eye showing the photoreceptors in blue, expressing the transgene for cyan fluorescent protein under a cone-specific promoter (Tg(TaCP:MmCFP)) and the secondary layer of neurons or bipolar cells in red, expressing yellow fluorescent protein under a bipolar-specific promoter, (Tg(nyc:mYFP)).

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Zebrafish have one other major advantage for the study of disease, which is the aqueous environment in which they reside. Methods are being developed for rapid high-throughput drug screens using zebrafish by adding compounds to their water and evaluating the effects. Most of the current studies with this method use fluorescent cell markers to indicate the presence or absence of various cells. This technique has been used with fluorescent hair cells to identify compounds that prevent hair cell death in the presence of the ototoxic agent neomycin. As zebrafish are small and easy to maintain, they are the best vertebrate model for this type of shotgun approach to drug development. Evaluating zebrafish vision

There are several ways of evaluating zebrafish vision. The simplest involves a manipulation of the fish instinct to maintain its position within a moving stream. In this test, groups of fish can be placed in a long dish with a series of moving bars along its side. As the bars move, the fish respond to the apparent current by swimming to maintain their position with the bars. Thus, shoals of fish can be tested simultaneously for their ability to see and respond to the moving lines. This is known as the optomotor response. A related test called the optokinetic response (OKR) is done with a single fish placed in a small dish in the center of a rotating drum decorated with vertical stripes. In this test, the fish’s eye will follow the stripes with periodic involuntary saccades. This is a very sensitive measure of an individual fish’s ability to perceive its visual environment. Several labs have identified blind fish in mutagenesis screens using the optomotor response and/or the OKR. These screens have yielded a variety of mutants that can be used as models for retinal disease.

Another method that has been used to measure fish vision is the electroretinogram (ERG), which is a stimulated measure of the electrical response of the eye to a flash of light. In these measurements, the fish are dark adapted and a small electrode is placed on the cornea. The eye is then stimulated with a light flash and the electrical response is recorded. This technique records both the primary photoreceptor response, which appears as a negative spike at the beginning of the recording known as the a-wave, and the response of the secondary neurons, which is a large secondary positive response following the a-wave, known as the b-wave.

The Visual System: Phosphodiesterase and Phototransduction Cyclic GMP (cGMP) phosphodiesterase (Pde) is an important enzyme in the process of phototransduction. During phototransduction the 11-cis retinal absorbs a single photon of light and alters the conformation of the opsin protein to activate the associated heterotrimeric guanosine triphosphate (GTP)-binding protein, transducin. The activated transducin removes the inhibitory gamma subunit from phosphodiesterase, which then degrades cGMP in the outer segments (for a schematic see Figure2). The lowering of cGMP levels stimulates the closure of cyclic-nucleotide-gated ion channels in the plasma membrane, causing hyperpolarization and a decrease in neurotransmitter release at the synapse, initiating light signaling to downstream neurons. Channel closure also interrupts Ca2+ influx leading to a decrease in intracellular [Ca2+]i. The drop in intracellular [Ca2+]i activates a Ca2+-sensitive guanylyl cyclase, restoring cGMP to presignaling levels. Both types of photoreceptors have a similar phototransduction cascade, but use different

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Figure 2 A schematic of the initial steps of phototransduction. The absorbance of a photon activates the 11-cis retinal of rhodopsin, which in turn activates the heterotrimeric GTP-binding protein, transducin. The Ga subunit of transducin binds to the inhibitory Pg subunit of the Pde6 holoenzyme and relieves the inhibition of the catalytic domain. Pde6 then cleaves cGMP, resulting in a drop in cGMP levels that causes the closure of cGMP-gated ion channels in the plasma membrane.


genetically encoded enzymes to accomplish each task. Therefore, a mutation in the cone phosphodiesterase will not affect rod phototransduction. The photoreceptor Pde (Pde6) is a multisubunit enzyme that differs slightly in rods and cones. In both cells the catalytic domain is a dimer that is bound and inhibited by the regulatory gamma subunit called Pg. During phototransduction, activated transducin binds to Pg and exposes the catalytic cGMP-binding site allowing the catalytic domain to cleave cGMP. In rod photoreceptors, two related but different proteins, Pa and Pb, form the Pde6 catalytic domain. In cones, the catalytic domain consists of a dimer of one protein Pa’, also known as Pde6c.

Retinal Degeneration in pde6 Mutants: Primary Degeneration Mutations in the Pde gene (pde6) are found in several families with RP and similar mutations have been used for many years as a model for RP in mice. The oldest and most commonly used mouse model of RP is the retinal degeneration 1 (rd1) mouse that contains a mutation in the Pb rod-specific subunit of Pde6, the pde6b gene. In this model, rod degeneration is apparent by postnatal day 8 and nearly all of the rods are lost by 3 weeks of age. Despite many years of study, the process of primary photoreceptor degeneration in the rd1 mouse is not well understood. Initial work has implicated programmed cell death pathways. The death of rods is associated with the extreme DNA cleavage that accompanies apoptosis, and this DNA cleavage can be detected with the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. However, several studies suggest that the standard apoptotic cascades, including a group of cysteine proteases known as the caspases, and other typical apoptotic effectors, are not involved. Instead, recent work implicates intracellular calcium concentration ([Ca2+]i) and the calpains, a calcium-activated set of proteases found in the mitochondria, as the relevant initiators of photoreceptor programmed cell death. In rd1 mice, the absence of Pde6 results in elevated levels of cGMP even under dark conditions, and it has been hypothesized that this results in the greater open probability of the cyclic-nucleotide-gated ion channels leading to increased [Ca2+]i levels and apoptosis. However, little is known about the distribution of [Ca2+]i during the death of cones. An initial report using the Ca2+ channel blocker, D-cis-diltiazem, to inhibit [Ca2+]i accumulation showed a decrease in apoptosis. However, other labs using the same and other Ca2+ channel blockers have been unable to repeat these results. Due to the difficulty of imaging a mouse retina in vivo over long time periods, none of these studies has examined the levels of [Ca2+]i within the cells in vivo. The translucence

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of the zebrafish retina enables in vivo imaging of both the morphological changes of degeneration as well as changes in levels of signaling molecules, such as Ca2+ or cGMP.

Retinal Degeneration in pde6 Mutants: Secondary Retinal Degeneration, the Bystander Effect – Models and Mechanisms In humans, RP is characterized by a lack of night vision, but often progresses over time to tunnel vision and, in some cases, complete blindness. This progression of the disease is due a gradual death of cones by apoptosis. Cell death begins in the peripheral retina where the number of rods is highest and moves toward the central retina. The cone photoreceptors are fully functional and do not use the mutated genes for phototransduction. However, the death of the mutant rods causes healthy cones to die apoptotically. This transmission of death to healthy neighboring cells is a process known as the bystander effect. The death of these cones represents the most debilitating part of this disease, and has significant potential for therapeutic intervention. In addition to retinal degeneration, the bystander effect has been seen in a variety of diseases, including cancerous tumors, and can be either beneficial or detrimental to therapeutic efforts. One of the first descriptions of the bystander effect occurred during studies of gene therapy for cancer in which malignant tumors were injected with viruses containing suicide genes, which convert a prodrug into a lethal compound inside cells. Researchers found that, although only a small population within the tumor expressed the detrimental genes, significant portions of the tumor mass still died, suggesting that virally transfected cells were able to induce death in untransfected neighboring cells. Recently, researchers have found a similar occurrence in cells exposed to radiation therapy. In this case, cells that have not been irradiated show the genetic instability associated with radiation exposure. In general, apoptosis does not affect the health of neighboring cells and it is unclear why in some instances there is a spread of death across a population. There are currently several hypotheses for how healthy cells are induced to die. One possibility is that live cells release a trophic factor that stimulates the growth and differentiation of their neighbors and is required for their proper maintenance. For instance, it has been suggested that rods release a factor that stimulates the growth of cones. Another possibility is that dying cells release toxic factors that kill neighboring cells. A potential corollary to both of these hypotheses is that either toxic or trophic factors are released to neighboring cells through gap junctions. It is also possible that the immune response triggered by the

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E Figure 3 Schematic of some of the possible sources of the bystander effect. (a, b) Live cells release trophic factors (happy faces) that help neighboring cells and are lost when the mutant cells (in red) die. These factors could be released exogenously (a) or through gap junctions between cells (b). (c, d) Dying cells release toxic factors (skulls) that kill neighboring cells, again either exogenously (c) or through gap junctions (d). (e) The immune response to the presence of dying cells could have a deleterious effect on the remaining healthy cells.

removal of apoptotic cells has a deleterious effect on the neighboring cells. See Figure 3 for a schematic of these possibilities. Understanding the source of the bystander effect will suggest other methods by which it might be prevented.

Levels of the Bystander Effect in Photoreceptor Degeneration The extent of bystander cell death is not the same for all cases of retinal degeneration. The death of cones in rod–cone dystrophies has been extensively studied in human patients and in a variety of animal models, including the rd1 mouse. Mutations in rod phototransduction, resulting in rod death, almost always lead to some cone degeneration. However, the number of cells that die within the central fovea varies significantly from patient to patient. Mutations in genes required for cone phototransduction and their effects on rods are less well studied. Cone–rod dystrophies are conditions in which degeneration of the cones causes a decrease in scotopic vision, low light vision that is produced exclusively by rod function. Little is known about the state of rods in these patients, but it is thought that there is some degeneration associated with the visual loss. In contrast, some patients with cone dystrophies have cone death without affecting the rods or scotopic vision. Differences in the connections between cells in a population might account for the variable levels of bystander death. In particular, it is thought that gap junctions

between rods and cones in mammals may be predisposed to allow the flow of materials from the rods to the cones, but not from the cones to the rods. Thus, it may be possible that the flow of information would be asymmetrical between cell types and this could lead to differences in the effects on neighboring cells. In order to better understand the bystander effect, an important first step is to determine how death progresses throughout the population and which populations of cells are capable of propagating apoptotic signals to healthy neighbors. For this type of analysis, zebrafish provide an ideal system. Not only are mosaic animals easy to generate, but the transparency of the embryo make it possible to analyze the transmission of death in vivo. This feature combined with the other tools described above provides a novel and powerful approach to examining the bystander effect within the photoreceptor population.

Zebrafish Models of Retinal Degeneration: Mutations in pde6c Mutations in the zebrafish cone phosphodiesterase have been identified in two separate genetic screens for fish lacking OKR at 5 dpf. One of the mutations is a null mutant (w59), while the other is a missense mutation in a conserved amino acid (els). Recently, recessive mutations in pde6c were also identified in mice and in three human families with achromatopsia. Preliminary results indicate that in humans this form of achromatopsia may be progressive, suggesting the possibility of a bystander-associated death of rods. pde6cw59 pde6cw59 is a mutation in the splice site between exons 11 and 12 in the pde6c gene. The abnormal splicing caused by this mutation introduces a premature stop codon generating a null mutant. Zebrafish, homozygous for the pde6cw59 mutation, are viable and form normal swim bladders, a marker of general fish health, but must be raised with higher-than-normal food concentrations, as their visual defects impair acquisition of food (Figure 4(a)). In these fish, cone photoreceptors initially develop normally. However, at 4 dpf, when fish first respond to light, the cone photoreceptors in the central retina begin to degenerate. The pde6cw59 mutants exhibit a flat ERG at 5 dpf, indicating a total lack of photoreceptor response to light and no signaling to downstream neurons. At this stage in development, zebrafish vision relies on cones, so a flat ERG is consistent with the rapid cone degeneration observed in these fish. As the cone photoreceptors die, they retract their synaptic connections and outer segments and become spherical. Time-lapse images of this process are shown in Figure 4(b). In the final stages of cell death, only the


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Figure 4 Images of the pde6cw59 mutant fish. (a) Homozygous pde6cw59 mutant fish have swim bladders, and are generally healthy. These fish contain the transgene Tg(TaCP:MmCFP), which causes expression of membrane-tagged cyan fluorescent protein (MmCFP) specifically in cone photoreceptors. The level of fluorescence in the mutant eye is decreased due to the degeneration of cone photoreceptors. (b) A time-lapse sequence of a single mutant cone photoreceptor undergoing apoptosis. The initial picture is an image of wild-type cones. The subsequent pictures are a single pde6cw59 mutant cell over a 5-h time course. As the cells die, they retract their synaptic connections and round up to form apoptotic bodies which are eventually removed by the immune system. (c) Apoptotic photoreceptors (green) viewed using the membrane label boron-dipyrromethene (BODIPY), which labels all cellular membranes in the fish. Apoptotic bodies are clearly visible in the outer nuclear layer (arrows). Bipolar cells express yellow fluorescent protein (Tg(nyc: mYFP)) and are shown here in red. (d, e) The dramatic loss of fluorescently labeled photoreceptors in the whole eye of Tg(TaCP:MmCFP) pde6cw59 mutant. Eyes were removed from euthanized and fixed 6-day-old Tg(TaCP:MmCFP) WT (d) and pde6cw59 (e) mutant animals. Fluorescently labeled cone photoreceptors were viewed with confocal microscopy. From Lewis, Wong and Brockerhoff, in preparation.

rounded apoptotic bodies remain (Figure 4(c), arrows). This cell debris travels out of the outer nuclear layer and is disposed of by the macrophages of the immune system. Within the central retina of the pde6cw59 mutant, a majority of cones die by 5 dpf. Figures 4(d) and 4(e) show the central retina of a wild-type (d) and mutant (e) eye, expressing membrane-tagged cyan fluorescent

protein (MmCFP) specifically in the cone photoreceptors. The MmCFP accumulates in the outer segments of the cones. In Figures 4(d) and 4(e), the eye is visualized by removing it from a day-6 zebrafish larva and inverting it onto a microscope slide. In the wild-type eye, the central retina is densely populated with cones that show a regular mosaic pattern (Figure 4(d)). In the pde6cw59 eye,

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most of the cone photoreceptors have died and been removed from the retina, and many of the remaining outer segments appear dystrophic (Figure 4(e)). Unlike humans or mice, fish continue to produce photoreceptor cells throughout their life. At the periphery of the eye is a region of cells known as the circumferential marginal zone, in which new photoreceptors are generated by multipotent stem cells. In this zone, young cones are constantly differentiating throughout the life of the animal. Even in adult pde6cw59 mutants, there are always cones in this region of the eye, indicating that there is no defect in cone morphogenesis or differentiation, but that cones die as they mature. The circumferential marginal zone is not visible in Figures 4(d) and 4(e) but can be seen in Figure 4(a) as a faint ring of fluorescence around the periphery of the pde6cw59 eye. The rods in the central retina also deteriorate during early development in pde6cw59 mutants but later recover. At 7 dpf, the rods appear normal and are slightly more clustered than in wild type, but not reduced in number. At 8–9 dpf, the number of rods begins to decrease in the central retina. The outer segments of the remaining rods in the central retina appear dystrophic. The deterioration of the rods continues through at least 6 weeks postfertilization. These data were the first evidence that zebrafish can undergo a bystander effect in the retina (i.e., mutant cones kill neighboring healthy rods). The multipotent stem cells in the circumferential marginal zones of the eye can differentiate into rods throughout the life of the animal. Additionally, in cases of damage or injury, the Mu¨ller glia can also enter a mitotic state and produce stem cells capable of forming all types of retinal neurons. In the pde6cw59 mutants, this continuous regeneration eventually replenishes the small number of rods that have died. Thus, by 3 months postfertilization, the retina is completely populated with rods. Interestingly the pde6cw59 mutants never develop a scotopic ERG or OKR, indicating that even the remaining rods are unable to form proper connections with the downstream neurons in the eye. Unlike human and mouse eyes, zebrafish do not have separate rod and cone bipolar cells. The bipolar cells that connect to rods also connect to cone photoreceptors. The lack of scotopic vision in the pde6cw59 mutant suggests that the cones are necessary for the proper connections of the rods with their bipolar targets. In support of this theory, the bipolar cells of the pde6cw59 embryos often show an altered morphology with axons that extend into the ganglion cell layer and dendritic branches that send filopodia into the photoreceptor layer. The pde6cw59 mutant allows for exceptional visibility of photoreceptor degeneration. Due to the rapid degeneration of the cones and rods in the central retina, it is possible to monitor many aspects of cell death as they are occurring. However, in the human diseases of RP and Achromatopsia, retinal degeneration can be a slow

process occurring over several years. Thus, although it is easier to visualize cell death and develop drugs that will combat the rapid loss of photoreceptors in the pde6cw59 mutation, it would also be useful to have a mutant where degeneration occurred over a more protracted period of time. Recently the els mutant was identified as a mutation in pde6c that results in slow cone degeneration. els The els mutation produces a single amino acid change; methionine (M) 175 is mutated to an arginine (R) (M175R) in the first GAF domain of Pde6c. At 5 dpf, when zebrafish vision is cone dependent, fish that are homozygous for the els mutation have a flat ERG and no OKR, although initially the cones appear morphologically normal. This indicates that phototransduction is disrupted but surprisingly cell death has not been triggered. This finding suggests that the els allele is not null for the Pde6c protein, but that secondary defects within the els cones may be disrupting phototransduction, resulting in a flat ERG. One finding in support of this idea is that, although all four types of cones are initially present in the els mutant, the localization of various opsins within the photoreceptors is abnormal. Generally, opsins are found only in the outer segments, but in the els mutant, opsin proteins are found throughout the cell. At this stage, there is also a small but significant increase in the number of apoptotic cells in the els retinas. By 3 weeks postfertilization, the cones and rods in the central retina have begun to show an altered morphology, and the number of cone cells has decreased. However, despite their slightly altered morphology, rod maturation occurs in these fish and by 3 weeks postfertilization the fish respond to OKR under scotopic conditions. As the els fish matures, the cones continue to deteriorate and by 6 months postfertilization the retina no longer contains cones, but consists entirely of rods. The rods are morphologically normal and, unlike those found in pde6cw59, functional. This suggests that the significantly slower death of cones in els compared to pde6cw59 fish allows rods to form proper synaptic connections with bipolar cells. Interestingly, the total number of cells in the inner retina is decreased in this mutant; however, the ratio of inner retinal cells to rods has increased over wild type, suggesting that the rods are forming more connections in the mutant than they do in the wild-type retina. The number of mitotically dividing cells in the retina is also increased in the mutant, suggesting that there is an increase in cellular proliferation probably resulting from the death of the cone photoreceptors. Pde6 Structure Recent work on the structure and function of Pde6c has shed some light on the potential effects of the els mutant


for the Pde6c holoenzyme. The catalytic subunit of the Pde6 protein consists of two domains: a regulatory domain with two GAF domains, one of which (GAF A) binds cGMP, and a catalytic domain that has phosphodiesterase activity. The inhibitory subunit Pg binds to the GAF A domain though its C terminus and the catalytic domain through interactions with its N terminus. Recent work with the rod Pde6 has suggested that the binding of cGMP to the GAF A domain may increase the binding affinity of the N terminus of Pg for the catalytic domain. Thus, the binding of cGMP to the GAF A domain may help to regulate the activity of Pde6c in vivo. Met175 is located within the allosteric cGMP-binding site of the GAF A domain of Pde6c (Figure 5(a)). A recently determined crystal structure of the GAF A domain from chicken Pde6c reveals that the side chain of Met175 (equivalent to Met179 in chicken Pde6c) is in close proximity to the cyclic phosphate group of cGMP, although it does not directly interact with the cyclic nucleotide. As in other cyclic-nucleotide-binding Pde GAF domains, the phosphate group of the ligand is stabilized through the positive dipole of helix a3 (Figure 5(b)). The mutation M175R (equivalent to M179R in chicken Pde6c) introduces a larger and positively charged side chain into the binding pocket and thereby changes the cGMP-binding environment and, potentially, the binding affinity for the allosteric regulator cGMP (Figure 5(c)). A model in which a methionine side chain is substituted for an

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arginine side chain suggests three potential consequences of the M175R mutation. First, the R175 side chain clashes with helix a3, which may disrupt the dipole interaction between the helix and the phosphate group. Second, the R175 side chain clashes directly with the phosphate group of cGMP. Third, R175 might form a salt bridge with the phosphate group. In the former two scenarios, R175 would disrupt cGMP binding and lower the binding affinity of the GAF domain for cGMP, whereas in the latter scenario, R175 may cause a higher affinity for cGMP. In either model, it is likely that the M175R mutation interferes significantly with cGMP binding and the sensitive regulatory mechanism of Pde6 through its GAF A domain. This, in turn, may lead to impaired Pde6 function and cause over- or underexpression of Pde6 protein, thereby disrupting the cone photoreceptor function. No data are available on the stability or functionality of the els mutant protein. However, the phenotypic comparison with the w59 mutant suggest that the els mutant is not a null mutation, but creates a more subtle effect on the levels or activity of the Pde6c protein. The proximity of the mutation to the cGMP-binding site in the GAF A domain implicates a possible disruption of the intramolecular allosteric regulation of catalytic activity ascribed to this portion of Pde6c. The els mutant represents a unique opportunity to understand more about the enzymatic functions of Pde6 and its role in retinal degeneration. The slower degeneration

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Figure 5 Structural consequences of M179R mutant in Pde6c GAF A. (a) Domain organization of Pde6c. The C-terminal catalytic domain is regulated by allosteric noncatalytic binding of cGMP to the N-terminal GAF A domain and binding of the inhibitory Pg-subunit. The methionine (M)179 to arginine (R) mutation in chicken Pde6c, for which there is a crystal structure, is equivalent to the els Met175R mutation in zebrafish. (b) Experimentally determined structure of wild-type cGMP-binding pocket of chicken Pde6c GAF A (pdb-code: 3dba). a-Helices are shown in red, b-strands are shown in blue. cGMP and Met179 are shown in sticks with carbon atoms in cyan. Met179 is also highlighted through spheres. The figure was prepared with PyMOL, a molecular modeling program. (c) Model of M179R mutant cGMP-binding pocket of Pde6c GAF A. M179 was mutated to R179 through the mutation-function of PyMOL. In the absence of some structural rearrangements in the binding pocket, the longer side chain of R179 clashes with the phosphate group of cGMP and helix a3. a-Helices are shown in red; b-strands are shown in blue. cGMP and R179 are shown in sticks with carbon atoms in cyan. R179 is also highlighted through spheres.

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of this mutation compared to the w59 allele more accurately reflects slower human forms of degeneration. These two zebrafish pde6c mutants will provide complementary tools for studying Achromatopsia and apoptosis due to phosphodiesterase deficiency.

Conclusion Zebrafish have gained prominence as a model for retinal disease. Cone-based vision, visual translucence, inexpensive maintenance, and rapid external embryologic development help make this model particularly exciting for retinal studies. Several genetic screens for blind fish have identified a variety of mutants that mimic human retinal disease. Among these, the pde6c mutants are a particularly good example of a retinal model that has been studied for many years in mice, and will benefit from the types of study available in the zebrafish system. In particular, the potential for live imaging of cells in vivo in an intact animal presents a novel opportunity to visualize and understand the source of photoreceptor degeneration. One of the largest differences between the eyes of zebrafish and humans is the continuous growth of the zebrafish eye, and its regenerative ability in response to damage. Although this regenerative potential can complicate the evaluation of zebrafish as a model organism, it also presents a novel possibility to understand and imitate a natural system of retinal stem cell regeneration. By studying the differences between the zebrafish and mammalian systems it may be possible to stimulate our own potential for retinal regeneration. The zebrafish model also offers an unprecedented potential for high-throughput drug screening. Using fluorescent cell markers and fluorescent plate readers, it will soon be possible to do large-scale screening of drugs that affect the levels of retinal degeneration. This method can also be used to test permeability and the toxicity of drugs. This is the first vertebrate animal model that provides a method for this type of rapid drug development. See also: Color Blindness: Inherited; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Primary Photoreceptor Degenerations: Terminology; Secondary Photoreceptor Degenerations; Zebra Fish– Retinal Development and Regeneration.

Further Reading Brockerhoff, S. E., Hurley, J. B., Janssen-Bienhold, U., et al. (1995). A behavioral screen for isolating zebrafish mutants with visual system defects. Proceedings of the National Academy of Sciences of the United States of America 92(23): 10545–10549. Cote, R. H. (2007). Photoreceptor phosphodiesterase (PDE6): A G-protein-activated PDE regulating visual excitation in rod and cone photoreceptor cells. In: Beavo, J. A., Francis, S. H., and Houslay, M. D. (eds.) Cyclic Nucleotide Phosphodiesterases in Health and Disease, pp. 165–193. Boca Raton, FL: CRC Press/Taylor and Francis. Doyon, Y., McCammon, J. M., Miller, J. C., et al. (2008). Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nature Biotechnology 26(6): 702–708. Goldsmith, P. and Harris, W. A. (2003). The zebrafish as a tool for understanding the biology of visual disorders. Seminars in Cell and Developmental Biology 14(1): 11–18. Hamada, N., Matsumoto, H., Hara, T., and Kobayashi, Y. (2007). Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects. Journal of Radiation Research (Tokyo) 48(2): 87–95. Martinez, S. E., Heikaus, C. C., Klevit, R. E., and Beavo, J. A. (2008). The structure of the GAF A domain from phosphodiesterase 6C reveals determinants of cGMP binding, a conserved binding surface, and a large cGMP-dependent conformational change. Journal of Biological Chemistry 283(38): 25913–25919. Morris, A. C., Scholz, T. L., Brockerhoff, S. E., and Fadool, J. M. (2008). Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retina. Developmental Neurobiology 68(5): 605–619. Muto, A., Orger, M. B., Wehman, A. M., et al. (2005). Forward genetic analysis of visual behavior in zebrafish. PLoS Genetics 1(5): e66. Nishiwaki, Y., Komori, A., Sagara, H., et al. (2008). Mutation of cGMP phosphodiesterase 6alpha’-subunit gene causes progressive degeneration of cone photoreceptors in zebrafish. Mechanisms of Development 125(11–12): 932–946. Paquet-Durand, F., Johnson, L., and Ekstrom, P. (2007). Calpain activity in retinal degeneration. Journal of Neuroscience Research 85(4): 693–702. Ripps, H. (2002). Cell death in retinitis pigmentosa: Gap junctions and the ‘bystander’ effect. Experimental Eye Research 74(3): 327–336. Sancho-Pelluz, J., Arango-Gonzalez, B., Kustermann, S., et al. (2008). Photoreceptor cell death mechanisms in inherited retinal degeneration. Molecular Neurobiology 38(3): 253–269. Stearns, G., Evangelista, M., Fadool, J., and Brockerhoff, S. E. (2007). A mutation in the cone specific pde6 gene causes rapid cone photoreceptor degeneration in zebrafish. Journal of Neuroscience 27(50): 13866–13874. Wissinger, B., Chang, B., Dangel, S., et al. (2007). Cone phosphodiesterase defects in the murine cpfl1 mutant and human achromatopsia patients. Investigative Ophthalmology and Visual Science 48(5): 4521. Zhang, X. J., Cahill, K. B., Elfenbein, A., Arshavsky, V. Y., and Cote, R. H. (2008). Direct allosteric regulation between the GAF domain and catalytic domain of photoreceptor phosphodiesterase PDE6. Journal of Biological Chemistry 283(44): 29699–29705.

Zebra Fish–Retinal Development and Regeneration T J Bailey and D R Hyde, University of Notre Dame, Notre Dame, IN, USA ã 2010 Elsevier Ltd. All rights reserved.

Glossary BrdU (bromodeoxyuridine) labeling – The synthetic nucleoside which is incorporated into nascent DNA during replication and is detectable by antibodies to indicate what cells have divided since exposure to BrdU. CMZ (circumferential marginal zone) – The region of the retina distal to the optic stalk and proximal to the ciliary margin and lens. Here, retinal cells are continually born throughout the life of the zebrafish. CMZ cells express genes found in the neuroretina of the developing zebrafish embryo. Homeobox transcription factors – The proteins that pattern tissue in that they regulate gene transcription by binding to a specific DNA sequence, the homeobox, in the target gene promoter. Morpholino – Similar to RNA, this polymerized oligomer with a morpholino (rather than ribose) backbone, can base pair with ribonucleic acid (RNA) molecules, and persists in the cell as it is not easily degraded by RNases. Morpholinos interfere either with ribosomal processing of the messenger RNA into protein or spliceosome processing of premessenger RNA into mRNA, thus depleting the amount of protein produced. Notch signaling – The plasma membrane-bound receptor that regulates the cell fate choice of individual neurons. Intracellular cleavage product can act as a transcription factor and regulate the expression of pro-neural genes such as basic helix–loop–helix (bHLH) transcription factors. Pcna (proliferating cell nuclear antigen) – A marker for DNA replication in that it is a protein that functions as a trimer to promote DNA polymerase d processivity. Shh (sonic hedgehog) signaling – The Shh family members act as morphogens to pattern tissue. Signaling pathway proteins and pathways are reutilized in more specific cell fate specification as tissues are patterned. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) – A hallmark of apoptotic cells is genomic DNA fragmentation by specific nucleases that cleave DNA between histones. This process results in semi-uniform lengths of DNA with overhanging hydroxyl groups. Terminal transferase efficiently polymerizes labeled nucleotides to DNA

hydroxyl groups without the need of a template. TUNEL is used to detect the genomic fragmentation of an apoptotic cell.

Introduction Zebrafish has rapidly become a leading model system to study a variety of developmental processes, due to its large clutch size, external development of transparent embryos, and the rapid development of the embryo. Large and small genetic screens identified hundreds of mutants that affect the development of various tissues, including the retina. The ease in generating transgenic zebrafish lines has permitted a detailed cellular analysis of organ development without harm to the embryo. For example, transgenes that label specific cells are used to follow cell fates in the transparent embryo during tissue development. Alternatively, transgenes that express molecules that lead to cell death have been used to ablate cells to study either the disruption of development or organization of a tissue. Furthermore, the nearly complete sequence of the zebrafish genome has allowed comparative analyses with other vertebrate genomes to predict the presence of orthologous genes and developmental processes. Combined with the ability to direct the transient reduction in expression of desired proteins, it is possible to functionally analyze the potential role of different signaling pathways in the development of various tissues. Our understanding of zebrafish retinal development, from a sheet of neuroepithelial cells to a laminated and functional neural tissue, has benefited significantly from all of these approaches. In addition to being an excellent model system to study early development, zebrafish has quickly become the premier model system to study tissue regeneration. In addition to exhibiting rapid and functional regeneration of the fin, liver, and heart, zebrafish also regenerate neuronal tissues, including the spinal cord, brain, and retina. Using mutants and transgenic lines that are readily available in zebrafish, a detailed comparison of the genes, molecules, and process that are required for retinal development and retinal regeneration is starting to be generated. While it may initially seem reasonable that regeneration would recapitulate the mechanisms that are involved in retinal development, recent studies revealed that regeneration may utilize the same genes and proteins as

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development, but in a different context. This becomes most obvious when one compares the development of a laminated retina from an unpatterned neuroepithelium to the regeneration of single neuronal cell type from an existing laminated retina. To fully appreciate the differences between these two processes and the mechanisms involved in regeneration, it is important to contrast our understanding of the general events involved in retinal development with our recently acquired knowledge of the processes underlying retinal regeneration.

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Zebrafish Eyes Form from a Single Field in the Anterior Neural Plate There is a large body of knowledge regarding the genes and genetic pathways that are required for the proper formation of the vertebrate eye. Around 10 h postfertilization (hpf ), several secreted proteins induce the most anterior region of the anterior ectoderm to become the neural plate. Several signaling pathways (wingless (Wnt), fibroblast growth factor, and insulin-like growth factor) then further subdivide the anterior neural plate to produce the presumptive eye field. Some anterior-most cells then begin expressing several homeobox transcription factors, such as the retinal progenitor genes visual system homeobox 2 (vsx2), paired box gene 6 (pax6), and retinal homeobox (rx), which further restrict their fates to be retinal progenitor cells. The midline of the underlying head mesoderm then expresses several secreted, signaling molecules (such as the sonic hedgehog-related protein, sonic-you), which induces the eye field in the anterior neural plate to split into two distinct regions. This yields the first morphological sign of the developing visual system, the bilateral evagination of a single-cell thick epithelium from the anterior end of the neural keel, which will develop into the optic lobe. The optic lobes are morphologically visible by 12 hpf (Figure 1(a)). Each optic lobe expresses diffusible signals that induce the overlying naı¨ve epithelium to commit to form the lens. As the lens placode starts to thicken (18 hpf, Figure 1(b)), it produces soluble signaling molecules that promote the underlying optic lobe to proliferate and invaginate, which results in the formation of a concave neuroepithelium – the optic cup. Expression of the transcription factor genes microphthalmia-associated transcription factor (mitf ) in the ventral optic cup and vsx2 in the dorsal optic cup commits those cells to develop into the retinal pigmented epithelium and neural retina, respectively. The region that lies at the junction of the mitf and vsx2 expressing cells later becomes one region of persistent retinal neurogenesis in the adult retina, the circumferential marginal zone (CMZ, discussed below).

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Figure 1 Zebrafish retinal development. (a) Cells evaginate bilaterally from the anterior neural keel (NK) to form the optic lobes (OL), which are morphologically distinguishable by 10–12 h postfertilization (hpf). (b) By 18 hpf, the proliferating cells of the presumptive neuroretina (PNR) have induced the overlying ectoderm to thicken into the lens placode (LP). The retina then invaginates to form the OC, with the thicker dorsal cells, or presumptive neuroretina, expressing vsx2 and the thinner ventral cells, or presumptive retinal pigmented epithelium (PRPE), expressing mitf. (c) The cells in the neural retina continue to proliferate until a wave of sonic hedgehog (Shh) signaling induces the first retinal ganglion cells (RGCs) in the most basal nuclear layer, the ganglion cell layer (GCL: between the arrowheads), to differentiate around 40 hpf in the center of the retina, whereas cells in the retinal margin remain in an uncommitted state. (d) Additional waves of Shh signaling produce three nuclear layers, the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). The ONL is immediately below the retinal pigmented epithelium (RPE), which is now darkened with pigment granules by 3 days postfertilization (dpf). Scale bars represent 20 mm. OC, optic cup; NK, neural keel; NR, neural retina; RPE, retinal pigmented epithelium; CMZ, circumferential marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; L, lens; LP, lens placode; OL, optic lobe; ONL, outer nuclear layer; PNR, presumptive neuroretina; PRPE, presumptive retinal pigmented epithelium.

The Laminar Structure of the Retina Forms as Cells Exit the Cell Cycle and Differentiate The number of cells in the NR increases through cell proliferation until the diffusible signaling protein sonic hedgehog (Shh) is produced in a ventronasal patch of the NR. The Shh protein initiates a wave of expression of the basic helix–loop–helix (bHLH) transcription factor atonal homolog 7 (Atoh7) that sweeps radially toward the dorsal retina that induces the differentiation of the retinal


ganglion cells (RGCs) (Figure 1(c)). Shh is then secreted from the newly specified RGCs to sequentially induce the apically located naı¨ve mitotic cells to exit the cell cycle and commit to the other retinal neuronal identities. Later, Shh signaling from the RPE is required for proper photoreceptor differentiation. This process results in the formation of the terminally laminated retina, which is composed of three nuclear layers (ganglion cell layer, inner nuclear layer (INL), and outer nuclear layer) and two synaptic layers (inner plexiform layer and outer plexiform layer) by 72 hpf (Figure 1(d)). Intrinsic commitment and specification of the different retinal cell types also requires the expression of homeobox transcription factors and pro-neural genes of the Notch signaling pathway. Experiments aimed at determining when the different retinal cell types are committed (neuronal birthdating) established a bias of early committing cells to the RGC, amacrine cell, cone, and horizontal cell classes, followed by the bipolar and rod photoreceptor cells, and lastly the Mu¨ller glial cells. The reproducible timing observed with these different cell types suggests that a molecular clock modulates their commitment and differentiation. This model proposes that the commitment of a retinal progenitor cell to a particular neuronal cell type corresponds to when the progenitor cell exits the cell cycle, with early committed cells localizing more basally in the retina. Thus, retinal progenitor cells divide and some daughter cells become ganglion cells, while the remaining daughter cells continue to divide. Some of these retinal progenitors exit the cell cycle and differentiate as amacrine cells and others continue as retinal progenitors. These progenitors continue their asymmetrical cell division to produce some retinal neurons with each round of cell division, until the final retinal progenitors are committed to become Mu¨ller glial cells. This suggests that the Mu¨ller glia is the retinal cell type that is most recently differentiated from the retinal progenitor cell. This model also allows for the presence of external signals to influence the commitment of the cell, which also changes over time. These mechanisms appear to be conserved across species. Addition of Retinal Cells Throughout the Life of a Zebrafish Unlike mammals, the zebrafish eye continuously grows throughout the lifetime of the fish. This growth requires the continual generation of new retinal neurons in a process called persistent neurogenesis. These additional retinal cells are produced from two adult stem cell niches, the CMZ (Figure 1(d) and Figure 2(a)) and an INL stem cell niche (Figure 2(a)). The stem cells within the CMZ continue to express the cell cycle genes and the embryonic retinal progenitor genes, such as orthodenticle homolog 2 otx2, pax6, and rx, throughout the life of the fish. These stem cells proliferate to yield daughter cells that ultimately

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differentiate into ganglion cells, amacrine cells, horizontal cells, cone photoreceptors, bipolar neurons, and Mu¨ller glial cells, but not rod photoreceptors. Rods arise from the INL stem cell niche as described below. The addition of these newly differentiated cells to the region adjacent to the CMZ (Figure 2(c) and 2(d)) results in the radial growth of the adult retina. The INL stem cells have recently been demonstrated to correspond to the Mu¨ller glial cells. Unlike the CMZ stem cells, however, the asymmetric division of the Mu¨ller glia ultimately produce only rod photoreceptors during persistent neurogenesis (Figure 2). Relatively few Mu¨ller glia are actively dividing at any given moment. The asymmetric division of the Mu¨ller glial cell produces neuronal progenitor cells (Figure 2(a)), which continue to proliferate as they migrate to the outer nuclear layer, where they are called rod precursor cells and continue to undergo cell division (Figure 2(b)). Unlike the pluripotent CMZ stem cells, these Mu¨ller glial-derived rod precursor cells are committed to differentiate into only rod photoreceptors during persistent neurogenesis (Figure 2(c)). As the adult eye enlarges, the distance between the originally differentiated rod photoreceptors increases. The Mu¨ller glialderived rods fill in this space. Thus, persistent neurogenesis in the zebrafish retina encompasses both the radial growth of the retina by addition of all retinal cell types by the CMZ and the slow production of additional rod photoreceptors by the Mu¨ller glia (Figure 2(d)). Regeneration in the Zebrafish Retina Zebrafish regenerate all retinal neurons Persistent neurogenesis involves the continual generation of new neurons in the adult retina, without any prior loss of retinal neurons that must be replaced. It should be noted that the scientific literature often uses retinal regeneration to define the reprojection of axons from viable neuronal soma to replace damaged axons, such as the reprojection of axons from RGCs subsequent to an optic nerve crush or severing. For this discussion, retinal regeneration refers to the replacement of entire neuronal cells that were lost through retinal insult or genetic causes. Zebrafish respond to the loss of retinal neurons by significantly increasing both the number of Mu¨ller glia that reenter the cell cycle and the rate of proliferation in the neuronal progenitor cells, relative to that observed during persistent neurogenesis. This amplified proliferation response appears to be proportional to the amount of damage suffered. In contrast to persistent neurogenesis, these Mu¨ller-glial-derived neuronal progenitors are not committed to become only rod photoreceptors. These neuronal progenitors proliferate, migrate to the retinal layer that contains the missing neurons, and differentiate specifically into the lost neurons. Thus, the damage response alters the persistent neurogenesis program to

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significantly increase the number of neuronal progenitors and allow them a greater breadth of cell differentiation potential. A variety of damage paradigms have been studied in the zebrafish retina. For example, simple surgical lesion induces cells proximal to the cut site to die, which usually results in the death of all the retinal cell types in a small area of the retina. Similarly, heat caused by either a high temperature probe or laser ablation, or high concentrations of ouabain (Na, K-ATPase inhibitor), often causes loss of cells from all three retinal layers. In contrast, other damage models exhibit more restricted cell-type loss. For example, intravitreal injection of low concentrations of ouabain causes the loss of ganglion cells and INL neurons, without the loss of significant numbers of rod and cone photoreceptors. Constant intense light, in contrast, causes apoptosis of only the rod and cone photoreceptors, primarily in the dorsal and central retina, with no detectable cell death in the INL or GCL layers. An advantage of the light damage model is that only two neuronal classes are lost, rod and cone photoreceptors, which limits the complexity of the regeneration response. Furthermore, the light damage extends across a large region of the retina, which induces the participation of a very large number of Mu¨ller glia and neuronal progenitor cells. This results in an amplification of the signals and processes that are required for regeneration, which should increase the likelihood of their identification. Constant Intense Light Kills Photoreceptors, Which Are then Regenerated by Mu¨ller Glia Zebrafish rods and cones die by apoptosis upon exposure to prolonged high-intensity light. Apoptosis is a genetically programmed cell death mechanism, in which the dying cell fragments its DNA and the cell body (blebs) to produce easily phagocytosed cell corpses. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), is a standard method to detect this fragmented genomic DNA. Fragmentation of the DNA produces large

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numbers of free 30 -hydroxyl ends that can be used by terminal deoxynucleotidyl transferase to add dUTP nucleotides that are covalently modified to a detectable molecule, such as biotin or fluorescein (Figure 3). The light-induced photoreceptor cell death is rapid, with TUNEL-positive cells first detected in the ONL as early as 3 h after initiating the light treatment. By 16 h of constant light, the photoreceptor cell death is evident based on the reduction in the number of ONL nuclei relative to undamaged retina and is striking by 3 days of constant light (Figure 4(a) and 4(b)). In addition to the strong TUNEL-positive signal that is detected in the ONL, a weaker TUNEL signal is observed throughout the Mu¨ller glial cells in the INL. Strikingly, this INL TUNEL signal is neither restricted to, nor predominantly localized in, the Mu¨ller glia nuclei, which demonstrates that the Mu¨ller glial cells are not apoptotic. Rather, the colocalization of some proteins derived from apoptotic rods with the TUNEL signal in the Mu¨ller glia suggests that the Mu¨ller glia selectively engulf dying rod photoreceptors. These data are consistent with results in the degeneration model of the Tg(Xops:mCFP) transgenic line, which expresses membrane-bound cyan fluorescent protein (mCFP) from the Xenopus rod opsin promoter (Xops) in only zebrafish rods. The Tg(Xops:mCFP) retina exhibits a persistent loss of only rod photoreceptors, even in the absence of any retinal insult, such as constant bright light. Furthermore, the Tg(Xops:mCFP) retina revealed TUNEL labeling in the INL in a morphology similar to Mu¨ller glia. Thus, the engulfment of apoptotic rod photoreceptors by Mu¨ller glia is a common early feature of the damaged zebrafish retina and may be required for regenerative Mu¨ller glial cell stimulation. TUNEL labeling in the Mu¨ller glia can be detected early, within the first 12 h of light treatment, and represents one of the earliest signs of Mu¨ller glial response to apoptotic photoreceptor damage. Robust regeneration of the light-damaged zebrafish retina follows and a normal complement of rod and cone photoreceptors reform within 28 days after terminating the constant light treatment (Figure 4(c)).

Figure 2 Schematic of persistent neurogenesis in the adult zebrafish retina. (a) In the adult retina, stem cells (SC) in the circumferential marginal zone (CMZ) continue to proliferate through asymmetric cell division to produce neuronal progenitor cells (pink circles), which then exit the cell cycle to become newly differentiated cells (blue circles). These CMZ-derived cells differentiate into cone, horizontal, bipolar, amacrine, and ganglion neurons, as well as Mu¨ller glia (not labeled), but not rod cells. Throughout the remainder of the retina, a limited number of Mu¨ller glial cells (MG) divide asymmetrically to produce a Mu¨ller glial cell and a neuronal progenitor cell (NP). (b) As the CMZ stem cells continue to produce neuronal progenitors (pink circles), there is a radial growth of the adult retina, with the most recently differentiated neurons (green circles) located closer to the CMZ than the older neurons (blue circles). In the central region of differentiated retina, the new NP cell continues to divide and migrates to the ONL. Once these NP cells reach the ONL, they are termed rod precursor cells (RP). (c) The CMZ continues producing new retinal cells (yellow circles), which are located closest to the CMZ. The RP cells in the ONL continue to proliferate, with some of the daughter cells differentiating into rod photoreceptors (purple circles). The newly differentiated rod cell intercalates between differentiated cone cells to maintain the density of rod photoreceptors during the radial expansion of the retina. (d) Persistent neurogenesis continues as the CMZ-based retinal stem cells and the Mu¨ller-glial-derived neuronal progenitors continue to produce new neurons at the margin and central retina, respectively. CMZ, circumferential marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; MG, Mu¨ller glial cell; ONL, outer nuclear layer; NP, neuronal progenitor; RP, rod precursor cell; SC, stem cell.


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Figure 3 TUNEL assay detects the fragmented DNA in apoptotic nuclei. (a) A healthy nucleus contains intact DNA, which contains only a single 30 hydroxyl group at the end of each chromosome. The terminal deoxynucleotidyl transferase enzyme (TdT), which can add polymerized stretches of deoxyuridine triphosphate (dUTP), without the need for a complementary strand of DNA with which to pair nucleotides (template-independent). TdT binds to both ends of the linear chromosomal DNA and adds only a few dUTPs per healthy nucleus. (b) Apoptosis results in the fragmentation of the DNA, which generates a very large number of free 30 hydroxyl ends. (c) TdT adds dUTPs to each of the now many generated 30 hydroxyl ends. The large number of dUTP molecules can either directly fluoresce because they are tagged with a fluorochrome or, in the case of biotinylated-dUTP, are detected by tagged Streptavidin molecule to generate a strong fluorescent signal in the apoptotic nucleus. Because of the absence of the genomic DNA in the cytoplasm, little, if any, fluorescent signal is present in the cytoplasm of an apoptotic cell.

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Figure 4 Cell-type restricted retinal damage in zebrafish. (a) Histological section of an undamaged zebrafish retina (control) with the following labeled: outer segments (OS), outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). (b) After 3 days of constant light, the loss of photoreceptor nuclei in the ONL and reduced outer segment integrity relative to the control are evident. (c) Photoreceptors are regenerated only 28 days post light treatment based on the nuclear density and thickness of photoreceptor nuclei in the ONL and the restoration of the photoreceptor outer segments. (d) A transgenic zebrafish line, Tg(gfap: eGFP), expresses EGFP in the Mu¨ller glia from the gfap promoter. Light-induced damage of this transgenic line induces proliferating cell nuclear antigen (Pcna-red) in a subset (arrowheads) of the Mu¨ller glial cells (green) after only 51 h of constant intense light. (e) Proliferating cell nuclear antigen (Pcna-red) immunolabeling Mu¨ller glial cells (arrowheads) at 2 days after a surgical lesion ( TO-PRO-3 labeling of nuclei-blue) is very similar to the response observed in the light-damaged retina (d). (f ) Three days after intraocular injection of ouabain, the damaged retina revealed lost retinal ganglion cells and INL neurons, without significant loss of ONL photoreceptors. (g) Sixty days after ouabain injection, the regenerated retina revealed a GCL and INL that is nearly indistinguishable from wild type (a). (h) The Tg(gfap:eGFP) line revealed that Pcna was detected in a subset of Mu¨ller glial cells (arrowheads) at 3 days after intraocular injection of ouabain.


In light-damaged zebrafish retinas, the Mu¨ller glia exhibit increased expression of proliferating cell nuclear antigen (Pcna), which is a component of the DNA replication machinery. The expression of this protein in the Mu¨ller glia indicates their reentry into the cell cycle (Figure 4(d)). To confirm that the Mu¨ller glia were proliferating, light-damaged Tg(gfap:EGFP) retinas, which express enhanced green fluorescent protein (EGFP) specifically in the Mu¨ller glial cells from the zebrafish glial fibrillary acidic protein (gfap) promoter, were coimmunolabeled for the expression of the proliferation marker Pcna. The number of actively dividing Mu¨ller glia, coexpressing EGFP and the red fluorescing anti-Pcna antibody, increases through 51 h of constant intense light treatment (Figure 4 (d)), at which point the Mu¨ller glial-derived INL neuronal progenitor cells continue to proliferate to produce clusters of 8–12 progenitor cells associated with a single Mu¨ller glial cell. While this suggests that all the neuronal progenitor cells in a cluster are derived from a single Mu¨ller glial cell and remain associated with that glial cell, this has not been formally demonstrated. This Mu¨ller glial cell-based regeneration response is conserved throughout a number of different damage models. For example, either surgical lesion or ouabain injection results in the death of many different neuronal cell types in the retina, and they both exhibit increased proliferation of the Mu¨ller glial cells (Figure 4(e)–4(h)). Curiously, not all of the Mu¨ller glial cells proliferate in response to constant intense light treatment. Earlier reports suggested that a threshold of rod cell death was required to induce the Mu¨ller glial proliferation response. However, intravitreal injection of a low concentration of ouabain into the zebrafish retina resulted in massive death of the neurons in both the ganglion cell and INLs, with minimal cell death of photoreceptors (Figure 4(f )). Regeneration of the ouabain-damaged retina takes longer than the light-damaged retina, but still produces a relatively normal retina by 60 days post ouabain injection (Figure 4(g)). This demonstrates that significant rod or cone cell death is not required to induce retinal regeneration from the Mu¨ller glia (Figure 4(h)). Furthermore, this suggested that the number of apoptotic neurons, rather than the type of neuron, was critical for inducing this regeneration response. To address if only rod photoreceptor cell death was sufficient to induce a Mu¨ller glial-derived regeneration response, the Tg(Xops:mCFP) transgenic line and the phosphodiesterase 6c (pde6c) mutant line were analyzed. The pdge6 mutant line fails to maintain cone cells, in contrast to the rod photoreceptor cell death in the Tg (Xops:mCFP) fish. While no detectable Mu¨ller glial proliferation response was observed in the Tg (Xops:mCFP) line, a small, but significant, Mu¨ller glial proliferation response was detected in the pdge6 mutant line. This suggested that loss of rods requires only increased proliferation of the ONL rod precursor cells

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that were derived from the neuronal progenitor cells during persistent neurogenesis, while loss of cones requires the increased proliferation of the pluripotent Mu¨ller glia. To further test this hypothesis, a Tg (zop:ntr) transgenic line was generated that expresses the bacterial nitroreductase b (ntr) gene from the zebrafish rhodopsin promoter (zop). The NTR enzyme converts the prodrug, metronidazole, into a cell-autonomous toxin within NTR producing cells. Exposing a Tg (zop:ntr) transgenic line, expressing NTR in all rod photoreceptors, to metronidazole results in the death of only rod photoreceptors and the induction of a Mu¨ller glial proliferation response. Addition of metronidazole to a similar transgenic line that expresses NTR in only a subset of rods, however, failed to induce the Mu¨ller glial response. These data suggest that it is the magnitude of the cell death that determines if the Mu¨ller glia exhibit a robust proliferation response. The failure of the Tg (Xops:mCFP) transgenic line to induce the Mu¨ller glial proliferation response may be due to the rod cell death being small and chronic relative to the massive and acute cell death observed in the metronidazole-treated Tg(zop:ntr) transgenic retinas that express NTR protein in all the rod photoreceptors. Discovery and Analysis of Candidate Genes Involved in Retinal Regeneration Several groups have performed microarray analyses of mRNA expression patterns in different retinal damage models to determine what genes might change their expression during specific points of regeneration. In both the surgical-lesioned and light-damaged models, signal transducer and activator of transcription 3 (stat3) and its negative regulator suppressor of cytokine signaling 3 (socs3) exhibited increased expression shortly after the retinal insult (e.g., within 16 h of starting the constant intense light treatment). The increased stat3 expression suggests that Gp130 receptor signaling is involved in the early damage response. Gp130 is a promiscuous receptor that binds a number of different extracellular signaling molecules, such as cytokines, to activate the Stat3 transcription factor in neuronal niches to promote cell proliferation in the adult vertebrate brain. The socs3 gene, which is transcriptionally activated by the Stat3 protein, encodes a protein that binds the activated receptor to prevent further Stat3 activation. Increased stat3 expression is also known to occur in RGCs following optic nerve crush, further supporting Stat3’s role in the damage response. Microarrays also revealed that both the achaetescute complex-like 1a (ascl1a) pro-neural gene and the notch pathway genes were upregulated in the surgicallesioned and light-damaged retinal models. By contrast, retinal progenitor genes, such as rx and vsx2, whose expression are maintained in the CMZ throughout life, are not significantly increased in expression in the damaged retina.

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Figure 5 Schematic comparison of zebrafish retinal development and regeneration. (a) The healthy retina is made up of rod (tall dark blue) and cone (short light blue) photoreceptors interdigitating with the processes of Mu¨ller glial cells (purple). Other retinal neurons are neglected for simplicity. Mu¨ller glial cells express genes indicative of their differentiated state, glial fibrillary acidic protein (gfap) and glutamine synthetase (glul). (b) As the retina expands, a Mu¨ller glial cell becomes stimulated to proliferate (brown), possibly due only to slow or sporadic progression through the cell cycle. (c) The stimulated Mu¨ller glial cell (green) expresses pcna and pax6 as it begins to divide. (d) The cell division in panel C produces a daughter Mu¨ller glial cell (brown) and a neuronal progenitor cell (green), which expresses pcna, cyclins, notch, delta, and hes genes as it migrates to the photoreceptor layer. (e) This neuronal progenitor cell reaches the ONL and is now called a rod precursor cell (yellow), as it expresses neurod and the rod specification genes nr2e3, crx, and rx1. (f) The rod precursor cell divides with one daughter cell differentiating into a rod photoreceptor (green). (g) The undamaged retina,


This suggests that Mu¨ller glia exhibit a state of competency that is downstream of the CMZ stem cells, but upstream of retinal neuron differentiation. The ability to test the function of these various candidate genes in the regenerating retina has been recently advanced by the development of a method to electroporate morpholinos into the adult retina. Morpholinos are modified oligonucleotides that contain a morpholine ring, rather than deoxyribose. The morpholinos, which are complementary to a specific mRNA sequence, can base pair with and transiently block the efficient translation of the target mRNA. Because some protein could still be correctly translated in the presence of a morpholino, these loss-of-function experiments and organisms are termed knockdowns and morphants, respectively. Morpholinos have traditionally been introduced into zebrafish embryos by direct injection into either the relatively large cells of the early embryo (1–32 cell stage) or the yolk. The morpholinos then diffuse into the daughter cells as they divide during embryonic development. This approach has tested the function of numerous proteins in zebrafish development. Recently, morpholinos that are covalently attached to a positively charged fluorochrome, lissamine, have been injected into the vitreous and then electroporated into the adult retina. These morpholinos can disrupt retinal regeneration if they are electroporated into the retina to knockdown the expression of a target protein prior to its role in regeneration. Proof-of-principle experiments to test the effectiveness of this method were shown for the requirement of Pcna in retinal regeneration. Morpholino-induced knockdown of Pcna expression resulted in the Mu¨ller glia failing to proliferate in the light-damaged retina, which led to the premature death of the stimulated Mu¨ller glial cells due to their inability to proceed through the S phase of the cell cycle. Retinas that were injected and electroporated with anti-Pcna morpholinos also failed to upregulate expression of the retinal progenitor cell marker pax6 in the Mu¨ller glial-derived neuronal progenitor cells. Similar morpholino knockdown studies revealed that Stat3 and Pax6 are also required at different steps in the regeneration process. Microarray analyses of mRNA expression at different time points during regeneration of the surgical-lesioned and light-damaged retinas

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have revealed numerous candidate genes for functional study. The electroporation of morpholinos will permit a relatively rapid loss-of-function analysis to elucidate the genes that are required for regeneration and the key steps and processes underlying retinal regeneration. Events Underlying Regeneration of the Light-Damaged Retina Adult Mu¨ller glial cells are characterized by the expression of cell-specific markers (Figure 5(a)), such as glutamine synthetase (Glul) and glial fibrillary acidic protein (Gfap) among others. During persistent retinogenesis, rods are added to the established repeating mosaic of cone and rod photoreceptors from the Mu¨ller glial cell population. It is not clear if there is a signal (arrow) to stimulate Mu¨ller glia proliferation or if a small subset of Mu¨ller glia remain in a slow cell cycle (Figure 5(b)). This limited number of proliferating Mu¨ller glia can be detected by Pcna and Pax6 expression (Figure 5(c)). The small number of dividing Mu¨ller glial cells produce a daughter Mu¨ller glial cell (Figure 5(d), brown) and a neuronal progenitor cell (green), which continues to express genes important in the cell cycle (Pcna and cyclins) and genes required for cell specification signaling (notch, delta, and hes). The neuronal progenitor continues to proliferate and migrates to the ONL. As the neuronal progenitor reaches the ONL (where it is now called a rod precursor), it continues dividing or begins to differentiate, expressing rod specification genes (neurogenic differentiation (neurod ), nuclear receptor subfamily 2e3 nr2e3, crx, and rx1) and giving rise only to rod photoreceptors (Figure 5(f ), green). Upon light-induced retinal damage (or other forms of retinal insult), Mu¨ller glia hypertrophy and transiently increase their expression of Glul and Gfap (Figure 5(h)). If the number of dying rods and cones (Figure 5(h), red) is sufficiently large, the Mu¨ller glial cells increase their expression of the ascl1a and signal transducer (stat3 genes (Figure 5(i)) through unclear mechanisms. Electroporation of morpholinos into the retina prior to inducing retinal damage revealed that both stat3 and ascl1a are independently required for the Mu¨ller glial proliferation response. The reentry of these Mu¨ller glia into the cell cycle is accompanied by the increased expression of the

identical as in 4A is repeated here for comparison. (h) Photoreceptors (light blue) begin to undergo apoptosis (red) within 6 h of entering constant intense light treatment (rod precursors have been ignored for simplicity). The damaged photoreceptors signal (arrow) a subset of the Mu¨ller glial cells (brown) to hypertrophy, increase expression of gfap, and glul, and phagocytose the apoptotic rod cell bodies. (i) At 35 h, responding Mu¨ller glial cells (green) increase expression of the early response genes, such as stat3 and ascl1a, and the cell cycle regulatory genes, the cyclins. Loss of Pcna expression by morpholino-induced knockdown, results in the failure of responding Mu¨ller glial cells to proliferate and regenerate the lost rods and cones. Similarly, morpholino-induced knockdown of Stat3 expression significantly reduces the number of proliferating Mu¨ller glial cells. (j) Pcna-positive neuronal progenitor cells are clustered around a Mu¨ller glial cell and exhibit increased expression of retinal progenitor genes, such as pax6, and genes involved in intracellular signaling pathways, such as Notch. (k) Pcna-positive neuronal progenitors display several neuronal and photoreceptor differentiation markers, including neurod, nr2e3, and crx, as they migrate to the damaged ONL. (l) One month after exiting the constant intense light treatment, regenerated photoreceptors (green) have differentiated and are indistinguishable from the undamaged photoreceptors.

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cell cycle regulatory proteins, the cyclins. The Mu¨ller glial cell divisions produce neuronal progenitor cells, which continue to proliferate (expressing Pcna and the cyclins) and begin to express the retinal progenitor gene pax6 and genes in the Notch and Delta neuronal signaling pathways (Figure 5(j)). As the neuronal progenitor cells migrate toward the ONL, they begin to express genes that are required for the commitment and differentiation of rod and cone photoreceptors (neuroD, nr2e3, and crx ; Figure 5(k)). Within 28 days of ending the constant intense light treatment, the rod and cone photoreceptors have regenerated (Figure 5(l)). Vestigial Retinal Regeneration Activity in Mammals The finding that the Mu¨ller glial cell acts as an adult neuronal stem cell in the regeneration of the damaged zebrafish retina suggests that it potentially could produce a similar regeneration response in the damaged mammalian retina. Recent studies support the hypothesis that mammalian Mu¨ller glia possess some of the features of adult neuronal stem cells. Transplanted rodent Mu¨ller glia into a damaged retina will produce rhodopsin-positive cells. As expected, intraocular injection of neurotoxic doses of N-methyl-D-aspartic acid (NMDA), which is a synthetic amino acid that binds a glutamate receptor, results in the death of many retinal cell types. However, NMDA damage in the rat retina is followed by a small number of Mu¨ller glia proceeding through one cell division to yield a limited number of cells that differentiate into photoreceptors and bipolar cells. This Mu¨ller glial cell proliferation and regeneration response can be slightly enhanced with the addition of various growth factors. Injection of the toxin N-methylN-nitrosourea (NMU) into the adult rat eye, which specifically kills photoreceptors, induces Mu¨ller glial hypertrophy and increased expression of GFAP and the neural stem cell marker, nestin, followed by the proliferation of some Mu¨ller glia. While BrdU labeling confirmed that the Mu¨ller glia actively divided in response to the NMU damage, only 42% of the BrdU-labeled cells remained 2 weeks after the NMU injection. Of these surviving BrdU-labeled cells, which were all located in the INL, 58% expressed glutamine synthetase (suggesting they corresponded to Mu¨ller glial cells that initially divided) and only 8% expressed rhodopsin. Thus, there were very few rhodopsin-positive cells produced by this Mu¨ller cell division and the ones that were generated, failed to properly migrate to the ONL. Expression or addition of various growth factors increased the number of proliferating Mu¨ller glia only to a small extent. Activation of various signaling pathways, such as Notch, similarly resulted in only a slight increase in the number of Mu¨ller glial-derived neuronal progenitors in the damaged rodent retina. While retinal damage induced some mammalian Mu¨ller glia to proliferate and various growth factors or

signaling pathways slightly increased that number, there remained an insufficient number of proliferating Mu¨ller glial cells and neuronal progenitor cells to properly regenerate all the neurons lost from the retinal damage. Analyzing the mechanisms underlying the robust Mu¨ller glial proliferation response in the light-damaged zebrafish retina will provide important clues as to why the regenerative capacity of the mammalian retina is so limited.

Similarities and Differences of Development with Retinal Ontogeny/Genesis As discussed above, Mu¨ller glia and the neuronal progenitor cells respond to retinal damage by increasing the expression of many of the retinal progenitor genes, such as pax6, that are expressed during embryogenesis and near the CMZ throughout the life of the fish. The inability of the mammalian Mu¨ller glial cells to mount a sufficiently robust proliferative and regenerative response may be due to differences in the signals that stimulate the Mu¨ller glia to respond. All attempts to stimulate sufficient levels of Mu¨ller glial proliferation in the mammalian retina have met with very limited success. Conversely, the failure to robustly proliferate may be due to intrinsic differences between the mammalian and zebrafish Mu¨ller glial cells. For example, transplanted mammalian Mu¨ller glial cells into a damaged retina will differentiate into rhodopsin-positive cells that are predominantly restricted to the INL, rather than repopulating the ONL. Thus, a further understanding of the signals that stimulate the zebrafish Mu¨ller glial and neuronal progenitor cells to proliferate and migrate may reveal insights into how to better manipulate the mammalian retina, and by extension, potentiate human retinal regeneration. See also: Color Blindness: Inherited; Injury and Repair: Light Damage; Retinal Histogenesis.

Further Reading Bailey, T. J., El-Hodiri, H., Zhang, L., et al. (2004). Regulation of vertebrate eye development by Rx genes. International Journal of Developmental Biology 48: 761–770. Bernardos, R. L., Barthel, L. K., Meyers, J. R., and Raymond, P. A. (2007). Late-stage neuronal progenitors in the retina are radial Muller glia that function as retinal stem cells. Journal of Neuroscience 27: 7028–7040. Fausett, B. V. and Goldman, D. (2006). A role for alpha1 tubulinexpressing Muller glia in regeneration of the injured zebrafish retina. Journal of Neuroscience 26: 6303–6313. Fimbel, S. M., Montgomery, J. E., Burket, C. T., and Hyde, D. R. (2007). Regeneration of inner retinal neurons after intravitreal injection of ouabain in zebrafish. Journal of Neurosciences 27: 1712–1724. Kassen, S. C., Ramanan, V., Montgomery, J. E., et al. (2007). Time course analysis of gene expression during light-induced

Zebra Fish–Retinal Development and Regeneration photoreceptor cell death and regeneration in albino zebrafish. Developmental Neurobiology 67: 1009–1031. Malicki, J. (2000). Harnessing the power of forward genetics – analysis of neuronal diversity and patterning in the zebrafish retina. Trends in Neurosciences 23: 531–541. Morris, A. C., Scholz, T. L., Brockerhoff, S. E., and Fadool, J. M. (2008). Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retina. Developmental Neurobiology 68: 605–619. Morris, A. C., Schroeter, E. H., Bilotta, J., Wong, R. O., and Fadool, J. M. (2005). Cone survival despite rod degeneration in XOPS-mCFP transgenic zebrafish. Investigative Ophthalmology and Visual Science 46: 4762–4771. Otteson, D. C. and Hitchcock, P. F. (2003). Stem cells in the teleost retina: Persistent neurogenesis and injury-induced regeneration. Vision Research 43: 927–936. Raymond, P. A., Barthel, L. K., Bernardos, R. L., and Perkowski, J. J. (2006). Molecular characterization of retinal stem cells and their niches in adult zebrafish. BMC Developmental Biology 6: 36.

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Raymond, P. A. and Hitchcock, P. F. (2000). How the neural retina regenerates. Results and Problems in Cell Differentiation 31: 197–218. Thummel, R., Kassen, S. C., Enright, J. M., et al. (2008). Characterization of neuronal progenitors in zebrafish adult retinal regeneration. Experimental Eye Research 87: 433–444. Thummel, R., Kassen, S. C., Montgomery, J. E., Enright, J. M., and Hyde, D. R. (2007). Inhibition of Muller glial cell division blocks regeneration of the light-damaged zebrafish retina. Developmental Neurobiology 68: 392–408. Vihtelic, T. S. and Hyde, D. R. (2000). Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina. Journal of Neurobiology 44: 289–307. Vihtelic, T. S., Soverly, J. E., Kassen, S. C., and Hyde, D. R. (2006). Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish. Experimental Eye Research 82: 558–575. Yurco, P. and Cameron, D. A. (2005). Responses of Muller glia to retinal injury in adult zebrafish. Vision Research 45: 991–1002.


Subject Index Note: Page entries followed by an ‘f ’ refer to Figures; those followed by ‘t’ refer to Tables. A AANAT (arylalkylamine N-acetyltransferase), circadian rhythms in chick retina 71–3, 72f, 73f ABCA4 648–9 ABC-type multidrug transporter definition 726 Drosophila white mutation and 727 Abetalipoproteinemia (Bassen–Kornzweig syndrome), retinitis pigmentosa and 703 Ablim 1 238 Absorbance spectra, butterfly visual pigments 150, 150f, 151t Accessory optic nuclei, definition 295 Acetazolamide, effects on RPE 762–3 Acetylcholine (ACh) retinal function, directional selectivity and 299 starburst amacrine cells 451 Achromatopsia 684, 698 acquired 137 definition 853 inherited 146 Actin LpMyo3 circadian phosphorylation and 424 retinomotor movement 220, 220, 222 Action potential(s) amacrine cell generation 25, 447 RGCs 301 rod phototransduction inactivation and 615 Activator protein-1 (AP-1), light-induced damage and 341 Active transport light-driven protein translocation (photoreceptors) 413 Acuity see Visual acuity Acyl-CoA:retinol acyltransferase (ARAT) 650 Adaptation definition 290 different definitions of 325 higher (non-retinal) level 325 retinal see Retinal adaptation Adaptive immunity definition 270 innate immunity relationship 379 mucosal system see Mucosal immunity; monochromatic aberration correction 8, 9f principle 8f T-cell-mediated see T-cell-mediated immunity visual correction 9 Adaptive optics (AO) 7–16, 528 benefits 526 definition 525 Adaptive optics OCT (AO OCT) 7, 9, 529–30, 529f, 530f clinical application 530–1, 531f Adaptive optics scanning laser ophthalmoscopy (AOSLO) 9 eye motion compensation 9–10, 9f see also Retinal imaging, adaptive optics Adenosine, retinomotor movement regulation 225 Adenylate (adenylyl) cyclase circadian rhythms in chick retina 70–2 neuropeptides and 478–9 Adherens junctions definition 58, 773 rod photoreceptors 677 Adult hippocampal progenitor cells (AHPCs) 367 Adult vasculogenesis, in choroidal neovascularization (CNV) 92 Afferent pupillary defect, definition 399 Agenesis, definition 198 Age-related macular degeneration (AMD) 270–5, 354, 830–5, 854 acquired color blindness 136 blood–retinal barrier breakdown 62, 63f cannabinoids and 719 choroidal neovascularization see Choroidal neovascularization clinical forms 270, 831 definition 87, 354, 717 drusen classification 831 formation/composition 271, 832 histology 832f epidemiology 270, 830

fundoscopy 831f genetics 272, 833 ARMS2/HTRA1 273, 834 CFH 273, 833 histopathology 271, 346–8, 832, 832f immunology/immune system role in pathogenesis 271, 274, 274f infectious agents and 272 inflammation role 271, 272, 341, 346–8 macrophages 271 mouse models 273 neoantigens 272 isoform-specific VEGF mouse model 757 light damage as model for 342 macular structure 831 oxidative stress and 766 iron role 767 smoking relationship 766–7 risk factors 832 smoking 766–7, 833 RPE role 761 oxidative stress and 766 treatment 271, 275, 834 lipoic acid and 766–7 visual acuity 4, 5f Aggressive posterior retinopathy of prematurity (APROP) 790, 796 Airline pilots, visual acuity requirement 4 Akt kinase, light protection mechanisms and 343 Albumin, immunohistochemical staining, blood–retinal barrier (BRB) assessment 53f, 55, 55f Allele(s), definition 726 Alpha cells, AII amacrine cells and 323, 323f Alstro¨m syndrome, retinitis pigmentosa 704 Alzheimer’s disease acquired color blindness 138 glaucoma and 740 Amacrine cells (ACs) 276 AII 448 dopaminergic regulation 497 gap junctions 25, 449–50 information processing and 323 lateral networks 25 looming detector 307–8, 323, 323f location 449–50 morphology 449–50, 450f numbers 450 rod pathway and position within rod pathway 450 signal transfer from rod bipolar cell to 843 action potential generation 25, 447 cell fate specification 750–1 classification 24, 276, 447, 448f lateral Acs 276–8, 277f multiple schemes 447–8 vertical Acs 276–8, 277f clomeleon-labeled 454 definition 276, 494 disease role 283 functional roles 24, 322, 451 structure–function relationship 447–8, 449 historical aspects 447 location 447 morphology 276, 322, 447–51, 448f axons 447 branching patterns 277f, 278, 448, 448f dendrodendritic synapses 276, 277f function relationship 447–8, 449 overlapping (coverage factor) 449 ribbon synapses 278 variability 447–8, 449 neuritic stratification 395 neurochemistry 278, 448–9, 451 cotransmitters/modulators 233, 279, 497 GABA 233, 278, 322, 448–9, 490 GAT-1 expression 230 glutamate receptor expression 278

875

876

Subject Index

Amacrine cells (ACs) (continued) glycine 278, 322, 448–9 melatonin receptor expression 502 peptide immunostaining 489, 490 polyaxonal 323 spacing 449 synapses/communication 278 basic connectivity 280f bipolar cells and 278, 286, 289 dendrodendritic synapses 276, 277f development 394f, 395 dopaminergic regulation 497 GABAergic 233, 278, 280–1, 448–9 gap junctions and electrical coupling 447, 449–50 glutamatergic input 278 glycinergic 278, 281, 448–9 interplexiform cell synaptic input 473, 473f lateral communication networks 24 modulation 279 output targets 278 remodeling and disease 283 ribbon synapses 278 see also Amacrine cells, information processing Amacrine cells, information processing 276–83 AC networks 280 directionally selective GC pathway 282, 282f, 322 glycinergic cells of rod pathway 281 midget BC to GC pathway 281 rod pathway 280, 281f, 450, 843 AII cells and 323 local edge detector 323–4, 324f looming detector 323, 323f contrast gain and 320 crossover inhibition and 322 disease and 283 lateral inhibition and 322 RGC regulation and 301 signal processing fundamentals 279 feedback/feed-forward 279, 279f retinal amplification control 279–80, 280f submotifs 280 see also specific cell types Amiodarone, nonarteritic anterior ischemic optic neuropathy and 403 AMP see Antimicrobial peptides (AMPs) AMPA receptors retina amacrine cells 278 horizontal cells and 311 Amphibian eye historical aspects 848 regeneration of optic nerve 848 unique retinal properties 847–8 see also individual species Amyloid deposition in corneal stromal dystrophies b-Amyloid-mediated apoptosis 740, 740f reduction 741 Amyloid precursor protein (APP) 740 abnormal processing 740 in glaucoma 740 Anastomosis, definition 28 Angiogenesis choroidal vasculature, developmental 179 definition 179, 541, 753 Angiogenic factor(s) 541–2, 542t Angiomatous, definition 87 Angiopoietin(s), proangiogenic activity 547, 548f Angiotensin, in retinal vasoconstriction 34 Animal models of vision 854 Aniridia, definition 212 Anophthalmia 215–16 definition 198, 212 Anterior segment in retinitis pigmentosa 690 Anterior segment OCT 525–6 Antiangiogenic factor(s) 541–2, 542t, 549 Antiangiogenic therapy 549 Antiglaucoma drugs, disadvantages 735 Antioxidant(s) light damage, protection mechanisms 344 as neuroprotective agents 740 retinopathy of prematurity treatment 799 Antiproliferative drugs, PVR management 714

Anti-VEGF therapy choroidal neovascularization 92–3 macular edema 83 AP20187 definition 847 drug-inducible retinal degeneration and 851 Apical junctional complex, definition 773 Apical membrane, definition 773 Apolipoprotein E (ApoE), AMD pathogenesis 274 Apo-opsin, opsin regeneration 651 Apoptosis 734 bystander effect see Bystander effect definition 338, 381, 536, 734, 853 light damage role 341 in optic neuritis 540 pathways 734, 735f retinal, induced by intense light 867, 868f RP role 857 secondary photoreceptor degenerations 839–40 see also Apoptosis in glaucoma Apoptosis in glaucoma 386, 734, 736 mechanisms 736 b-amyloid-mediated 740, 740f excitotoxicity 737, 738f extracellular matrix degradation 742 glial–neuronal interactions 741, 741f neurotrophic factor withdrawal 734, 737f reactive oxygen species 739, 739f Approach-sensitive ganglion cells 304–5, 307 AII amacrine inhibitory inputs 307–8 receptive field 307 Aqueous humor, innate immunity inhibition 379 Arachidonoyl ethanolamide (AEA) 717, 718f inactivation/metabolism 717, 718f synthesis/release 717 as TRPV1 ligand 720 2-Arachidonoyl glycerol (2-AG) 717, 718f inactivation/metabolism 717, 718f release from bipolar cells voltage-dependent 723 voltage-independent (mGluR1a-mediated) 723 synthesis/release 717 ARAT (acyl-CoA:retinol acyltransferase) 650 Arf4 definition 847 rhodopsin targeting/trafficking 679, 680f, 682f Arf GTPases family members 679 rhodopsin targeting/trafficking 679, 680f Argus II 357 Arhabdomeral lobes 583f definition 582 Arhabdomeric cell, definition 416 ARMS2/ARMS2, AMD and 834 Arrestin circadian regulation of expression in Limulus 422–3 cone inactivation and 606, 627–8 complex stability and 606–7 light-dependent translocation 412–13 rod inactivation vs. 606 definition 412, 582, 605, 610 light-driven translocation in photoreceptors 412, 413f, 614–15 active transport vs. diffusion 413–14 mechanism 415 possible functions 413 return to dark-adapted locations 415 threshold light intensity 412–13 metarhodopsin inactivation in Drosophila 242, 244f mutation and retinal degeneration 731 rhodopsin interactions and rod inactivation 611–12, 644–5, 645f light adaptation and 601 light damage and 339–40 light-dependent translocation 412–13 rod vs. cone 606 squid (sArr) 584f, 587 Arteritic anterior ischemic optic neuropathy (A-AION) 405 clinical features 405 laboratory investigations 405, 408f signs 405, 405f, 406f symptoms 405 systemic 405 fundus photography 405f, 406f

Subject Index management 406 GCA management as 406 steroid therapy and 407 timing and outcome 407 nonarteritic vs. 407 pathogenesis 400f, 405 GCA 405 see also Giant cell arteritis (GCA) Arteritic posterior ischemic optic neuropathy (A-PION) 408 management 410 Artificial silicon retina (ASR) 355–6 Artiodactyls, horizontal cell morphology 465, 466f Arylalkylamine N-acetyltransferase (AANAT), circadian rhythms in chick retina 71–3, 72f, 73f Ascl1 (Mash1), retinal histogenesis 750 Aspirin therapy CRVO and 80f, 82, 83f macular edema 82 NA-AION 404–5 Astrocyte(s) inner blood–retinal barrier (iBRB) 46 in neovascularization 349f, 352 PVR and 710 Atoh7/Ath5 retinal expression regulation 235, 236f suppression 236–7 retinal ganglion cell development role 235 activation of Pou4f/Brn3 expression 237, 237f Atonal, definition 235 ATP-binding cassette transporter 4 (ABCA4) clearance of all-trans-RAL from OS disks 648–9 Atrophy, definition 753 Autoimmune retinopathy, retinitis pigmentosa vs. 693–4 Autologous RPE grafts, retinitis pigmentosa treatment 697 Autoradiography definition 487, 815 neuropeptide receptors in retina 491 Autoregulation 34 definition 28 Autosomal dominant vitreochoroidopathy (ADVIRC) 259 Avascular retina definition 790 retinopathy of prematurity pathophysiology 796 Avastin see Bevacizumab Avian models, glaucoma 42 Axonal cells (AxCs) 276 information processing 282 neurochemistry 282 Axonemes definition 219, 575 in photoreceptor outer segments 576, 577f, 578f, 579f Axons definition 381 optic nerve see Optic nerve axons

B BAC (bacterial artificial chromosome), definition 38, 487 Bacterial artificial chromosome (BAC), definition 38, 487 Bad 736, 738, 738f definition 734 Bak, definition 734 Balanus phototransduction, early studies 240 Bailey–Lovie chart 2 Bardet–Biedl syndrome 816–17 BBS genes and 681 cilia and 680–1 definition 847 retinitis pigmentosa and 686, 687f Xenopus laevis models 849–50 Bare light perception (BLP), definition 354 Basal bodies, definition 575 Basal junctions, bipolar cells 662 Base interval of stimulation, definition 506 Basic helix–loop–helix (bHLH), definition 235 Basic helix–loop–helix-Per-ARNT-Sim (bHLH-PAS) domain transcription factors in chick retina 68, 69f, 70f definition 68 Basolateral membrane, definition 773 Bassen–Kornzweig syndrome (abetalipoproteinemia), retinitis pigmentosa 703

Bassoon (protein) 664 Bathorhodopsin 641 Batten disease (neuronal ceroid lipofuscinosis), retinitis pigmentosa and 704 Bax 738 definition 38, 734 B-cell, AMD role 272 Bcl-2 736 definition 734 BDNF (brain-derived neurotrophic factor) 736–7 Behr’s syndrome 336 Benzodiazepines, GABAA receptor binding 231 Bevacizumab therapy AMD 271, 834 choroidal neovascularization 92–3 macular edema 83 BGT-1 230 retinal expression 229t Bicarbonate ions (HCO3-) RPE fluid regulation and metabolic load 762–3, 764, 765f, 766f transporters 764 Bid 738f definition 734 Bilaterian animals, definition 205 Binary stimulation, definition 506 Bioassays, retinal neuropeptides 487 Biomechanical engineering studies, optic nerve axons, effects of elevated intraocular pressure 382, 383f, 384f Bionic implants, remodeling and 365 Bipolar cells (BCs) axon terminal development 392, 392f basal junctions 662 classification/types 389, 452 blue cone (S-cone selective) 453 diffuse 23, 455 functional 23, 285–6, 285f morphological 23, 284, 285f, 452, 453f inner plexiform layer and 284, 452, 453f, 456–7, 456f co-stratification of pre-/post-synaptic partners 458, 458f synaptic morphology 458 type-specific differences 457–8, 457f morphology 452–60 clomeleon-labeled 454 functional characteristics and 284–5, 285f, 287f immunocytochemistry 455, 456f, 459, 460 layer-specific 284, 285f, 287f mammalian types 452, 453f mouse vs. primate 453, 453f, 454f nonmammalian types 459, 459f number of cell contacts 455 ribbon synapses 458, 458f transgenic mouse lines 456f, 457, 459 neurochemistry dopaminergic regulation 497 endocannabinoids and 719–20, 721 GABA receptors 233, 289, 313 GAT expression 231 glycine receptors 289 species differences fish vs. mammals 107 mouse vs. primate 453, 453f, 454f species-specific markers 455–6, 456f synapses amacrine cells 278, 286, 289 development 393–4, 393f GABAergic input 233 horizontal cells and 286, 313 interplexiform cell synaptic input 473, 473f mixed rod/cone 284–5 rod synapses see Rod bipolar cells zebrafish pde6 mutants 860 see also OFF bipolar cells; ON bipolar cells Bipolar cells, information processing 284–9 amacrine cells and 278, 286, 289 center-surround organization and 284, 286, 287f BP cell type and input used 286 glutamatergic inputs and 286 hypothetical pathways 286, 288f membrane resistance changes 286 contrast gain and 320 directional selectivity and 299, 459 horizontal cells and 286 feedback control 286–9

877

878

Subject Index

Bipolar cells, information processing (continued) feed-forward control 289, 313 morphology-specific light responses 284–5, 285f, 287f Bird models, glaucoma 42 Birthdating, pulse-chase procedure 745 BK channels, rod photoreceptors 821 Blindness, nonvisual light responses 113, 116 Bloch–Schulzberg syndrome (incontinentia pigmenti), retinitis pigmentosa and 704 Blood flow autoregulation, definition 653 ocular see Ocular blood flow Blood–ocular barriers 44, 45f see also Blood–retinal barrier (BRB) Blood–retinal barrier (BRB) 44–50, 51, 659, 773 breakdown 46f, 48, 48f, 51–7 assessment 54 in diabetic retinopathy 783 disease associations 51 future prospects 56 inhibition 55 after injury 348 molecular mechanisms 53 role of inflammation 52 SRS composition and 767 tight junctions 51, 52f vesicular transport 52, 53f clinical evaluation 48 definition 653, 753 drug permeability 49 fluorescein angiography 48, 48f function 44, 58, 659 improvement studies 67 inner see Inner blood–retinal barrier (iBRB) macular edema and 49 ocular immune privilege and 48 outer see Outer blood–retinal barrier (oBRB) polarity 47 retinal disease treatment and 49 see also Tight junctions (TJs) Blowfly (Calliphora vicina), diurnal ERG changes 129 Blue-cone monochromacy (BCM) 146 epidemiology 146 genetic basis 146 Blue field entoptic technique, ocular blood flow measurement 36 Blue–yellow color defects 144 definition 134, 140 epidemiology 144–5 glaucoma and 136 penetrance 144–5 red–green color defects vs. 145–6 S-cone gene mutations 142f, 144–5 Bmal1/BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator 1) 68, 69f, 70f Bolwig’s organ 132 Bone spicule pigmentation, retinitis pigmentosa 699, 700f B opsin, butterfly 150–1, 151–2, 151t, 154 Bovine model, glaucoma 42 Bradyopsia (slow vision) 329 Brain-derived neurotrophic factor (BDNF) 736–7 Branch retinal vein occlusion (BRVO) 75 Branch Vein Occlusion Study, macular edema treatment 434 BrdU (bromodeoxyuridine) labeling, definition 863 Brn3 see Pou4f/Brn3 Bruch’s membrane 753, 761 age-related changes 758, 759f in AMD 832 definition 87 development 755 function 756 injury of leading to neovascularization 348, 348f OCT tomograms 529–30, 530f structure 756, 756f Buffer device 276 Bull’s eye maculopathy mfERG results 510, 512f, 513f retinitis pigmentosa 699, 701f Butterflies color vision 148–55 ecological significance 154–5 filtering pigments 153, 154 genetic diversification of opsins 151, 152f, 153f, 154 mechanism 149 ommatidial structure 149, 149f

visual pigments 149–50, 150f, 151t sexual dimorphism 154 spectral heterogeneity 150, 151t evolution 148, 149f, 152f, 153f polarization sensitivity 674 Bystander effect 857 definition 853 occurrence in disease 857 cone dystrophies 858 levels of in photoreceptor degeneration 858 rod–cone dystrophy 854 possible mechanisms 857–8, 858f

C Calcineurin737–8, 738f Calcium (Ca2+) molluskan photoreceptors calpain-like protease activation 586 ciliary 445 microvillar 439–40, 440f phototransduction role cone restoration of levels after 607, 627 light adaptation in cones and 607, 629 light-dependent retinal degeneration and 731 Limulus see Limulus phototransduction rod restoration of levels after 612 retinomotor movement regulation in RPEs 226 ribbon synapses channels 664 currents 664–5, 664f intracellular calcium 663, 665 role in vesicular release 665–6 Calcium (Ca2þ) binding proteins, ribbon synapses CaBP4 665 intracellular 663, 665 Calcium-calmodulin kinase II (CaMKII), circadian regulation of CNGCs 120 Calcium (Ca2+) channels circadian rhythms in cAMP formation 70 in channel regulation 74, 121 definition 118 imaging and neuropeptide function 479, 480f, 481f in ribbon synapses 664 structure 121–2 Calcium imaging physiology, definition 477 Calcium-induced calcium release (CICR) definition 661 ribbon synapses 665 Calcium (Ca2+) microdomains, definition 661 Calliphora vicina (blowfly), diurnal ERG changes 129 Calmodulin, light adaptation in rods 600, 602 Calnexin, retinal degeneration and 732 Calpain-like protease 586 definition 582 Calpains 737–8, 738f Calycal processes definition 219 retinomotor movement 220 structure 813f Cancer, retinopathy and 693–4 Cancer-associated retinopathy (CAR), retinitis pigmentosa vs. 693–4 Canine models, glaucoma 42 Cannabinoid(s) 717 ocular tissue effects 719 Cannabinoid receptors 717 CB1 receptors 717–18 photoreceptor modulation 721–2 retinal CB1R-IR 719–20 CB2 receptors 717–18 distribution/function 718 retinal localization 719, 721t metabotropic vs. ion channel 717–18 Carbachol 226 Carbonic anhydrase (CA) diabetic macular edema role 428 RPE role 762–3, 765f Carbonic anhydrase inhibitors (CAIs) acetazolamide effects on RPE 762–3 in improvement of blood–retinal barrier function 67 Carboxyethyl pyrrole (CEP)–protein adducts, AMD pathogenesis 272 Cardiotrophin-like ligand (CLC), light damage, protection mechanisms 343–4

Subject Index Car drivers, visual acuity requirement 4 Cartridges, fly optic lamina 124–5, 125f Cascade, definition 156 Caspase-9, definition 847 Cataract, etiological factors retinitis pigmentosa and 690, 695 Stickler syndrome and 253, 257f Cathelicidin (LL-37) 376 CCL2 (chemokine ligand 2) AMD pathogenesis 272–3 CD59 (MIRL), antiangiogenic activity 549 Cecocentral scotoma, definition 333 Cell birth 175 cell cycle exit and 176 linking mechanisms 177 definition 169 Notch and 175, 176f Cell birthdate definition 745 identification (birthdating) 745 Cell competence, definition 567 Cell culture retinal pigment epithelial (RPE) cells 778–9, 778f Cell cycle G0, definition 169 G1, definition 169 G2, definition 169 M phase, definition 169 regulation 170, 171f cyclin-dependent kinase inhibitor role 171, 171f cyclin role 170, 171f Rb (retinoblastoma) family role 170, 171f retinal progenitor cells vs. retinal transition cells 172, 172f R phase, definition 169 S phase, definition 169 Cell cycle exit 175 cell birth and, linking mechanisms 177 definition 169 Notch and 176f retinoblastoma proteins and 173–4, 174f Cell-cycle rate 175 definition 169 Cell death apoptosis see Apoptosis retinal remodeling 364 Cell extrinsic signaling, definition 263 Cell fate, definition 745 Cell fate determination definition 567 rod vs. cone 570–1, 570f Cell fate specification amacrine cell 750–1 definition 263, 567, 745 Cell interactions in retinal vascular development 546 Cell intrinsic signaling, definition 263 Cell migration definition 360 neuronal migration 360, 363 proliferative vitreoretinopathy and 713 Cell polarity definition 676 rod photoreceptors 676 Cellular retinaldehyde-binding protein (CRALBP) 650 Cellular retinol-binding protein type 1 (CRBP1) 650 Center-surround antagonistic receptive field(s) (CSARF) bipolar cells see under Bipolar cells, information processing definition 284 horizontal cells and 309, 310f, 316 RGC spatial processing 302 difference of Gaussians modeling 302, 303f summary of effects 302 Central retinal artery (CRA) 28, 29, 29f, 75–6, 76f, 181f, 183–4, 653, 654f, 655f definition 28, 179 intraretinal branches 654, 655f controversy 655 retinal layers and 654f, 655 origins 653 sympathetic innervation 658, 659f Central retinal vein (CRV) 29–30, 75–6, 76f, 184, 655f, 658, 658f, 659f surgical decompression 83 Central retinal vein occlusion (CVRO) 75–86 clinical features 77, 77f complications 79

879

cilioretinal artery occlusion 80, 81f conversion of nonischemic CRVO to ischemic CRVO 81, 82f macular edema 77f, 79 ocular neovascularization 79, 80f vitreous hemorrhage 80, 80f, 81f course 79 demographic characteristics 76 diagnosis 77–8 ischemic vs. nonischemic CRVO 78, 78f, 79t hematological risk factors 75–6 investigations 85–6 local risk factors 75–6 management 81–2 ischemic CRVO with neovascular glaucoma 85 medical 82 surgical/invasive 82–3 natural history of visual outcome 85 pathogenesis 75 site of occlusion 76, 76f systemic risk factors 75–6 Central serous retinopathy (CSR) mfERG results 510, 511f RPE barrier breakdown 65, 66f Centrifugal fibers, interplexiform cell synaptic input 473 Centrioles definition 575 in photoreceptor outer segments 576, 577f Cephalopods polarization sensitivity 671–2 behavior based on 673–4 see also Squid Cerebelloculorenal syndrome (Joubert syndrome), retinitis pigmentosa and 704 Cerebral achromatopsia 137 Cerebral cortex acquired color blindness 137 prosthetic devices 354–5 Cerebrospinal fluid (CSF) analysis, optic neuritis 538 Change probability definition 551 perimetry 556 Channels, definition 163 Cheliceral ganglia, circadian regulation in Limulus 420, 421f Chelicerate, definition 416 Chemical barriers (innate immunity) 376 lactoferrin 377 lipocalin 377 secretory PLA2 376 Chemical-induced color blindness 139 Chemoattractant, definition 541 Chemokine(s) innate immune response and 379 lacrimal gland and light damage role 342 RPE and 767, 768t, 769f Chimeras, zebrafish 856 Chlamydia pneumoniae, AMD pathogenesis 272 Chloride/bicarbonate (Cl--/HCO3-) exchanger RPE and metabolic load reduction 764, 766f Chloride ions (Cl–), RPE transport and 764, 766f Chloroquine, acquired color blindness 138 CHM gene/CHM protein, retinitis pigmentosa 703 Choriocapillaris f, 755, 756f definition 28, 87, 179 embryology 179 fenestrae 182 functional role 182 OCT tomograms 529–30, 530f other capillaries vs. 180 pathology 182 RPE role in survival/maintenance 757 Choriocapillaris pillars, definition 87 Choroid definition 87 development 754, 754f diseases 346–8 function/functional importance 755 impact of changes on RPE 757 inner 30 OCT imaging, three-dimensional wide-field 527, 528f outer (Haller’s layer) 30 receptor expression 757 structure 755, 756f vasculature see Choroidal vasculature

880

Subject Index

Choroidal neovascularization (CNV) 87–95, 182, 271 AMD and dry AMD 182 wet (exudative) AMD 182 animal models 90 growth factor models 90 histopathology 89–90 laser-induced CNV 90 clinical detection 87, 88f fluid/blood leakage 87, 88f fluorescein angiography 87, 89f indocyanine green (ICG) angiography 87, 89f optical coherence tomography 89, 90f definition 753 future directions 95 histopathology 89 pathobiology 91–2 angiogenesis 91 angiogenesis inhibitors (natural) 91 complement 92 inflammation 91 matrix metalloproteinase 91 monocytes/macrophages 91 transforming growth factor-b (TGF-b) 91 vascular endothelial growth factor 91 vasculogenesis 92 therapy 92 Choroidal vasculature 30, 179–82 anatomy cilioretinal artery and 653–4 gross anatomy 179–82, 180 ophthalmic arteries and 180–1 embryology 179 pathology 182 physiology 182 regulation 34–5 Choroideremia, retinitis pigmentosa 703, 704f Choroid plexus epithelium, retinal pigment epithelium (RPE) vs. 774 Chromatic adaptation 325, 330, 330f definition 330 pi mechanisms 330 two-stage models 330 Chromatic function of cones 96–104 eccentricity and 102 isolation of responses rod intrusion 101 selective chromatic adaptation 100, 100f silent substitution 100–1, 101f spatial densities and 101 individual variations 101 retinal variations 101 Chromophore(s) butterfly 149–50, 150f absorbance spectra 150, 150f, 151t spectral heterogeneity 150 definition 17, 205, 668 polarization sensitivity 670 Chronic progressive external ophthalmoplegia (CPEO) Kearns–Sayre syndrome and 704 retinitis pigmentosa and 704 Cilia definition 575, 811 human disease and 680–1 photoreceptors 812f, 813–14, 814f rod photoreceptors 677 ROS as modified cilium 680 Ciliary, definition 205 Ciliary axonemes definition 219 in outer segments of photoreceptors 576, 577f, 578f, 579f Ciliary body (CB), proliferative vitreoretinopathy and 711 Ciliary neurotrophic factor (CNTF) light damage prevention 343–4 retinal remodeling reversal/prevention 365 retinitis pigmentosa management 696 in rod photoreceptor development 268–9 Ciliary (Gt) opsins 208 Ciliary photoreceptors 567–8 definition 438 Cilioretinal artery 184, 653 choroidal origins 653–4

definition 653 occlusion 80–1, 81f, 653–4, 656f occurrence 184 Cilium see Cilia Cip/Kip cyclin-dependent kinase inhibitors in cell cycle regulation 171, 171f retinal development role 172f, 174 Circadian clock(s) 112 cell autonomous 127 central (pacemaker) 126, 127 definition 105, 106, 416 entrainment 105 fly circadian system(s) 126 peripheral 126, 127 photoreceptors as 129 synchronization 126 see also Circadian rhythm Circadian oscillators, definition 118 see also Circadian rhythm(s) Circadian photoreception 112–17 blindness and 113, 117 dopamine role 497, 498 interplexiform cells and 475 health implications 475 nonvisual opsins 113 mammalian 113, 115t nonmammalian 113 nonvisual photoreception 112–13 historical aspects 113 mammalian 112–13 nonmammalian 112 photoreceptors 113 classical 116–17 extraocular 112 see also Circadian rhythm(s) SCN and central projections 112, 113f, 115 Circadian rhythm(s) 116, 118 chick retina 68–74 cAMP 69, 72, 72f clock genes 68–9, 69f, 70f, 71f iodopsin 73–4 melanopsin 73 melatonin synthesis 71, 72f, 73f phospholipid metabolism 73 clock genes 126–7, 128f definition 68, 112, 494, 500 dopaminergic neuron regulation 108, 495 entrainment, definition 105, 124 eye components 116 ion channels in photoreceptors 118–23 cGMP-gated channels 74, 119 dopamine 120–1 signaling pathways 120, 121f somatostatin 121 channel types 119 L-type voltage-gated calcium channels 74, 121–2 potassium channels 122 regulatory function of circadian rhythms 118, 122–3 retinal disorders caused by abnormalities in 122–3 phase resetting 112, 114f retinomotor movements 219–20, 225 RPE effects 502 Circinate lipid exudates, definition 426 Circle of Zinn 179, 180, 180f, 181f Circular polarization definition 668 mantis shrimp 674 Circumferential marginal zone (CMZ) 860 definition 863 persistent neurogenesis 865, 867f Circumpallial nerve 439–40 definition 438 Class B scavenger receptors, definition 726 Claudins definition 773 tight junctions 51–7 RPE 762, 775f, 776, 776f, 778 Clinically significant macular edema 785, 787f, 787t diabetic 428 Clock-controlled genes, definition 68 Clock (clk) gene/protein 127, 128f

Subject Index Clock genes/proteins chick retina 68, 69f, 70f, 71f definition 68, 124 Drosophila 126–7, 128f see also Circadian clock(s) Clomeleon, definition 452 Cnidaria definition 205 opsins (cnidops) 206 Cnidops (Cnidaria opsins) 206–8 Coat’s disease, retinitis pigmentosa and 700, 701f Co-duplication definition 205, 210 opsin evolution 210 Coelocanth 154 Collagen(s) disorders vitreoretinopathies 252, 260 Coloboma, definition 198 Color blindness acquired 134–9 changes with time 135 classification 134–5 cortical defects/injury 137 inherited vs. 135–6, 135t ocular diseases 136 range 139 toxin-induced 138 clinical assessment 135–6 inherited 140–7 achromatopsia 146 acquired defects vs. 135–6, 135t blue-cone monochromacy 146 blue–yellow (tritan) defects 144 epidemiology 143, 144–5, 146 genetic basis 141, 142f red–green defects 143, 144f, 145f perceptual consequences 134, 135f, 143–4, 145f, 146 Color constancy, chromatic adaptation and 330 Color Doppler imaging (CDI), ocular blood flow measurement 35, 35f Color-matching functions, definition 96 Color vision 134, 624 adaptive optics (AO) 11 genetic basis 141–3, 142f variability and 141 impaired see Color blindness insect 209–10 see also Butterflies, color vision light level and 565 opsins and 208 prostheses and 358 Stell chromatic processing model 316, 316f turtle 460 vertebrate vs. insect 209–10 Commission Internationale d’Eclairage (CIE), definition 96 Competence, definition 169, 745 Complement 377, 378 definition 346 ocular immune responses and active defense 378 as chemical barrier 377 immune surveillance 378 ocular inflammation and disease drusen formation 271 factor H (CFH) 272–3, 833 neovascularization 350 neovascularization, choroidal (CNV) 92 proangiogenic activity 548 Complement factor H (CFH), polymorphism in AMD 272–3, 833 Compound eyes definition 416 insect (fly) 124, 729, 729f, 730f circadian rhythms 127 Limulus lateral eyes 416, 418f polarization sensitivity 671–2, 672f distinguishing from color vision 672–3 Concentric retinitis pigmentosa 701 Conditional knockout mouse, definition 567 Cone bipolar cells 22–3, 157f, 158, 285–6, 452, 455 cone signaling pathway 23–4, 23 glutamate receptors 23

types/classification 23 functional 23 morphological 23 Cone dystrophies 136, 684, 698, 853–4 bystander effect 858 Cone-isolated ERG responses 706, 706f Cone matrix sheath 813f Cone mosaic, adaptive optics retinal imaging 11, 11f, 12f Cone opponency 102, 103f advantages 102–3 Cone pathways definition 105 dynamic range and 845 Cone pedicle, definition 461 Cone photoreceptor(s) 17, 22, 96–7, 140, 156–62, 558, 567, 854 biophysical properties 158 degeneration 136, 684, 698 zebrafish pde6 mutants 860–1 development 568, 569f from cone precursor 569f, 570f, 571 distributions 101, 568 Drosophila photoreceptors vs. 729, 730f electrical coupling 159, 160–1 cone–cone 160–1 negative feedback and 161 evolution 22, 841 functional properties 159, 624, 625f functional roles 156, 624, 841–2 importance 162 goldfish retina 106, 106f imaging, adaptive optics (AO) 11 light sensitivity/dynamic range 105–6, 159, 589 efficacy of activation 589 importance to vision 589 rapid responses 589 rods vs. 589 saturation avoidance 589, 594 mosaic 568 adaptive optics retinal imaging 11, 11f, 12f mouse retina 568 numbers 625–6 photopigment 97, 141, 626 dominant negative interactions 145–6 genetics 141–3, 142f Mu¨ller cells and 18 photobleaching 327 rods vs. 626 variability of 141, 142f polarized light and 670–1, 672 retinal remodeling and 366 retinomotor movements 219, 220, 220f function 226 regulation by adenosine 225 regulation by cAMP 222–3, 223f, 224f regulation by dopamine 223–4 rod:cone densities 589 rods vs. 18, 589, 605–6, 607, 624, 625f, 626, 628, 628f spatial densities 101 individual variations 101 retinal 101 structure 156, 157f axon 156 gap junctions 159 invagination and triad 158 membrane proteins 158–9 morphology/topology 156 outer segment 626, 648 spectral sensitivity and 159 synapses 22–3, 158, 462 terminal 156 types/subclasses 140, 141f, 159, 568 Cone phototransduction 22, 97, 159, 603, 624–30 achromatopsia and 146 activation 627 PDE and cGMP hydrolysis 627 transducin and 627 adaptation 589–95 bandpass adaptation filter (spatiotemporal) 162 predictive coding and 159 cGMP channels and Ca2+ electrical coupling effects 160, 161

881

882

Subject Index

Cone phototransduction (continued) genetically modified mice models and 630 negative feedback 161 ephaptic theory 161 GABAergic theory 161 pH theory 161 noise 160 nonlinear synaptic transfer function 159, 160f obstacles to study of 625 outer segments and 626 photopigment and 626 distribution 626 mammalian photoreceptors 626–7 rods vs. 626 stability 626 vesicle release 158, 160 Cone phototransduction inactivation 605–9, 608f, 627 arrestin binding and opsin shutoff 606, 627–8 GRK1/GRK7 phosphorylation and 606, 627 light adaptation and 593, 593t opsin stability/kinetics and 606 PDE inactivation 607 restoration of Ca2+ and cGMP levels 607, 627 upregulation of GC 628 time constants 593, 593t transducin inactivation by RGS9 607, 628 Cone–rod dystrophy 853–4 congenital, rhodopsin mutations 645–6 symptoms 690 terminology/definitions 684, 698 Cone spectral sensitivities 97, 97f, 140, 141f estimation 98, 99f, 99t cone gains 99 templates 99, 100f von Kries scaling 99 fundamental 97, 97f postreceptoral spectra 102–3 prereceptoral attenuation 98 Confocal microscopy Limulus phototransduction studies 620, 620f Congenital stationary night blindness (CSNB) 684, 698 Connecting cilia, in photoreceptor outer segments 578, 578f Connexin(s) cones 159 definition 156, 461 Connexin36 (CX36) 24 Connexin57 (CX57), knockout effects on horizontal cell processing 316 Contact lens electrodes, use in mfERG 507 Contrast definition 290, 325 detection 163, 164f image 163, 164f intensity vs. 290 Contrast adaptation 290, 325, 329–30 contrast-modulated stimuli 329, 329f definition 329 spatial 290, 329 temporal 291, 329 flicker adaptation 329 multiple sites 329–30 Contrast constancy, definition 163 Contrast-detection threshold 163 definition 163 Contrast sensitivity 163–8, 290–4 contrast processing and adaptation 290 definition 163 physical limits 293 information loss and 293 statistical properties and 293 synaptic release rates and 293–4 threshold measurements 292 receptive field properties and 292, 292f retinal pathway disruption and 292 brain pathways and 292–3 LGN magnocellular lesions 293 LGN parvocellular lesions 293 spatial receptive field 290 light level and 561 linear vs. nonlinear RFs 291 ON vs. OFF bipolar cell synapses 290–1

RF properties and 292, 292f see also Contrast sensitivity function (CSF) temporal receptive field 166, 168f, 291–2 RF properties and 292, 292f temporal filter concept 291–2, 291f Contrast sensitivity function (CSF) 165, 167f definition 558 light level and 562, 563f flicker perception 563, 564f Corneal epithelium, immune privilege, 375 Cortical prostheses 354–5 Corticosteroids A-AION and GCA management 407 guidelines 407 NA-AION management 404–5 nonarteritic PION management 410 optic neuritis therapy 539–40 Cotton wool spots 784f, 785 definition 653, 781 radial peripapillary capillaries and 656 Coverage factor amacrine cells 449 definition 447 Cow model, glaucoma 42 CRALBP (cellular retinaldehyde-binding protein) 650 CRB1 gene/CRB1 protein, retinitis pigmentosa 701–2 CRBP1 (cellular retinol-binding protein type 1) 650 C-reactive protein (CRP) AMD pathogenesis 273 GCA and A-AION 405–6, 408f Creatine kinase (CK), extraocular muscle isoforms and ATP Critical flicker frequency, definition 163 Critical fusion frequency (CFF), multiple rod pathways and 25, 26f Crossover inhibition 321, 321f amacrine cells and 322 Crustaceans, polarization sensitivity 671–2, 672–3 circularly polarized light 674 mantis shrimp 674, 674f Cryotherapy, as cause of neovascularization 346–8 Cryotherapy for Retinopathy of Prematurity (CRYO-ROP) study 792 Cryptochromes (CRY/Cry) CRY1/CRY2 68, 69f, 71f definition 124 light entrainment role 127, 128f CSME (clinically significant macular edema) 785, 787f, 787t see also Macular edema C-terminal-binding protein 2 (CtBP-2) 663 CX3CR1, AMD pathogenesis 273–4 Cyanopsia 137 Cybrid cells, definition 333 cycle (cyc) gene/protein 127, 128f Cycles per degree, definition 1 Cyclic AMP (cAMP) circadian rhythms chick retina 69, 72, 72f Limulus eyes 422t, 423 in photoreceptors 120 definition 219 IFNg signaling 770–1 neuropeptides and 478–9 retinomotor movement regulation 222–3, 223f, 224f, 226 Cyclic GMP (cGMP), in molluskan ciliary photoreceptors excitation 443, 444f light adaptation 445, 446f Cyclic GMP (cGMP) and phototransduction achromatopsia and 146 cone phototransduction 158–9, 626 Ca2+ regulation and light adaptation 630 hydrolysis and activation 627 saturation avoidance in light adaptation 594 synthesis and inactivation 627, 628 definition 853 Limulus phototransduction and 622 melatonin effects on 503 rod phototransduction 610–11, 635, 821, 842–3 synthesis and inactivation 612 zebrafish phototransduction 856–7, 856f Cyclic GMP (cGMP)-gated cation channels (CNGC/CNG channels) circadian regulation in photoreceptors 74, 118–19 dopamine 120 signaling pathways 120, 121f

Subject Index somatostatin 121 definition 118 in molluskan ciliary photoreceptors 445 structure and function 119 Cyclic nucleotide-gated ion channels (CNGs) definition 205, 438 rods phototransduction and 635 Cyclin(s) in cell-cycle regulation 170, 171f retinal development role 172, 172f Cyclin-dependent kinase inhibitors (CDKIs/CKIs) in cell cycle regulation 171, 171f retinal development role Cip/Kip 172f, 174 Ink4 172f, 174 p19Arf 174 Cyclin-dependent kinases (CDKs) in cell-cycle regulation 170, 171f retinal development role 172, 172f Cyclopamine 214 Cyclophilin, definition 726 Cyclopia 214, 215f Cystic fibrosis transmembrane conductance regulator (CFTR) 770–1, 771f Cystoid macular edema 76 CRVO-associated 77f, 79 definition 426 fluorescein angiography 428–30, 429f treatment in RP 696 Cytochalasin D, definition 219 Cytokeratins, definition 708 Cytokines definition 346, 761 innate immune response and 379 in neovascularization 348, 349f, 350, 351, 351f RPE and 767, 768t, 769f Cytoskeleton axonemes in photoreceptor outer segments 576 retinomotor movement in photoreceptors 220, 220f retinomotor movement in RPE cells 223 ribbon synapses 664 rod photoreceptor structure 677

D Dark adaptation 325, 330 characteristics 331 cones 590–1, 590f, 624–5, 628, 628f factors affecting 628–9 recovery of sensitivity 331, 331f rods vs. 628, 628f definition 330–1, 596, 624, 705 disadvantageous nature 596, 603 equivalent background concept 331 flicker detection threshold 331 horizontal cells and 314–15 hue (Lie-specific) threshold 331 human gene mutations/genetics and 332 mechanisms 332 photopigment regeneration 332 postreceptoral factors 332 visual cycle regulation 644, 651 retinitis pigmentosa 690, 691 rods 602, 603, 628, 628f, 644 cellular model 603 human plot 603, 604f melatonin and 503–4 recovery of sensitivity 331, 331f time course 331, 331f transient tritanopia 331–2 Dark current definition 631 rod phototransduction and 635 Dark light, definition 631 Dark membrane potential, rods 842 Dark noise, rod phototransduction and 633 ‘Dark thermal events’ 821–2 Ddx39, definition 847 Defensins 376 Deformable mirror 8f definition 7

883

Demographic, definition 75 Demyelinating disease, mfERG/mfVEP results 522f, 523, 523f Dendrodendritic synapses 277f definition 276 11-Deoxyjervine 214 Detection acuity 1, 2f measurement 1, 2f see also Visual acuity Detection of apoptosing retinal cell (DARC) 734–5 Deutan defects 134, 143–4, 144f optic neuritis and 137 Deuteranomalous trichromats 143–4, 144f Deuteranopia/deuteranopes 143–4, 144f definition 134 Devic’s disease, optic neuritis vs. 539 Dextromethorphan 739 DGAT1 (diacylglycerol acyltransferase type-1) 650 Diabetes control and complications trial (DCCT) definition 781 glycemic control as risk factor 782f, 783f Diabetes mellitus (DM) classification 781 complications 781–2 definition 781 epidemiology 781 NA-AION 402, 404f Diabetic macular edema 77–8, 428, 429f carbonic anhydrase role 432 fluorescein angiography 428–9, 429f VEGF role 432, 434–5 Diabetic retinopathy (DR) 781–9 acquired color blindness 137 angiogenesis/neovascularization 185 pathogenesis 783–4 VEGF and ischemia 185 blood–retinal barrier breakdown 64, 65f classification 784, 786t definition 781 inflammation role 341 interplexiform cells and 475 pathogenesis 782 pericytes 783 PDGF effects on RPE 767–9 risk factors 782 duration of DM 782, 782f glycemic control 782, 782f, 783f screening 788 treatment 785–6, 787–8 Diabetic Retinopathy Clinical Research Network, clinical trials, diabetic macular edema therapies 432, 435 Diacylglycerol (DAG) Limulus phototransduction and 619–20 in molluskan microvillar photoreceptors 440–1, 441f Diacylglycerol acyltransferase type-1 (DGAT1) 650 Dichoptic stimulation, retinal adaptation studies 325 Dichroism 669–70, 669f definition 668 Dichromacy definition 140 horizontal cell cone selectivity and 467 Dielectric surfaces 669–70, 669f definition 668 Diet, protective against AMD 833 Differentiation, definition 263, 745 Diffuse bipolar cells 23, 454f, 455 Diffuse unilateral neuroretinitis (DUSN), retinitis pigmentosa vs. 693, 696f Diffusion active transport vs. (photoreceptors) 413 Digitalis, acquired color blindness 138 3,4-Dihydroxyphenylacetic acid (DOPAC) circadian changes in levels 495 light-induced changes in levels 494–5 Directional selectivity 295–300, 322 bipolar cells 299, 459 definition 276 integration of multiple cooperative mechanisms 299 Direction-selective ganglion cells (DSGCs) 295–300, 305–6, 322 acetylcholine and excitatory input 296, 299 circuitry 296, 298f, 305, 305f, 323f amacrine cell connections 282, 282f, 306, 322 bipolar cell connections 459 classification/types 304–5

884

Subject Index

Direction-selective ganglion cells (DSGCs) (continued) cooperative integration and 299 definition 305, 318 inhibitory control (GABAergic) 296, 298, 305 geometrically asymmetrical inhibition 296–7, 306 presynaptic (bipolar cell) inhibition 306 suppression during preferred movements 306 morphology 296–7, 297f null direction vs. preferred direction 295, 296f, 305 physiological functions/types 295 ON–OFF type 295, 304–5 stratification and 296–7 ON type 295, 304–5 Disinhibition, definition 228 Disk membranes, photoreceptors 812f renewal 815–18 catabolism 817–18, 817f in cones 815–16, 816f disk formation 817 opsin transport 816–17, 816f in rods 815, 816f see also Photoreceptor(s) Distal retina, definition 284 Diurnal rhythm, definition 500 see also Circadian rhythm(s) Docosahexanoic acid (DHA), retinitis pigmentosa treatment 696 Dog models, glaucoma 42 Dominant-negative, definition 753 Dominant-negative effect 252 Dominant optic atrophy (DOA) 335 clinical features 335, 336f epidemiology 335 genetics 335 gene identification 335 hearing loss and 335 histological features 335 Donder’s curve, definition 333 Dopamine/dopaminergic neurons 494–9 definition 105 melatonin colocalization 495, 502, 503 neuronal activity regulation 494 circadian control 495 dopamine synthesis/metabolism and 494 light responses 494 retina/phototransduction role 451, 494, 496 amacrine cells and 279, 497 bipolar cells and 497 circadian regulation and 108–9 circadian regulation of CNGCs in photoreceptors 120 developmental 498 horizontal cell modulation 315, 496 Mu¨ller cells and 497 neuromodulatory functions 473 nonphotopic vision 497, 498 photopic vision 498 photoreceptors and 496 projections 494, 495f retinomotor movement regulation 223, 225–6 RGCs and 497 RPE and 496 synthesis/metabolism 494 Dopamine receptors 495 as GPCRs 495–6 retinal expression 496, 496t amacrine cells 497 bipolar cells 497 circadian regulation and 108 ganglion cells 497 horizontal cells 496–7 Mu¨ller cells 497 photoreceptor cells 496 RPE 496 Dot-blot hemorrhages, in diabetic retinopathy 784f, 785 Double-strand break (DSB) mutagenesis, zebrafish 855–6 Drivers, visual acuity requirement 4 Drosophila melanogaster circadian (clock) genes/proteins 126–7, 128f developmental control genes 727 human similarities 729 behavior 728 functionally equivalent genes 727

metabolic/signaling pathways 727–8 phototransduction cascade 730 importance as a model organism 727–8, 732–3 transgenic flies 729 Drosophila models, glaucoma 42 Drosophila models of retinal degeneration 726–33, 854 ABC-type multidrug transporter and 727 historical aspects 727 large scale mutagenesis screens and 728–9, 730 mechanisms of degeneration 731 dual role proteins 732 light-dependent degeneration 731 light-independent degeneration 731 other models vs. 727 retinitis pigmentosa 730 visual assessment DPP 730 ERG analysis 728–9, 730 Drosophila phototransduction 242, 617–18, 622, 623, 729–30 early studies 240 genetic screens for mutants defective in phototransduction proteins 242, 243f Gq protein 244 light-dependent phospholipase C activation 245, 246f ina mutants 242, 243f metarhodopsin inactivation 242, 244f nina mutants 242, 243f phosphoinositide (PI) pathway 246f, 248, 248f photochemical cycle 242, 244f quantum bumps 243f, 250–1 Drosophila visual system 125f, 729, 854 circadian regulation clock genes/proteins 126–7 larval 132 locomotor activity relationship 130–1 second-order lamina neurons/glia 130, 130f serotonin role 131 tetrad synapses and 126f, 129 compound eyes 729, 729f, 730f morphology 241f developmental gene expression 727 larval 132 ocelli 124, 729 ommatidia 124, 241f, 729, 729f optic lobe 124, 125f, 126f photoreceptors 124, 240, 241f, 729, 730f opsins 729 single photon sensitivity 250 tetrad synapses 125, 126f, 129 spontaneous mutants 728f, 854 UV sensitive opsin 209–10 Drug-induced color blindness 138 Drug-induced retinopathies RP vs. 693 Xenopus laevis models of retinal disease 851 Drug toxicity, blood–retinal barrier and 64 Drug transport, across blood–retinal barrier (BRB) 49 Drusen 758, 759f classification 831 definition 270, 830 formation/composition 271, 832 fundoscopy 831f histology 832f retinitis pigmentosa 699, 700f Dry age-related macular degeneration 831, 832 acquired color blindness 136 choroidal vasculature and 182 fundoscopy 831f Duplicity theory 811 Dyad bipolar cell synapses 457f, 458 definition 452 Dynamic range of vision 105, 290, 318, 325, 589, 596, 841–6 definition 841 divergent rod/cone systems and 105–6 evolution 841 extending, light adaptation and 598 higher level adaptation 325 intrinsic retinal processes 105–6 pupillary responses 326, 841 Dynamic visual acuity 5

Subject Index Dynein definition 219 retinomotor movement 221 Dyschromatopsia, definition 333

E E2F in cell cycle regulation 171, 171f retinal development role 175 Early patterning 200 neural retina 201f optic cup 201f, 203 optic vesicle 201–2, 201f retinal pigment epithelium 201f transcription factors 199f, 200–1 Early Treatment Diabetic Retinopathy Study (ETDRS) 428, 785–6 definition 781 macula edema treatment 432–4, 435f Early Treatment for Retinopathy of Prematurity (ETROP) study 792 EAU see Experimental autoimmune uveitis (EAU) Eccentric cell definition 416 Limulus lateral eyes 416–17 Eccentricity, retinal, chromatic function and 102 Ectopia lentis, Marfan syndrome 256–7, 259f Ectopic eye, definition 212 Electrical conductance, definition 105 Electrical coupling amacrine cells 447 dopamine and 498 dopaminergic (IPC) modulation 473–4 Electron microscopes, definition 811, 815 Electron microscopy, blood–retinal barrier (BRB) assessment 55 Electroretinogram (ERG), definition 240 Electroretinography 705–6 b-wave, definition 661 cone-isolated responses 706, 706f definition 75, 124, 853 diurnal variation in flies 128–9 Drosophila models of eye disease 728–9 full-field 705 historical aspects 848 ischemic vs. nonischemic CRVO 79, 79t mixed rod–cone responses 706 multiple rod pathways and 25–7 neuropeptides 482 retinal adaptation studies 325 rod-isolated responses 706 RP assessment 691, 705 classification and 692, 693f, 694f, 695f utility 691–2 zebrafish models of retinal disease 856 ELISA definition 761 RPE analysis 762 Ellipsoid definition 219 structure 811–12, 812f Embryogenesis, definition 541 Emmetropia, dopamine role in establishing 498 Endocannabinoids 717 definition 228, 717 inactivation/metabolism 717, 718f retinal enzymes 720, 721t structures 718f synthesis/release 717 Endocytosis, definition 374, 819 Endophthalmitis, definition 374 Endostatin, CNV pathogenesis role 91 Endothelin(s), retinal vasoconstriction role 34 Enhanced S-cone dystrophy 258, 261 ENSLI amacrine cells circadian rhythms and somatostatin 121 definition 118 Entrainment (circadian), definition 105, 124 Enzyme-linked immunosorbent assay (ELISA) definition 761 RPE analysis 762 Eomes 238

Ephaptic feedback theory 161 Ephaptic transmission definition 156, 309, 461 horizontal cell–photoreceptor 314 Eph receptor(s) proangiogenic activity 546, 547f Ephrins, proangiogenic activity 546, 547f Epidermal growth factor (EGF) family goblet cells and inhibition of rod development 268–9 Epigenetic, definition 836 Erythrocyte sedimentation rate (ESR), GCA and A-AION 405–6, 408f Erythropoietin (Epo) angiogenic activity 548 retinopathy of prematurity and 798 Ethambutol, acquired color blindness 138–9 Ethical issues, stem/progenitor cell therapy 368 ETROP study 792 Eumetazoa, definition 205 Evans blue assay, blood–retinal barrier (BRB) assessment 54–5 e-vector 668 definition 668 Evolution amphibian retina 847–8 butterflies 148, 149f, 152f, 153f dynamic range of vision and 841 rod bipolar pathway 842 horizontal cell morphology and 467–8 intrinsically photosensitive retinal ganglion cells 115 melatonin, retinal synthesis 501 photoreceptors 22 rods 22, 841 retina circuitry 22 pineal gland relationship 501 Excitation, definition 301 Excitotoxicity, definition 734 Excitotoxicity-induced apoptosis, in glaucoma 737, 738f Extracellular matrix (ECM) definition 346 degradation, apoptosis and 742 injury-induced angiogenesis 350, 351f proliferative vitreoretinopathy and 711, 712f, 713 retinal cell therapy/transplantation barrier 371 glial scarring and 372, 372f myelin and 372 Extrastriate cortex (V4), acquired color blindness and injury 137 Extrinsic factor, definition 684, 698, 836 Eye(s), cannabinoid effects on 719 Eye development Bruch’s membrane 755 dopamine role 498 Drosophila 727 early 198–204, 199f Limulus visual system 418 retinal pigment epithelium (RPE)–choroid interactions during 756 Xenopus models 851 zebrafish 864, 864f Eye field, definition 212 Eye-field transcription factors (EFTFs) 212–18 discovery 212 homologs, names/null phenotypes 213t, 214–15 mutations, eye abnormalities associated with 215–16 role in normal eye development 214, 215f coordinated expression and 216, 217f effects of environmental toxins/genetic mutations on 214, 215f self-sustaining feedback network 216, 217f structural features 212, 213f Eyelashes as part of the passive defense system 374 eyeless gene/protein 727 Eyelids(s) as part of the passive defense system 374

F Familial exudative vitreoretinopathy (FEVR) 259, 261f Fas, pathogen clearance 379 FDT (frequency-doubling technology) perimeter 554 Feedback basic principles 279, 279f definition 276

885

886

Subject Index

Feedback synapse definition 284 Feed forward, definition 276 Fenestrae choriocapillaris 182 definition 179 Fenestration(s), definition 28, 51, 753 FEVR (familial exudative vitreoretinopathy) 259, 261f Fibrinolytic therapy, CRVO 83 Fibroblast(s), proliferative vitreoretinopathy and 710f, 711 Fibroblast growth factor(s) (FGFs) Fibronectin(s), proliferative vitreoretinopathy and 713 Fibrous tissue envelope (FTE) 75–6, 76f Filter, definition 290 Filtering pigments butterfly 153–4 definition 148 First-order kernel, definition 506 Fish, polarization sensitivity 672 Flap tears 801, 802, 802f Flicker adaptation 329 Flicker–fusion frequency, rapid photocurrent recovery 593 Flicker perception 563, 564f, 565f multiple rod pathways and 25 Flippases, definition 726 Floaters, rhegmatogenous retinal detachment and 801–2 Fluocinolone, intravitreal, macular edema treatment 82 Fluorescein fundus angiography blood–retinal barrier (BRB) 48, 48f, 55 choroidal neovascularization (CNV) 87, 89f CRVO, ischemic vs. nonischemic 77f, 78–9, 78f definition 75, 653 macular edema 428–30, 429f cystoid 428 diabetic 428 ocular blood flow measurement 35 RPE barrier assessment 61, 61f, 62f 5-Fluorouracil, PVR management 714 Fly circadian system(s) 126–7 Drosophila clock genes/proteins 126–7, 128f light entrainment 126 light input 127 other species 127 Fly visual system(s) 124–5 ocelli 124, 729 optic lobe and optic neuropil 124, 125f cartridges 124–5 photoreceptors 124, 125, 126f retinal functions 124–5 ultraviolet detection 209–10 Fly visual system, circadian rhythms 124–33 circadian circuitry 132 crustaceans vs 127–8 first visual neuropil (lamina) 129–30 driving factors 129 photoreceptor terminal organelles 131 second-order neurons/glia plasticity 130–1, 130f synaptic contact plasticity 129 functional roles 132–3 larval visual system 132 locomotor activity relationship 130–1, 132–3 neurotransmitter regulation 131 FMRFamide 131 GABA 131 histamine 131 PDF 131 serotonin 131 predictive nature 133 retina of compound eye 127–9 ERG patterns and 128–9 gene expression and 129 photoreceptors as peripheral clocks 129 photoreceptor size 127–8 pigment changes 128–9 stress and 132–3 FMRFamide, fly circadian plasticity and 131 Focal adhesions, definition 58 Forced-choice paradigms, definition 163 Four-alternative forced-choice (4AFC) detection task 163, 164f Fourier analysis, definition 163

Fovea, definition 426 Foveal avascular zone (FAZ), definition 426 Foveal region, capillary bed 657, 657f Foveola 427 definition 426 FOXN4, amacrine cell specification and 750 Frequency-doubling technology (FDT) perimeter 554 Friedreich’s ataxia optic neuropathy 336 retinitis pigmentosa and 704 Full-field electroretinography 705 Fundamental spectral sensitivities, definition 96 Fundus albipunctata 702, 703f Fundus examination/fundoscopy AMD 831f ischemic vs. nonischemic CRVO 78f, 79 NPDR 784f PDR 788f retinal injury/neovascularization 347f mouse 348f

G GABA fly circadian plasticity and 131 as major inhibitory transmitter 228 retinal 228–34, 229t amacrine cells 233, 278, 322, 448–9 cellular signaling 479, 482f co-localization with neuropeptides 485 cone negative feedback and 161 horizontal cells 228–9, 232, 312, 313 lateral inhibition role 228 metabolism 230f GABAA receptors 231–2 amacrine cells 233 benzodiazepine binding 231–2 bipolar cells 233 horizontal cells 232 Mu¨ller cells 234 neuropeptide co-localization and 490 photoreceptors 232 RGCs 233 GABAB receptors 231–2 amacrine cells 233 functional role in light response 233 RGCs 233 GABAC receptors 231–2 bipolar cells 233 horizontal cells 232 photoreceptors 232 salamander RGCs 233 GABA receptors 228, 231 classes 231–2 retina 228–34, 229t, 230f amacrine cells and 233, 278–9 bipolar cells 233, 289, 313 horizontal cells 232 Mu¨ller cells 234 photoreceptor terminals 232, 313–14 RGCs and 233 species differences 232–3 zinc ions and 232–3 GABA transporters 228 plasma membrane (GATs) 228, 230 functional role 231 reverse operation 231 subtypes 230 retina 229t, 230f bipolar cells 231 functional role 231 GATs 230 horizontal cells 228–9, 231 Mu¨ller cells 230 neuronal localization 230 RPE 231 VIAAT 228 vesicular (VIAAT) 228 Gabors 164–5, 165f definition 163

Subject Index Gadolinium, definition 536 GAF domains definition 853 zebrafish pde6 mutants 861, 861f Gain control definition 309 reduction of 845 definition 276, 325 Ganglion cell layer (GCL), definition 235 Gap junctions 24 definition 105, 309, 461, 853 GAT-1 230 retinal expression 229t, 230f amacrine cells 230 GAT-2 230, retinal expression 229t, 230f GAT-3 230 retinal expression 229t, 230f Mu¨ller cells 230 GDP (guanosine diphosphate), rod phototransduction and 634 Gene co-option definition 205, 210 opsin evolution 210 Gene duplication butterfly opsins 151–2, 152f, 154 co-duplication definition 205, 210 opsin evolution 210 crystallin evolution definition 148 opsin evolution 209 Gene homology, definition 212 Gene therapy primary 364 retinal disorders and retinal remodeling and 364, 365 retinitis pigmentosa 696 secondary 365 Genetic factors/genetics retinal regeneration 869 retinopathy of prematurity 795 Genetic recombination cone photopigment genes 141 Genuine S-cones, definition 452 Geographic atrophy (end-stage dry age-related macular degeneration) 831–2 fundoscopy 831f Giant cell arteritis (GCA) clinical features 405 laboratory investigations 405, 408f signs 405 symptoms 405 systemic 405 definition 399 ischemic optic neuropathy 405 management 406 steroid therapy and 407 timing and outcome 407 Glaucoma 381 acquired color blindness 136 Alzheimer’s disease and 740 compartmentalized self-destruct pathways in 386 definition 75, 734 diagnosis 734 drug therapy/prevention 735 early detection 381–2 elevated intraocular pressure as risk factor 381 Xenopus laevis model 849 Glaucoma Hemifield Test (GHT) 556 Glaucoma models 736 animal/in vivo 38–43, 736 nonmammalian 42 selection criteria 38 neuroprotective agents 743, 743t in vitro 736 Glial cells definition 381 Drosophila visual system, circadian regulation 130, 130f optic nerve head, response to elevated intraocular pressure (IOP) 382 proliferative vitreoretinopathy and 710 Glial fibrillary acidic protein (GFAP), definition 708

Glial–neuronal interactions, apoptosis and 741, 741f Glial scar/gliosis proliferative vitreoretinopathy 709 retinal cell therapy/transplantation barrie 372–3, 372f Glial seal, definition 360 Global indices, perimetry 555 Glutamate excitotoxicity 737 Glutamate/glutamatergic signaling retina BC–amacrine synapses, 278 melatonin effects 503 Glycemic control, DR risk factor 782, 782f, 783f Glycine/glycinergic transmission amacrine cells 278, 322, 448–9 melatonin effects 503 Glycoconjugates, definition 811 Goldfish retina (rod/cone pathways) 106, 106f circadian regulation 106 mammalian retina vs. 107 Goldmann–Favre syndrome 258, 261 Goldmann perimeter, ischemic vs. nonischemic CRVO 79 Golgi apparatus, definition 676 Go opsins 208 G-protein(s) definition 205, 240, 637 Go 205, 208 Gq 205, 208 Drosophila 244 light-dependent phospholipase C activation 245, 246f squid 585 Gs 205, 206–8 Gt 205, 208 molluskan ciliary photoreceptors 443–4 rhodopsin signaling and 600, 612, 644 G-protein-coupled receptor(s) (GPCRs) definition 205, 477, 637 dopamine receptors 495–6 melatonin receptors 500, 501 neuropeptide receptors as 487, 491 photopigments as 648 type II opsins 206 G-protein-coupled receptor kinase(s) (GRKs) definition 605 opsin inactivation and 606 G-protein-coupled receptor kinase 1 (GRK1) cone opsins and 606 definition 582, 610, 637 GRK7 vs. 627 rhodopsin inactivation and 611, 644, 644f, 645, 645f, 648 light adaptation and 601 light damage and 339–40 multiple sites 612 squid (SQRK) 584f, 586, 588 G-protein-coupled receptor kinase 7 (GRK7) cone inactivation and 606, 627 GRK1 vs. 627 Green fluorescent protein (GFP), definition 124 Growth factors, RPE and barrier breakdown 66 secretion 757, 758f GTP (guanosine triphosphate), rod phototransduction and 634 Guanylyl (guanylate) cyclase (GC) definition 438 in intraflagellar transport (retinal GC1) 580–1 in molluskan ciliary photoreceptors 444 Guanylyl cyclase activating protein (GCAP) cone phototransduction and 605–6, 607 definition 605, 610 rod phototransduction and 612–14 light adaptation and 600, 601, 601f Gyrate atrophy, retinitis pigmentosa 702, 703f

H Hair cells, ribbon synapses 662 Haller’s layer 30 Haploinsufficiency, definition 252 Head injury, acquired color blindness 137 Hearing loss, dominant optic atrophy and 335 Heavy metals, acquired color blindness 139

887

888

Subject Index

Hedgehog signaling pathway 265–6, 266f retinal histogenesis 266 Heijl–Krakau technique 554–5 Hematological, definition 75 Hematological disorders, central retinal vein occlusion and 75–6 Hemi-central retinal vein occlusion (HCRVO) 75 Hemodynamics, definition 28 Hemorrhage, vitreous, in CRVO 80, 80f, 81f Henle fiber 156–8 definition 156 Henle’s fiber layer 156–8, 430 definition 426, 825 Heterochromatic flicker photometry, definition 96 Higher order aberrations (HOAs) definition 558 light level and 563, 564f High-pass resolution perimetry 553, 554f High-throughput drug screening, zebrafish models of retinal disease 856 Histamine, fly circadian plasticity and 131 Histogenesis, definition 745 Histopathological, definition 75 Histoplasmosis, as cause of neovascularization 346–8 3H-mannitol assay, blood–retinal barrier (BRB) assessment 54–5 Hofbauer–Buchner (H–B) eyelet 127 Holoprosencephaly 216 Homeobox genes/proteins definition 726, 863 discovery in Drosophila 727 retinal development 864 Horizontal cell(s) A-type 309, 461 cone-specific synapses 310, 462–3, 463f functional role 468 morphology 461, 462f vesicular GABA release 228–9 B-type 309, 461 cone synapses 310, 462–3, 463f functional role 468 gap junctions 464, 464f morphology 461, 462f rod synapses 310, 463, 823–4 species differences 467 vesicular GABA release 228–9 chromaticity (C-type, H2, H3) cells 310 definition 105 functional roles 314, 461–2 inhibitory RF surrounds and 309, 310f type-specific differences 468 luminosity (L-type, H1) cells 310 GABA and 231 neurochemistry AMPA/KA receptors 311 cannabinoid receptors 720 GABAergic activity 228–9, 231, 233, 312, 313 GAT expression and GABAergic activity 231 see also Horizontal cells, information processing neuromodulators and 310–11, 314–15 all-trans-retinoic acid and 315 dopaminergic regulation 315, 496 nitric oxide and 315 possible third type 467 retinal population density 464, 464f synapses 310, 319, 319f cones 22–3, 24, 157f, 158, 462 development 393f, 394f, 396 as feedback mechanism 24, 461–2 GABA and 228–9, 233, 312, 313 indirect vs. direct photoreceptor pathways 313 rods 820–1, 820f, 823–4 type B axon terminal (HBat) 823–4 see also Horizontal cells, information processing Horizontal cell morphology 461–9 basic morphology 461, 462f A- vs. B-type cells 461, 462f dendritic spread 462 terminals 461 general morphology/connectivity 461 photoreceptor contacts 462 cones 157f, 158, 462, 463f rods 462f, 463, 463f, 820–1, 820f, 823–4

population properties 464, 464f species diversity 464, 468 artiodactyls 465, 466f evolutionary/phylogenetic perspective 467–8 perissodactyls 465, 466f primate types 24, 465, 465f, 466f rodents 466–7, 466f selective cone contacts 467 shape variations 465 third type of cell and 467 tree shrew (Tupaia belangeri) 465–6, 466f Horizontal cells, gap junctions/electrical coupling 24, 319, 464, 464f B-type cells 464, 464f DA effects 315 homotypical 464 retinal adaptation and 314–15 syncytium formation 310–11 uncoupling by nitric oxide 315 uncoupling via all-trans-retinoic acid 315 Horizontal cells, information processing 309–17 cellular mechanisms 312 chromatic processing 316 Stell model 316–17, 316f feed-forward onto bipolar cell dendrites 289, 313 GABA role 312, 313 inhibitory feedback 24, 309, 312, 313, 461–2 bipolar cells and 286–9 ephaptic transmission and 314 GABA and 312, 313 protons and 312, 314 RF surrounds and 309, 310f ionic conductances and light response 311 AMPA/kainate-mediated 311 light-driven hyperpolarization 311–12, 312f voltage-gated 311, 311f local gain control 309, 315, 319, 320f functional implications 316 high-gain synapses 315–16 neuromodulators and 310–11 protons role 312 roll back 310, 310f, 316 spatial processing 316 synaptic interactions 310, 319, 319f temporal processing 316 Horseshoe tears 801, 802, 802f HTRA1/HTRA1, AMD and 273, 834 Hues, unique, definition 96 Human fetal RPE culture model (hfRPE) 762, 764f Humphrey Matrix 554 Hyaloid artery arborization 182 regression 182–3 Hyaloid vascular networks 182 functional role/physiology183 pathology 183 Hyaloid vascular regression 182–3 Hydroxychloroquine retinopathy, mfERG results 510, 512f, 513f 5-Hydroxytryptamine (5-HT; serotonin), fly circadian plasticity and 131 Hyperacuity 4–5, 5f Hyperplasia, definition 198 Hypomorphic mutant, definition 212 Hypoxia, definition 541 Hypoxia-inducible factor-1a (HIF1a) retinopathy of prematurity and 798

I Iatrogenic disorders, surgical posterior ischemic optic neuropathy (PION) 408 IDDM (insulin-dependent diabetes mellitus) definition 781 Image contrast 163, 164f Immune effector cells, innate immune response and 379 Immune privilege blood–retinal barrier (BRB) and 48 corneal epithelium and 375 innate immunity and 379 Immune privilege, maintenance/aqueous humor role 379 Immune system, mucins and 374–5 Immunohistochemical staining, albumin, blood–retinal barrier (BRB) assessment 53f, 55, 55f Immunohistochemistry, definition 708 Immunosurveillance, complement role 378

Subject Index Immunotherapy, AMD 271, 834 Inactivation no afterpotential D (INAD) protein 249 discovery 249 signaling complex 249, 250f Incontinentia pigmenti (Bloch–Schulzberg syndrome), retinitis pigmentosa and 704 Increment threshold, light adaptation and 326, 326f Individual variations, cone numbers 101 Indocyanine green (ICG), angiography, choroidal neovascularization (CNV) 87, 89f Induced pluripotent stem cells 368, 369f Infection, role in blood–retinal barrier (BRB) breakdown 62 RP differential diagnosis 693, 695f Inflammation/inflammatory response AMD 271, 272, 341, 346–8 blood–retinal barrier (BRB) breakdown role 52, 62 CNV pathogenesis role 91 definition 338 innate immunity and 374, 379 light damage 342 Inhibition, definition 301 Inhibitory postsynaptic current (IPSC) bipolar cell GABA receptors and 233 definition 228 Inion, definition 506 Ink4 in cell cycle regulation 171, 171f retinal development role 172f, 174 Innate immunity 374–5 adaptive immunity relationship, 379 AMD and 272 aqueous humor and inhibition 379 components/principles 374 definition 270 inflammation and 374, 379 primary function 379 role in CNV pathogenesis 91 as two-tiered system 374 Innate immunity, active mechanisms 377 adaptive immunity and 379 immune privilege and 379 pathogen elimination and immune response 379 initiation/amplification 379 pathogen clearance 379 proinflammatory cytokines 379 pattern recognition receptors see Pattern recognition receptors (PRRs) Innate immunity, passive mechanisms 374 anatomical/physical barriers 374, 375f corneal epithelium 375 eyelids/lashes 374 posterior lens capsule 375 RPE 375 tear film 374, 376f Inner blood–retinal barrier (iBRB) 44, 45, 45f, 58, 659, 773 astrocytes 46 definition 44, 58 Mu¨ller cells 46 pericytes 46 polarity 47 retinal endothelial cells 45, 46f transport mechanisms 45–6, 46f retinal endothelial tight junctions 44–5, 46 Inner nuclear layer (INL) IPCs 470 melatonin receptor expression 502 persistent neurogenesis 865, 867f Inner plexiform layer (IPL) amacrine cells 447 bipolar cells see Bipolar cells melatonin receptor expression 502 organization 321–2, 322f sublamina 470 synaptogenesis 393–4, 393f, 394f, 395 Inner segments of photoreceptors organization 811–14 cilia 812f, 813–14, 814f proteins 812–13 structure 811, 812f, 813f Inositol 1,4,5-triphosphate (IP3) definition 438 light protection mechanisms and 343

Insects color vision 209–10 evolution butterflies 148, 149f, 152f, 153f opsins 150 polarization sensitivity 671–2, 672f behavioral responses 673, 674 distinguishing from color vision 671–2 ultraviolet and 210 in situ hybridization (ISH), definition 487 Insulin-dependent diabetes mellitus (IDDM), definition 781 Insulin-like growth factor-1 (IGF-1), retinopathy of prematurity and 798 Insulin-like growth factor-1 binding protein 3 (IGF-1BP3), retinopathy of prematurity and 798 Insulin receptor signaling, light protection role 343 Integrin(s), proangiogenic activity 548 Interferon g (IFNg) inflammatory disease pathogenesis 770–1 autoimmune uveitis and 770–1 PVR 770–1 regulation/signaling pathways 770–1 RPE effects 770 CFTR and 770–1 proliferation/migration and 769–70 regulation/signaling pathways 770–1 Interferon-inducible protein 10 (IP-10), RPE secretion 767 Interferon-inducible T-cell a-chemoattractant (I-Tac), RPE secretion 767 Interkinetic nuclear migration (INM) 174f cell birth and 176, 176f definition 169 Interleukin-1b (IL-1b) blood–retinal barrier (BRB) breakdown and 54 Internal limiting membrane (ILM), definition 825 Interneurons, dopaminergic modulation 473 Interphotoreceptor retinoid-binding protein (IRBP) 17, 628–9, 649–50 alternative visual cycle and 20 functional role 20–1 Interplexiform cells (IPCs) 451, 470–6 definition 494 dopamine secretion 470 basal release under dark conditions 475 light conditions inducing 474 functional roles/physiology 470, 473 chemical transmission modification 474 DA reconfiguration of retinal circuits 473 electrical coupling and 473–4 heterogeneity 474 intraretinal feedback 470, 471f, 473–4 novel and conventional light-driven pathways 474 ON-sustained cells 474–5 ON-transient cells 474–5 retinal circadian clock and 475 morphology 470, 472f, 473f axon-like processes 472 dendrites 471 distribution 471 homogeneity 474 soma 471 retinal degeneration and 475 diabetic retinopathy 475 Parkinson’s disease 475 retinal clock and 475 synaptic input 472, 473f amacrine cells 473 bipolar cells 473 centrifugal fibers 473 ipRGCs 473 Intraflagellar transport (IFT) definition 575 photoreceptors 578, 580f cargo 580 kinesin KIF17 579 rod outer segment 677 Intraocular pressure (IOP) cannabinoid effects 719 as glaucoma risk factor 381 Intraretinal cysts, chronic rhegmatogenous retinal detachment 803

889

890

Subject Index

Intraretinal layers, optical coherence tomography 528, 530f Intraretinal microvascular abnormalities (IRMA) 785 definition 781 Intraretinal proliferative vitreoretinopathy (iPVR) 708, 713 masses/tumors and 713, 713f, 714f Intravitreous neovascularization avascular retina and 796–7 definition 790 Intrinsically photosensitive retinal ganglion cells (ipRGCs) 114 central projections 115 definition 112 dopamine role 497 Drosophila phototransduction similarity 730 evolution 115 functions 115 circadian oscillations and 116 classical photoreceptors and 116 genetic mouse models 115–16, 116f interplexiform cell connections 473, 473f morphology 114–15 Intrinsic factor, definition 684, 698, 836 Invagination cone photoreceptors 157f, 158 definition 156, 819 rod photoreceptor(s) 820f, 821 Inverse retinitis pigmentosa 700 Invert, definition 156 Invertebrate phototransduction studies early 240 genetic 240–51 vertebrate phototransduction vs. 240 Iodopsin, circadian rhythms 73–4 Ion channels cone cells 158–9 definition 477 rod cells 821 Ionotropic receptors definition 228 IP-10, RPE secretion 767 Iris, definition 212 Iron deposition, AMD and 767 Irvine–Gass syndrome 432, 435 definition 426 Ischemia definition 541 retinal 346–8 retinopathy, VEGF and diabetic retinopathy 185 Ischemic optic neuropathy (ION) 399–411 as acute optic nerve disorder 399 arteritic vs. nonarteritic 407 blood supply schematic 400f classification 399 classification/definition 399 incipient nonarteritic 405 nonarteritic vs. 407 posterior ION 408, 410 posterior ION 408, 410 surgical 408, 410 Islet1, in retinal ganglion cell development 238 Isoelectric focusing, definition 536 Isoform(s), definition 487 I-Tac, RPE secretion 767

J Jak–STAT pathway IFNg signaling 770–1 secondary photoreceptor degeneration 840 Jansen syndrome 257–8 Joubert syndrome (cerebellooculorenal syndrome), retinitis pigmentosa and 704 Junctional adhesion molecules (JAMs), RPE inflammation and 771–2, 771f Juvenile X-linked retinoschisis, mfERG results 510, 514f

K Kainate (KA) receptors, horizontal cells and 311 Kd (dissociation constant), definition 219

Kearns–Sayre syndrome CPEO 704 retinitis pigmentosa 704 Kinesins definition 219, 575 intraflagellar transport (KIF17) 579, 580f opsin transport 817 retinomotor movement 220–1 in ribbon synapses (KIF3A) 663–4 Kinetic perimetry 551, 552f definition 551 Kniest dysplasia 253, 258f, 260 Knobloch syndrome 256 Knock-out mouse conditional, definition 567 definition 567

L Lactate, RPE transport and 765, 766f Lactoferrin, innate immunity and 377 Lambda-max 208–9 definition 148, 206 Lamina cribrosa 180 definition 75, 381, 506 IOP-related stress/strain 382, 383f vascular supply 31, 33f Lamina fusca 180, 181f Landolt C 1, 2f Landolt club, definition 452 Laser demarcation, rhegmatogenous retinal detachment 804 RRD due to retinal holes/dialysis 805 RRD due to retinal tears 805 Laser Doppler flowmetry 36, 36f Laser Doppler velocimetry (LDV) 36 Laser-induced chorioretinal venous anastomosis, nonischemic CRVO 83 Laser-induced injury definition 346 neovascularization and 346–8 animal models 348 Laser speckle technique 36 Laser therapy diabetic retinopathy 785–6, 787–8 macular edema 432–4, 434f Lateral geniculate nucleus (LGN) 292–3 contrast sensitivity role 293 definition 354 layers/organization 293 prosthetic devices 354–5 Lateral inhibition definition 416 GABAergic 228 amacrine cells 322 Limulus lateral eye 417–18 L-cones 134, 140, 159 diffuse bipolar cell connections 455 L-opsins 209 photopigment genes 141, 142f blue-cone monochromacy and 146 red–green color defects and 143 variability and 141 S-cones vs. 145–6 species differences in horizontal cell selectivity 467 spectral sensitivity 141f Leber’s congenital amaurosis (LCA) 649, 650, 687–9 definition 637 fundus appearance 689f gene therapy 364–5 rhodopsin mutations 645–6 Leber’s hereditary optic neuropathy (LHON) 333 clinical features 333–4 acute stage 334–5 clinical examination 335 fundus changes 334, 334f genetics 333 homoplasmic vs. heteroplasmic mutations 334 mtDNA mutations 333–4 penetrance 334 pathophysiology 334

Subject Index recovery 335 treatment 335 Lecithin:retinol acyl transferase (LRAT) 642, 650 knockout effects 646, 646f LED (local edge detector) 318, 323–4, 324f Lens capsule, posterior, innate defense and 375 Lens development 198–9, 200f Leukemia inhibitory factor, light damage, protection mechanism 343 Leukostasis, definition 51, 87 Lhx2 213 structural domains 213, 213f Light l-max 206, 208–9 retinopathy of prematurity pathophysiology 799 visual vs. nonvisual responses 113 Light adaptation 290, 325, 326 benefits/adaptive nature 596 Ca2+ role cone phototransduction and 607, 629 characteristics 326, 326f definitions 326, 412, 589, 596, 624, 705 functional role 290, 598 contrast information and response kinetics 598 dynamic range extension 598, 598f high-frequency linearity 327–8 mechanisms 328 human mutations/genetics and 329 multiplicative adaptation 328, 328f sites 329 subtractive adaptation 328 molluskan photoreceptors ciliary 445, 446f microvillar 441, 442f Naka–Rushton equation and 328, 328f saturation and 327f avoidance of 598, 598f cones 327 rods 327, 596–7, 597f Light adaptation, cone phototransduction 327, 329, 590, 624–5, 629 background illumination effects desensitization/acceleration 590, 590f, 591f flashes on background procedure 590–1, 590f incremental responses 591, 591f sensitivity and Weber’s law 591, 591f dynamic range and 590 intracellular Ca2+ and 607, 629 mechanisms 629–30 molecular basis 593, 594f human model 594 reaction steps underlying rapid recovery 593, 594f saturation avoidance and 594 shut-off time constants and 593, 593t photobleaching and 327, 592, 594 rapid photocurrent recovery 592 flicker–fusion frequency and 593 human ERG studies 592, 592f shut-off reaction speed 593, 593t Light adaptation, rod phototransduction 327, 327f, 329, 598 accelerated recovery 598, 598f Ca2+-dependent mechanisms 598, 598f, 600 Ca2+-sensitive proteins 600, 601 channel reactivation 602 guanylyl cyclase activation and 601, 601f negative feedback loop 600 resensitization and saturation prevention 600 shortened R* lifetime 601 Ca2+-independent mechanisms 600, 602 cGMP turnover acceleration 602 pigment depletion 602 response compression 602 desensitization/acceleration 597f, 598, 598f rod adaptation pool 328–9 saturation 596–7, 597f at high background intensities 598f, 600 prevention (range extension) 598, 598f, 600 slow changes 602 dominant time constant changes 602 Light damage 338–45 acute model 338–9, 339f chronic model 338–9, 340f genetic factors 339–40

injury mechanisms 340 apoptosis 341 inflammation 339f, 341 microarray studies 340–1 oxidative stress 341 rhodopsin role 338, 339 tissue remodeling 342 transcription factor expression 341, 342 protection mechanisms 342 antioxidant mechanism 344 as important area of research 343 LIF and 343 preconditioning effects 343 retinal plasticity 343 rhodopsin-activated 343 therapeutic potential 344 as retinal degeneration model 338, 342 Light-dependent ion channels, molluskan 445, 586 Light-driven protein translocation (photoreceptors) 412–15, 603, 614 active transport vs. diffusion 413 light-dependency of 412 neuroprotection and 413 possible functional roles 413 protein-specific mechanisms 414 return to dark-adapted locations 415 rod adaptational state and 603 Light-evoked activity, development 396, 397f Light levels 558, 559f high 558, 559f higher order aberrations (HOAs) and 563, 564f intermediate 558, 559f low 558, 559f pupil diameter and 559f, 563 quality of vision and 561 color vision 565 flicker perception 563, 564f, 565f spatial acuity 561, 562f spatial contrast sensitivity 561, 563f Limenitis butterflies, opsin diversity 152, 153f Limulus phototransduction 616–23 amplification and 617–18 early studies 240 graded depolarizations 417, 418–19, 617, 618–19, 618f kinetics 621, 621f intracellular Ca2þ increases 620 adaptation and 622 caged IP3-photolysis studies 620–1, 621f cGMP-gated channels and 622 confocal microscopy 620, 620f inward current activation 621, 622 IP3-mediated from SER 620, 621f potential mechanisms of action 622 pre- vs. post-electrical response 621, 621f timing/kinetics 620, 621, 621f TRP channels and 622 ion channel summation and 618 microvillus as site of 616, 617f phosphoinositide cascade and 619, 619f DAG 619–20 Drosophila phototransduction vs. 617, 622, 623 inositol 1,4,5-triphosphate (IP3) 619 second messengers 619, 619f reversal potential 617 ventral photoreceptors as model 617, 617f, 618f study methods 617, 618–19, 618f Limulus polyphemus 416, 617f Limulus visual system 416–25, 616, 617f differentiation 418 lateral compound eyes 416, 418f, 616 eccentric cells 416–17 lateral optic nerve 417, 419f neural plexus and lateral inhibition 417–18 ommatidium 416–17 median eyes (ocelli) 418, 419f, 616 optic nerve 417, 418, 419f organization 416, 417f, 616 photoreceptors 419 lateral eyes 416–17, 419–20, 616 median eyes 418, 419–20 microvilli 616, 617f

891

892

Subject Index

Limulus visual system (continued) rudimentary eyes 418, 419–20 structure 418f, 419, 419f ventral eyes 618f rudimentary eyes 418, 616 utility as model system 416 ventral eyes 616, 617f, 618f Limulus visual system, circadian regulation 420, 422t biochemical processes mediating 423 clock-driven phosphorylation 418f, 423, 423f octopamine and cAMP cascade 422t, 423 flies vs. 127–8 gene expression changes 422 arrestin 422 other photoreceptor proteins 423 organization 420 brain (cheliceral ganglia) oscillators 420, 421f eye type-specific 420 nocturnal efferent activity 420 vertebrates vs. 420 physiological changes 421 rhabdom shedding 422 light-driven vs. transient 422, 422f structural changes 420, 421f LE ommatidia 420 pigment granule migration 420–1 rhabdom 420 Lineage, definition 745 Lineage commitment, definition 263 Lineage tracing, definition 745 Linear polarization, definition 668 Lipid(s), rhodopsin–phospholipid co-transport 677 Lipofuscin, definition 648 Lipofuscin autofluorescence, definition 7 Lipoic acid, AMD treatment 766–7 Local edge detector (LED) 323–4, 324f definition 318 Long posterior ciliary arteries (PCAs) 179, 180 Looming detector AII amacrine cells and 307–8, 323, 323f approach-sensitive ganglion cells 304–5, 307 as hard-wired response 307 L-opsins 209 Low-molecular-weight heparin, PVR management 714 Low-pass filter, definition 156 LpMyo3 biochemical properties 423–4 circadian phosphorylation in Limulus 418f, 423 actin affinity and 424 LpMyo3 kinase activity and 424 sites 423f, 424 domain structure 423, 423f Lipopolysaccharides (LPSs) L-type calcium currents definition 661 in ribbon synapses 664–5, 664f L-type voltage-gated calcium channels (L-VGCC) circadian rhythms 74 cAMP formation 70 channel regulation 121–2 definition 118 in ribbon synapses 664 structure 121–2 Luminance, definition 325 Lumirhodopsin 641–2 LW opsin, butterfly 150–1, 151t genetic diversification 152, 153f Lysosome, definition 815 Lysozyme 376

M M2 macrophages AMD role 271 definition 270 Mach bands 320 Macrophages AMD role 271 light damage injury mechanisms 342 neovascularization role 351

choroidal 91 pathogen clearance 379 proliferative vitreoretinopathy and 711 Macula anatomy 427, 427f definition 426, 825, 830 histology 427, 427f retinitis pigmentosa 699, 701f rhodopsin mutations 645–6 Macular edema 426–37 blood–retinal barrier (BRB) and 49, 51, 56 causes 432 clinical findings 427 in CRVO 77f, 79 intravitreal triamcinolone therapy 82 macular grid photocoagulation 83 definition 51, 426 diagnosis 427 disease associations 427 fluorescein angiography findings 431 multifocal electroretinogram (mfERG) 435–7, 436f in NPDR 785, 785f optical coherence tomography (OCT) 427 findings 427–8, 429–31f unexpected/puzzling 435–6 treatment 432–3 anti-VEGF agents 82 aspirin 82 Branch Vein Occlusion Study results 434 Diabetic Retinopathy Clinical Research Network results 435 Early Treatment Diabetic Retinopathy Study results 432, 435f intravitreal fluocinolone 82 intravitreal triamcinolone 82 laser 432–5 VEGF role 432 Macular grid photocoagulation, macular edema 83 vitreous role in pathogenesis 825–9 confounding observations 827 vitreoretinal interface 825, 826, 827f, 828f vitreous traction 825 Macular pigment, optical density 98, 98f Macular pigmentary degeneration, CRVO-associated 79, 79f Macular region, capillary bed 655f, 657 Macular telangiectasia, type 2, adaptive optics OCT (AO OCT) 530–1, 531f Magnetic resonance imaging (MRI) blood–retinal barrier (BRB) assessment 55 optic neuritis 538 gadolinium 538, 538f Magnocellular (M) cells of the LGN 293 Magnocellular (parasol) RGCs 25 Malignant melanoma, retinopathy and 693–4 Mammalian models glaucoma 38–43, 736 cow 42 dog 42 pig 41–2 primate 38–9, 42 rabbit 42 3H-Mannitol assay, blood–retinal barrier (BRB) assessment 54–5 Mantis shrimp, polarization sensitivity 674, 674f circularly polarized light 674 Marfan syndrome 256–7, 258f, 259f, 261 Marijuana 717 ocular effects 719 prenatal effects on development 724 Marshall syndrome 253–4 Mash1 (Ascl1), retinal histogenesis 750 Math3, retinal histogenesis 750–1 Matrix metalloproteinase(s) (MMPs) angiogenic activity 548 in choroidal neovascularization (CNV) 91 neovascularization role 350 Matrix metalloproteinase-2 (MMP-2) inhibitory ECM digestion and cell transplants 372–3 M-cones 134, 140, 159 diffuse bipolar cell connections 455 M-opsin 208–9 photopigment genes 141, 142f blue-cone monochromacy and 146 red–green color defects and 143 variability and 141

Subject Index S-cones vs. 145–6 species differences in horizontal cell selectivity 467 spectral sensitivity 141f MCP-3, RPE secretion 767 Mean arterial pressure (MAP) 34 Medium-field amacrine cells 447, 448f Melanoma-associated retinopathy (MAR), retinitis pigmentosa vs. 693–4 Melanopsin 114–15 circadian rhythms in chick retina 73 definition 112 dopamine and 497 expression pattern/distribution 114 in ipRGCs 114–15 homologies 115 knock-ut mice 115–16, 116f mammalian 73, 114 nonmammalian vertebrates 114 phototransduction cascade 115 Melatonin 500–5 biosynthetic enzymes/steps 500 circadian rhythms release 122 synthesis 71, 72f, 73f definition 105 dopamine colocalization 502 paracrine functions 500 pineal synthesis 500 retinal functions 503, 504f cGMP effects 503 circadian clock and 109–10 dark adaptation 503–4 dopaminergic neuron effects 495, 502, 503 glutamatergic neuron effects 503 glycinergic neuron effects 503 modulation of transmitter release 495, 503 photoreceptor modulation 503 retinal synthesis 500 evolutionary perspective 501 inner retinal neurons 500 photoreceptors 500 rhythmicity 116 Melatonin receptors 500–5 dimerization 501 retinal location 501, 504f inner layers 502 photoreceptors 501 RPE 502 subtypes/classification 500, 501 specific cyclic rhythms and 503 Memantine 739 Membrane inhibitor of reactive lysis (MIRL) antiangiogenic activity 549 Membrane trafficking rod photoreceptors 677 Mercury, acquired color blindness 139 Mertk gene 818 Mesenchyme, definition 198 Mesopic, definition 558 Mesopic luminous efficiency 559 Mesopic vision 558, 559f, 560f definition 105, 156, 559, 705, 819, 841 rod/cone contribution 589 Metabolic disorders, optic neuropathies 333 Metabolism human–Drosophila pathway similarity 727–8 metabolic load and the RPE/subretinal space 763 Metabotropic glutamate receptors (mGluRs) mGluR6 284 retina rod–bipolar cell signaling 843, 844f Metabotropic receptors, definition 228 Metaretinochrome, definition 582 squid 585 Metarhodopsin definition 582 inactivation in Drosophila 242, 244f squid 584–5 Metarhodopsin I 611, 641–2 Metarhodopsin II 611, 641–2, 648 inactivation by rhodopsin kinase 648 Metarhodopsin III 611 MFAT (multifunctional O-acyltransferase) 650

Microaneurysms, diabetic retinopathy 784–5, 784f Microbiomics, definition 836 Microelectrode implants, retinitis pigmentosa treatment 697 Microglia in AMD 273–4 in neovascularization 352 Microneuroma(s) definition 360 retinal remodeling 363 Microphthalmia 215 definition 212, 753 Microsurgery, PVR management 714 Microtubules definition 156, 575 retinomotor movement 221, 222 Microvillar photoreceptors definition 438 excitation 439 action potentials 439f calcium ion flux 439–40, 440f diacylglycerol stimulation 440–1, 441f segregation to microvillar lobe 440f single-channel currents 441, 442f light adaptation 441, 442f phylogenetic distribution 575 squid photopigments 585 Microvilli definition 575, 668 Limulus 616 phototransduction generalization 623 polarized light and 671–2, 672f Midget bipolar cells 23, 453, 454f amacrine cell networks and 281 definition 452 dendritic contacts 453, 454f IPL contacts 457–8, 458–9 Midget (Pb/parvocellular) retinal ganglion cells 25 MIG, RPE secretion 767 Migration see Cell migration Minimum angle of resolution (MAR) 1, 2f definition 1 visual acuity reporting 3, 3t Minute of arc, definition 1 Mitochondria definition 811 Mitochondrial disorders optic neuropathies and 333, 336–7 DOA and 335 LHON 333 reactive oxygen species generation and 739, 739f Mitogen(s) 169 Mitogen-activated protein kinase (MAPK) signaling circadian regulation of CNGCs 120 IFNg signaling 770–1 Mollusks ciliary photoreceptors cGMP role in excitation 443, 444f cGMP role in light adaptation 445, 446f excitation 442, 442f, 443f, 444f function 442 guanylate cyclase 444 hyperpolarizing receptor potentials 442f light adaptation 445, 446f light-dependent ion channels 445 membrane conductance 443f photopigment 443 microvillar photoreceptors excitation 439, 439f, 440f, 441f, 442f light adaptation 441, 442f photoreceptors 438–46 Pecten irradians 439f Monkey models, glaucoma 38–9, 42, 736 Monoamine oxidase (MAO), definition 717 Monocarboxylate transporter(s) (MCTs) RPE metabolic load reduction 765, 766f Monocarboxylate transporter 1 (MCT1), human RPE 763–4, 766f Monochromacy blue-cone monochromacy 146 definition 134, 140 rod monochromacy (achromatopsia) 146

893

894

Subject Index

Monochromatic aberration, correction, adaptive optics (AO) 8, 9f Monocyte chemoattractant protein 3 (MCP-3), RPE secretion 767 Monocytes choroidal neovascularization role 91 oxidative stress-induced RPE invasion 767 Monokine induced by g interferon (MIG), RPE secretion 767 M-opsin 208–9 Morpholinos 855, 871 definition 863 Motion detection, importance 295 Mouse models 854 AMD 273 glaucoma 40–1 developmental 41 normal-tension 41 optic nerve axon degeneration 386, 387f pressure-induced 40 isoform-specific VEGF 757 retinitis pigmentosa (rd1 mouse) 857 Mouse photoreceptors 568 development 568, 569f, 570f M-sequence stimulation 508 definition 506 Mucin(s) immunological 374–5 secreted 374–5 surface-associated 374–5 TLRs and 374–5 Mucopolysaccharidoses retinitis pigmentosa and 704 Mu¨ller cells cannabinoid receptors 720 definition 17 dopamine role 497 endothelial cell interactions, retinal vascular development 546 GABA receptors 234 GAT-3 expression 230 inner blood–retinal barrier (iBRB) 46 neovascularization role 352 persistent neurogenesis 865, 867f, 870f, 871 remodeling/injury response role 360, 364 mammals 872 PVR and 710 zebrafish 867, 868f, 870f, 871–2 visual cycle and see Mu¨ller cells, alternative visual cycle zebrafish pde6 mutants 860 Mu¨ller cells, alternative visual cycle 17–21 cone chromophore source 18 IRBP and 20–1 pigment regeneration in cones 20, 20f rods vs. cones 18–19 11-cis-ROL dehydrogenase activity 19–20 retinoid isomerase in cone-dominant species 19 role in rod-dominant species 21 visual pigment regeneration role 17–18 Multifactorial, definition 75 Multifocal electroretinography (mfERG) 506–24, 706 central serous retinopathy 510, 511f data handling analysis 508 collection 508 noise reduction 508 recording 507 duration of tests 508, 509f hydroxychloroquine retinopathy 510, 512f, 513f juvenile X-linked retinoschisis 510, 514f macular edema 436, 436f neuro-ophthalmology asymmetric glaucoma 520f, 521, 521f demyelination 522f, 523, 523f mfVEP 512, 516f ONHC component of mfERG 514, 518f, 519f optic neuritis 514, 517f patient positioning 508 principles 506, 507f mfVEP 512, 516f protocol selection 506, 509, 517–19 small scotoma 511, 515f stimulus production 508 Multifocal visual-evoked potentials (mfVEP) asymmetric glaucoma 520f, 521, 521f demyelination 523f

noise reduction 508 optic neuritis 514, 517f principles 507f, 512, 516f stimulus production 512f Multifunctional O-acyltransferase (MFAT) 650 Multiple sclerosis (MS) acquired color blindness 137 definition 536 optic neuritis 537 Multipolar neurons 276 Multipotency, definition 263 Multipotent progenitor, definition 745 Musca domestica (housefly) visual system 124 circadian changes circuits underlying 132 in inter-receptor invaginations 131 in pigment granules 131 plasticity of L1/L2 monopolar cells 130–1 serotonin role 131 Myelin, ECM and transplantation barrier 372 Myelination, definition 506 Myofibroblast(s), definition 708 Myoid definition 219 retinomotor movement 220, 220f, 221 structure 811–12, 812f Myopia dopamine role 498 retinopathy of prematurity and 795 Stickler syndrome and 253 Myopic regression, post-LASIK 4:11 Myosin(s) circadian phosphorylation in Limulus 418f, 423, 423f class III domain structure 423, 423f Limulus lateral eye 616 retinomotor movement photoreceptors 220, 221 RPE cells 222 VIIa, Usher syndrome and 682

N National Academy of Sciences, standard for visual acuity measurement 1 Natural killer (NK) cells. pathogen clearance 379 Navigation, using polarized light 673–4 Neoantigens AMD pathogenesis 272 definition 270 Neofunctionalization, definition 148 Neovascular age-related macular degeneration (NVAMD) 87, 831–2 acquired color blindness 136 blood–retinal barrier breakdown 62–4, 63f, 64f, 65f fundoscopy 831f treatment 834 variants 93–5 polypoidal choroidal vasculopathy (PCV) 93, 93f retinal angiomatous proliferation (RAP) 93, 94, 94f Neovascular glaucoma, in ischemic CRVO 80–1 management 85 Neovascularization definition 75, 541 intravitreous avascular retina and 796–7 definition 790 ischemia-mediated 543, 543f in ischemic CRVO 79, 80f panretinal photocoagulation 83–5, 84f Neovascularization, injury-induced 346–53 acute responses angiogenic mediators 350 blood–retina barrier breakdown 348 complement activation 350 cytokines 348, 349f, 350, 351 ECM modulation 350, 351f animal models 348, 348f, 349f causes 346 cellular responses 350–1 astrocytes 349f, 352 macrophages 351 microglia 352

Subject Index Mu¨ller cells 352 neutrophils 351 stem/progenitor cells 351 to VEGF-A receptor binding 352 definition 346 in diabetic retinopathy 783–4, 786, 788f fundoscopy 347f, 348f Nerve of Tiedemann 658, 659f Nested feedback, definition 276 Neural progenitor cells, retinal repair/regeneration brain-derived 367 retinal 368 Neural retina (NR) development 199, 200f, 200t, 201f, 202 Neuritogenesis definition 360 retinal remodeling 363 NeuroD1, retinal histogenesis 750–1 Neurodegenerative disease, acquired color blindness 137 Neurokinin A (NKA) retina mRNA localization 488 receptor autoradiography 491 signaling pathway 491 Neurokinin B (NKB) retina receptor autoradiography 491 signaling pathway 491 retinal 478 Neurokinin receptor(s), retina NK1 478, 492, 492f NK2 492–3 NK3 478, 492–3, 493f receptor autoradiography 491 receptor immunohistochemistry 492 signaling pathway 491 Neuromodulation/neuromodulators dopaminergic interneurons and 473 melatonin role 503 neuromodulator definition 494 Neuronal ceroid lipofuscinosis (Batten disease), retinitis pigmentosa and 704 Neuronal migration definition 360 retinal remodeling 363 Neuropeptide receptors 487, 491 cloning 491 Neuropeptide Y (NPY), retina GABA co-localization 490 immunostaining studies 489, 489f mRNA localization 488 neurotransmitter release 484 receptor mRNA expression 491 signaling pathway 479, 480f, 491 Neuroprotection 736 definition 734, 736 light-driven protein translocation 413 retinitis pigmentosa management 696 stem/progenitor cell therapy and 369 Neuroprotective agents glaucoma 736, 738 antioxidants 740 cannabinoids 724 neurotrophic factors 736 NMDA-antagonists 738 study models 743, 743t vaccine 734 VEGF 798 Neuropsin 208 Neurotransmitters catecholamines 494 definition 22 endocannabinoid effects 720 receptors retinal remodeling 362 retinal, neuropeptides and 479, 483, 485 Neurotrophic factors definition 734 light damage, protection mechanisms 343–4 neuroprotective effects 736 retinal remodeling reversal/prevention 365 retinal transplantation and 373 withdrawal 736, 737f

Neurotrophin hypothesis 386 Neutrophils neovascularization and 351 pathogen clearance 379 New vessels elsewhere (NVE) 786–7, 788f definition 781 New vessels on disk (NVD) 786–7, 788f definition 781 Night blindness retinitis pigmentosa 690 Xenopus laevis model 849 Nitric oxide (NO) horizontal cell modulation 315 retinal neuropeptides and 479 vascular autoregulation role 34 NK1 receptor 478, 492, 492f NK2 receptor 493 NK3 receptor 492–3, 493f NMDA receptor(s), amacrine cells 278 NMDA receptor antagonists, as neuroprotective agents 738 Nocturnal arterial hypotension, NA-AION 399, 400f NOD-like receptors (NLRs) 377–8 eye distribution 378, 378t immune response initiation/amplification 379 ligand specificity 378t No light perception (NLP), definition 354 Nonarteritic anterior ischemic optic neuropathy (NA-AION) 399 arteritic AION vs. 407 clinical features 401 amiodarone and 403 bilateral (NA-AION) 402 diabetics 402, 404f familial 403 phosphodiesterase-5 inhibitors and 403 recurrence in same eye 403 symptoms/signs 401–2, 402f, 403f, 404f fundus angiograms 401f, 402f, 403f, 404f incipient 405 management 403 controversy 404–5 natural history and 403–4 techniques used 404–5 pathogenesis 399, 400f embolic lesions of vessels serving nerve head 399 nocturnal arterial hypotension 399, 400f transient nerve head circulation disruption 399, 400f risk factors 399 nocturnal arterial hypotension 399–401, 400f predisposing vs. precipitating factors 399–401 reduction of 404–5 stroke vs. 401 Nonarteritic posterior ischemic optic neuropathy (PION) 410 management 410 Non-insulin-dependent diabetes mellitus (NIDDM) definition 781 Nonlinear response, definition 506 Nonproliferative diabetic retinopathy (NPDR) 784 classification 785, 786t definition 781 fundoscopy 784f macular edema 785, 785f clinically significant 785, 787f, 787t Nonvisual photoresponses 112 definition 112 visual responses vs. 113 Norrin, retinopathy of prematurity and 795 Notch signaling pathway 263 definition 863 proangiogenic actions 545, 546f retinal histogenesis 175, 176f, 263, 264f, 750, 751f, 865 Nr2e1 213 structural domains 213–14, 213f NRL disease associations 572 in photoreceptor cell fate determination 570–1, 570f rods 570–1, 570f Nystagmus searching 333 recessive optic atrophy 335–6

895

896

Subject Index

O OAT gene/protein, retinitis pigmentosa 702–3 Obesity, risk factor for AMD 833 Object motion sensitive (OMS) ganglion cells 305, 306 excitatory receptive field 306 inhibitory input 306–7 mechanism of action 307 Occludin 46 in diabetic retinopathy pathogenesis 783 tight junctions 51–2 Ocellus (ocelli) definition 416 insects 124 Drosophila 729 photoentrainment and 127 Limulus 418, 419f, 616 Octopamine circadian regulation of Limulus eyes 422t, 423 definition 416 Ocular blood flow anatomical aspects 28, 29f, 30f future studies 37 measurement 35 angiography 35 blue field entoptic technique 36 color Doppler imaging 35, 35f laser Doppler flowmetry 36, 36f laser Doppler velocimetry 36 laser speckle technique 36 optical Doppler tomography 37 pulsatile flow 36 retinal vessel diameters 36 regulation 34 Ocular drug delivery, retinal, BRB permeability and 49 Ocular fluid flow 47, 59, 59f mechanisms 59–60 Na, K-ATPase pump 60 Ocular inflammation light damage role 339f, 341 retinopathies 341 RP differential diagnosis 693 Ocular vasculature blood flow autoregulation 653, 659 development 179–85 choroidal network 179 retinal network 183, 795 pathology hyaloid vascular network 183 proliferative vitreoretinopathy and 711, 712f OFF bipolar cells 284, 290–1, 319, 319f, 389, 452 axon terminal development 392–3 center-surround organization and 286, 287f definition 661 GABAergic regulation by horizontal cells 313 OFF-cone bipolar cells 23–4 horizontal cell interactions 319, 319f mixed rod/cone 285–6 neuropeptides and 482–3 OFF-cone bipolar cells 23–4 glutamate receptors 23 rod synapses 820–1, 820f photoreceptor cell contacts 662 polarization in phase with photoreceptors 319 OFF pathway 319, 319f definition 318 ON pathway interaction (crossover inhibition) 320, 321f amacrine cells and 322 rod–OFF pathway 845 OFF-RGCs 301 contrast sensitivity and 291 direction-selective (DS) cells 305–6 OFF-starburst cells 451 Oguchi disease 332 Old World primates, horizontal cell morphology 465, 465f, 466f Olfactory granule cells, amacrine cell homology 276, 277f OMIM (Online Mendelian Inheritance In Man) database, definition 252 Ommatidium/ommatidia butterfly structure 149, 149f variation 150f

definition 416 Drosophila 124, 241f, 729, 729f Limulus lateral eyes 416–17 circadian changes 420, 421f ON bipolar cells 284, 290–1, 319, 319f, 389, 452 axon terminal development 392–3, 392f center-surround organization and 286, 287f definition 284, 661 dopaminergic neuron regulation 494 GABAergic regulation by horizontal cells 313 glutamate receptors ON-cone bipolar cells 23–4 horizontal cell interactions 319, 319f melatonin effects 503 mixed rod/cone 285–6 neuropeptides and 482–3 ON-cone bipolar cells 23 polarization out of phase with PRs 319 Online Mendelian Inheritance In Man (OMIM) database, definition 252 ON–OFF-RGCs 301 direction-selective (DS) cells 305–6 ON pathway 319, 319f definition 318 OFF pathway interaction (crossover inhibition) 320, 321f amacrine cells and 322 ON-RGCs 301 contrast sensitivity and 291 direction-selective (DS) cells 305–6 ON-starburst cells 451 ON-sustained interplexiform cells 474–5 ON-transient interplexiform cells 474–5 OPA1 gene 335 Ophthalmic artery 28, 29f, 179, 653 branches 179, 180f definition 28 Ophthalmoscopy, definition 75 Opsin(s) butterfly 149–50, 150f genetic diversification 151, 152f, 153f, 154 spectral heterogeneity 150, 151t cone opsins 605–6, 626 evolution 209 genetics 141, 142f human 209 non-human209 rhodopsin vs. 18 thermal stability and 606 definition 17, 206, 648 Drosophila 729 evolution 205–11, 605–6 convergent evolution of type I and II 206, 207f co-option and 210 evolutionary relationships of type II opsins 207f gene duplication 209, 210 gene loss and 209 modes 210 functions 206 human cone opsins 209 RGRs 208 non-visual mammalian 113, 115t nonmammalian 113 photoisomerization 206 regeneration 651 rod/cone homologies 605–6, 626 structure 206 transport within photoreceptor 816–17, 816f type I definition 205 occurrence 206 type II vs. 206 type II ciliary (Gt) 208 cnidops 206 color vision and 208 definition 205 as GPCRs 206 major classes 206–8, 207f, 209t retinal G-protein receptor/Go clade 208 rhabdomeric (Gq) 208

Subject Index type I vs. 206 wavelength sensitivity (l-max) 206, 208–9, 210 Opsonization, definition 270, 374 Optical aberrations, monochromatic, correction with adaptive optics 8, 9f Optical coherence tomography (OCT) 525–35 advantages 525 choroidal neovascularization (CNV) 89, 90f three-dimensional wide-field 527, 528f definition 525, 781, 847 development/historical aspects 525, 526f optic neuritis 539 retinitis pigmentosa diagnosis 692 see also Anterior segment OCT Optical density, definition 96 Optical Doppler tomography, ocular blood flow measurement 37 Optic cup development 199, 200f dorso-ventral patterning 200t, 201f, 203 retinoic acid role 203, 268 Optic lobe (insect) 124, 125f, 126f neuropil 124, 125f cartridges 124–5 Optic nerve anatomy 30, 31f, 536 definition 381 demyelinating disease, mfERG/mfVEP results 522f, 523, 523f Limulus visual system 417, 418, 419f pallor, waxy in RP 699, 700f prosthetic devices and 355 regeneration in amphibians 848 as site of occlusion in CRVO 76, 76f transection, Xenopus laevis glaucoma model 849 vasculature 30–1, 31f, 32f histology 32–4 lamina cribrosa region 31, 33f prelaminar region 31, 32f retrolaminar region 31, 33f superficial nerve fibre layer 31, 32f Optic nerve axons elevated IOP-induced damage 381–8 biomechanical engineering studies 382, 383f, 384f ganglion cell axon dysfunction 382, 386 mouse model 386, 387f Optic nerve head (ONH) edema GCA and A-AION 405, 405f NA-AION 402, 402f, 403f glial cells, elevated IOP-induced modulation 382, 385f vascular supply 30–1 Optic nerve head component (ONHC) of mfERG 514, 518f, 519f comparison with mfVEP in asymmetric glaucoma 520f, 521, 521f demyelination 522f, 523 Optic nerve sheath decompression, NA-AION management 404 Optic neuritis 536–40 acquired color blindness 137 definition 536 diagnosis 537 cerebrospinal fluid (CSF) analysis 538 clinical examination 537 magnetic resonance imaging (MRI) 538, 538f optical coherence tomography 539 retinal nerve fiber layer (RNFL) 539 visual-evoked potential 537–8, 538f differential diagnosis 539 Devic’s disease 539 epidemiology 537 etiology 537 multiple sclerosis 537 immunopathogenesis 536 T lymphocytes 536–7 mfVEP results 514, 517f prognosis 540 Snellen chart 540 symptoms 537 treatment 539 corticosteroids 539–40 neuroprotective treatment strategies 540 apoptosis 540 retinal ganglion cells (RGCs) 540 optic neuritis treatment trial (ONTT) 539–40

Optic neuropathy acute conditions 399 hereditary 333–7 clinical features 333 genetics 336–7 as metabolic disorders 333 treatment options 337 Optic neuropil (insects) 124, 125f lamina 124, 125f cartridges 124–5 daily morphological changes 130, 130f lobula 124, 125f medulla 124, 125f Optic stalk, development 200f, 201f Optic vesicle, development 198, 199f proximo-distal patterning 200t, 200–1, 201f Optokinetic nystagmus, definition 295 Optokinetic response (OKR) definition 853 zebrafish models of retinal disease 856 Optomotor responses, zebrafish models of retinal disease 856 Optophysiology definition 525 in vitro rabbit retina 531–2, 532f in vivo human retina 532–3, 533f Optotype(s) 1, 2f definition 1 National Academy of Sciences standard for visual acuity measurement 1 Orbital vascular pathology, PVR and 713, 713f, 714f Orofacial abnormalities, Stickler syndrome 253, 257f Orthodenticle protein homolog 2 (OTX2) 569–70, 570f, 571f Ortholog, definition 500 Osmotic avoidance abnormal protein (OSM-3) 579–80 Outer blood–retinal barrier (oBRB) 44, 45f, 47, 58, 654f, 659, 773–80 assessment methods 60 clinical assays 61 electrophysiologic 60 ex vivo assays 61 fluorescein angiography 61, 61f, 62f occludin immunolocalization 60, 60f optical coherence tomography (OCT) 61, 61f in vitro assays 60–1 in vivo assays 61 culture models 778 definition 44, 58 diseases and 779 function 774 assessment methods 60 cellular level 775 improvement studies 67 molecular level 775 tissue level 774 molecular/fluid movement across 47, 59, 774f, 775 mechanisms 59–60 Na, K-ATPase pump 60 polarity 47 structure 58, 59f, 774 assessment methods 60 cellular level 775 molecular level 775 tissue level 774 Outer blood–retinal barrier (oBRB) breakdown 58–67 clinical associations 61 age-related macular degeneration 62, 63f neovascular 62–4, 63f, 64f, 65f central serous retinopathy 65 diabetes mellitus 64 drug toxicity 64 inflammation/infection 62 proliferative vitreoretinopathy 64 retinitis pigmentosa 66 role of growth factors 66 Outer limiting membrane (OLM) structure 812 Outer plexiform layer (OPL) GABA release 228–9 melatonin receptor expression 502 synaptogenesis 393–4, 393f, 394f, 396 see also specific cell types Outer segments (OS) of photoreceptors 412, 626, 648 definition 17, 648 membrane renewal 576, 815–18

897

898

Subject Index

Outer segments (OS) of photoreceptors (continued) catabolism 817–18, 817f in cones 815–16, 816f disk formation 817 opsin transport 816–17, 816f in rods 577f, 815, 816f organization 575, 811–14 cilia 812f, 813–14, 814f proteins 812–13 structure 811, 812f, 813f as sensory cilia 575–81 development and structure 576, 577f, 578f, 579f intraflagellar transport 578, 580f cargo 580 kinesin KIF17 579 Oxidative stress AMD relationship 766 light damage role 341 mitochondrial damage 766 retinopathy of prematurity and 798 RPE and 761, 766 trabecular meshwork and see under Trabecular meshwork (TM) Oxygen, retinal development and 795, 798 Oxygen-induced retinopathy (OIR) definition 790

P p19Arf, retinal development role 174 P100 latency, definition 536 Paired domain, definition 726 Panretinal photocoagulation (PRP) CRVO 83 visual field effects 83, 84f definition 75 Papillomacular fibers definition 333 hereditary optic neuropathies 333 LHON 334 Paracellular space, definition 773 Paracrine regulation definition 477 Paracrine signaling definition 487, 500 melatonin 500 retinomotor movements 223–4 Parasol (Pa/magnocellular) retinal ganglion cells 25 Parkinson’s disease (PD) acquired color blindness 137–8 interplexiform cells and 475 Parvocellular (P) cells of the LGN 293 Parvocellular (midget) RGCs 25 Patch-clamp recording, definition 295, 477 Pathogenesis, definition 75 Pathological retinal angiogenesis see Retinal neovascularization Pathways concept (dynamic range) 326, 842 adaptation to mean background light 845 cone pathways 159, 841–2, 845 adaptation to mean background light 846 RGCs and 846 rod–cone pathway 844 principles/background 842 rod pathways 610, 611, 841 adaptation to mean background light 845 rod–cone pathway 844 rod–OFF pathway 845 Pattern deviation plots, perimetry 555 Pattern recognition receptors (PRRs) 377 classification/types 378t discovery 377 immune response initiation/amplification 379 NOD-like receptors 377 Pax6/Pax6 212, 727 eye development role 214–15 retinal histogenesis 750, 750f mutations, eye abnormalities associated with 215–16 structural domains 212, 213f Pcna (proliferating cell nuclear antigen) definition 863 in retinal regeneration 869, 871 PCR (polymerase chain reaction), definition 219

PDE6B gene, retinitis pigmentosa 686, 857 Pecten irradians (scallop), double retina 439, 439f Pedicle, definition 156 Pegaptanib, CNV therapy 92 Penetrance (genetic), definition 333 Pericyte(s) diabetic retinopathy loss 783 inner blood–retinal barrier (iBRB) 46 Pericyte–endothelial cell interactions, in retinal vascular development 547 Perimeter, definition 75 Perimetry 551–7 analytical techniques 555 change probability 556 global indices 555 linear regression 556 total deviation/pattern deviation plots 555 definition 551 frequency-doubling technology (FDT) 554 Goldmann stimulus sizes 552, 552t reliability estimates 554 false-negative responses 555 false-positive responses 555 fixation accuracy 554 techniques 551 test targets 552 Peripheral severe retinopathy of prematurity (PSROP) 790, 796 Peripherin/RDS, definition 847 Perissodactyls, horizontal cell morphology 465, 466f Peropsin 208 per/PER (period gene/protein) 68, 69f, 71f, 127, 128f Persistent hyperplastic primary vitreous (PHPV) 183 Persistent neurogenesis, zebrafish retina 865, 867f, 870f, 871 pH cone negative feedback and 161 horizontal cell feedback and 312, 314 Phagocytosis definition 374, 753, 815 photoreceptor disk membranes 818 Phenotype, definition 198, 252 Phosducin 414 definition 412 Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) definition 438, 676 Limulus phototransduction and 619–20, 619f molluskan microvillar photoreceptors 440–1 Phosphene, definition 354 Phosphodiesterase (PDE) cone phototransduction and 607, 627 definition 438, 605, 610, 726 pde6 mutants 857 bystander effect and 857, 858f RP and 686, 857 structural effects 861, 861f rod phototransduction and 610–11, 612, 634 zebrafish phototransduction 856–7, 856f Phosphodiesterase-5 inhibitors acquired color blindness 138 nonarteritic anterior ischemic optic neuropathy and 403 Phosphoinositide (PI) pathway 619, 619f definition 240 Drosophila phototransduction 246f, 248, 248f Phospholipase C (PLC) definition 240, 438 light-dependent activation by Drosophila Gq protein 245, 246f squid 585, 586 Phospholipid(s) circadian rhythms in metabolism 73 Phospholipid A2 (PLA2), innate immunity 376 Phosphorylation, signaling regulation of rhodopsin 644, 644f Photobleaching 327 cone light adaptation and 327, 592, 594 dark adaptation and 596 light damage and 338 Photochemical cycle definition 240 Drosophila phototransduction 242, 244f Photodynamic therapy (PDT), choroidal neovascularization (CNV) 92 Photoentrainment 112 definition 112 dopamine role 498 fly circadian system(s) 126–7

Subject Index Photoisomerization 648 definition 338, 637 opsin(s) 206 Photophobia, retinitis pigmentosa and 690 Photopic, definition 558 Photopic vision 558, 559–60, 559f, 560f, 589, 596, 631 definition 22, 105, 205, 494, 705, 841 major contribution to vision 589 Photopigments butterfly 149–50, 150f absorbance spectra 150f opsin diversification 151, 152f, 153f, 154 spectral heterogeneity 150, 151t definition 134, 140, 148, 624 genetic basis 141, 142f rods vs. cones 626 stability 626 Photopsia 363 rhegmatogenous retinal detachment 801–2 Photoreceptor(s) 648, 854 amphibian 848 circadian rhythmicity 116 definition 830 evolution 22, 567–8 rods 22, 841 horizontal cells and feedback control 313 ephaptic transmission and 314 hyperpolarization 637 maturation 569f, 573 melatonin and functional effects 503 receptor expression 501 synthesis of 500 neurochemistry/neuromodulation dopaminergic regulation 496 GABA receptors and 233, 313–14 oxygen consumption 763–4 polarization sensitivity 670 rhabdomeric 567–8 rod vs. cone pathways 22–7 sensitivity 413 synaptic triads 309 visual cycle stages 648, 649f transfer of all-trans-retinol to RPE 649 see also Cone photoreceptor(s); Opsins; Rod photoreceptor(s) Photoreceptor degenerations 853–4 animal models mechanistic value of 840 cone dystrophies 136, 684, 698, 853–4, 858 mixed model 684, 698 primary 698 pde6 mutants 857 secondary retinal degeneration with 684, 698, 836–7, 837t secondary vs. 836–7 retinal detachment and 836–7 rod dystrophies 853–4 secondary 836–40 bystander effect and 857, 858f etiology 836 extrinsic type 685, 699, 837–9, 838t intrinsic type 685, 699, 837–9, 838t mechanisms 839 mixed intrinsic/extrinsic etiology 684, 698, 837–9 prevention 836–7, 839 primary degenerations vs. 836–7 primary retinal degeneration with 684, 698, 836–7, 837t terminology 698–707 Photoreceptor development 567–74 circuitry 393 cone see under Cone photoreceptor(s) early stages 569 from RPC to photoreceptor precursor 569, 570f, 571f factors affecting genesis 266, 570f, 571f human retina 267, 569f mouse retina 267–8, 569f, 570f signaling molecules 268, 268f Photoreceptor retinol dehydrogenase (prRDH) 649 Photoresponse, in squid see Squid, photoresponse Photorhodopsin 641–2 see also Rhodopsin Photostasis, definition 338 Phototaxis, definition 567

Phototransduction definition 22, 205, 240, 624, 631 light-dependent retinal degeneration and 731 Limulus see Limulus phototransduction melanopsin and nonvisual photoresponses 115 pde6 mutants and 857 protein transport, intraflagellar 580 rod vs. cone pathways 22–7 vertebrate vs. invertebrate 240 zebrafish 856, 856f Phylogeny, definition 148 Piccolo (protein) 664 Pigment-dispersing hormones definition 124 fly circadian plasticity and 126–7, 131, 132 Pigmented paravenous retinochoroidal atrophy (PPRCA) 702, 703f Pigment epithelium-derived factor (PEDF) RPE secretion 757, 758f Pig models, glaucoma 41–2 Pilots, visual acuity requirement 4 Pineal gland, retina relationship 501 Pinealocyte(s) definition 500 melatonin synthesis 500 retina relationship 501 Pituitary adenylate cyclase-activating peptide (PACAP) retina glutamate co-expression 490 immunostaining studies 490 receptor mRNA expression 491 signaling pathway 491 Placental growth factor, proangiogenic action 544 Planar cell polarity (PCP) pathway 264, 265f Plasma membrane, definition 811 Plasmapheresis (plasma exchange), definition 536 Platelet-derived growth factor (PDGF) diabetic retinopathy 767–9 proangiogenic action 544, 544f, 545f proliferative vitreoretinopathy 767–9 receptors, RPE location 769–70, 770f retinal vasculature development 183 Plus disease, retinopathy of prematurity 790–1, 792f PMNs see Polymorphonuclear leukocytes (PMNs) Pneumatic retinopexy rhegmatogenous retinal detachment 805 complications 809 contraindications 806 outcomes 807 RRD due to retinal holes/dialysis 806 RRD due to retinal tears 806 Poikilotherm, definition 819 Point-spread function (PSF) definition 7 diffraction-limited eye 7, 8f normal human eye 8f Poiseuille’s law 34 definition 28 Polarized light 668–75 definition 668 generation of 669, 669f skylight 669f underwater 670f overview 668 polarization sensitivity 670, 671f circularly polarized light 674 in compound eyes 671–2, 672–3, 672f distinguishing from color sensitivity 672 effect on animal behavior 673 neural analysis 671 polarization vision 672 in vertebrates 670–1, 672 Poly-ADP-ribose polymerase (PARP), inhibition, in prevention of diabetic Polyaxonal amacrine cells 323 Polymerase chain reaction (PCR), definition 219 Polymer scaffolds, stem/progenitor cell therapy and 370 Polypoidal choroidal vasculopathy (PCV) 93, 93f Porcine models, glaucoma 41–2 Positive selection, definition 148 Posterior ciliary arteries (PCAs) 28, 30f, 179 branches of main PCAs 179 long 180 lamina fusca 180, 181f

899

900

Subject Index

Posterior hyaloid, definition 825 Posterior ischemic optic neuropathy (PION) 408 classification/definition 399, 408 clinical features 409 signs 409, 409f, 410f symptoms 409 diagnosis 408 incidence 408 management 410 arteritic 410 nonarteritic 410 surgical 410 pathogenesis 408 arteritic 408 nonarteritic 408 surgical 408 Postgestational age, definition 790, 791–2 Post-Golgi transport carriers (TCs) definition 676 rhodopsin (RTCs) 677 Postnatal vasculogenesis, in choroidal neovascularization (CNV) 92 Postoperative endophthalmitis 375 Potassium (K+) channels circadian rhythms 122 cone photoreceptors 158–9 molluskan ciliary photoreceptors 442–3, 445 rod photoreceptors 821 Pou4f/Brn3, retinal ganglion cells activation by Atoh7/Ath5 237, 237f Islet1 and 238 regulatory role 238 POU-domain transcription factors definition 235 see also Pou4f/Brn3 Precursor, definition 567 Pregnancy, diabetic retinopathy and 782 Preprotachykinin (PPT), retinal mRNA localization 488 Prestriate cortex (V2), acquired color blindness 137 Preterm birth, epidemiology 790 Primate(s) bipolar cell morphology 453, 453f, 454f glaucoma models 38–9, 42 horizontal cell morphology 465, 465f, 466f Primordial retina, vasculature 542 Progenitor cells, definition 567, 745 Programmed cell death (PCD), definition 853 see also Apoptosis Progressive cone dystrophies 136 Progressive restriction model definition 745 retinal histogenesis 749, 749f Proinflammatory cytokines, innate immune response and 379 Proliferative diabetic retinopathy (PDR) 786 classification 786t, 787–8 definition 781 fundoscopy 788f Proliferative vitreoretinopathy (PVR) 64, 708–16 cells involved 709, 710f, 712f ciliary body epithelial cells 711 fibroblastic cells 711 glial cells 710 macrophages 711 RGCs 711 RPE cells 710f, 711, 767–9, 770–1 T-cells 711 vascular elements 711 clinical classification 714, 803, 804t definition 708 incidence 713 intraretinal (iPVR) 708, 713 masses/tumors and 713, 713f, 714f management 714 antiproliferative drugs 714 microsurgery 714 tamponade 714–15, 715f membrane formation and retinal traction 708, 709f clinical significance 709 closed funnel formation 709, 709f extension into vitreous base 709 severity 708–9 membrane location 708 outcomes 715

pathogenesis/natural history 712 adhesive glycoproteins 713 cell loss 713 cell migration 713 cell proliferation 713 ECM build-up 711, 712, 712f, 713 IFNg 770–1 retinal detachment and 708, 712, 803, 804f, 804t, 809 time course 712–13 vitreous degeneration 712 risk factors 713 Prolonged depolarizing afterpotential (PDA) 242, 243f definition 240 Prosencephalon, definition 198 Prostheses (visual) 354–9 approaches 354 brain prosthetic devices 354 challenges 357 color vision and 358 future prospects 358 rationale 354 retinal prosthetic devices 355 clinical trials 356 optic nerve stimulation 355 power output and 355–6 retinal position and 355 retinal remodeling and 355, 357–8 variety/types 356 safety issues 358 Protan defects 134, 143, 144f Protanomalous trichromats 143, 144f Protanopia/protanopes 143, 144f definition 134 Protein kinase A (PKA), retinal movement regulation 223 Protein kinase C (PKC) definition 438 light adaptation in molluskan photoreceptors 441, 442f Protein misfolding 740 reduction 741 Proton(s) definition 309 horizontal cell inhibitory feedback 312, 314 Proximal retina, definition 284 PS (pico-siemens), definition 438 Pseudoexotropia definition 252 Wagner syndrome 261f PSROP (peripheral severe retinopathy of prematurity) 790, 796 Psychophysical assessment, vision 163, 164f Psychophysical studies, multiple rod pathways and 25 Ptf1a, retinal histogenesis 750–1 Pulsatile ocular blood flow, measurement 36 Punctum-plug occlusion Pupil, light level and 559f, 563 Pupillary light reflex (PLR) 326

Q Quantum bump definition 416 Drosophila phototransduction 243f, 250–1 Limulus phototransduction 617–18 Quantum efficiency, definition 631 Quantum units, definition 96 Quiescence, definition 753

R Rab6, membrane trafficking and 678 Rab8, membrane trafficking and 678–9 Rab11, membrane trafficking and 678 Rabbit models glaucoma 42 in vitro optophysiology 531–2, 532f Rab GTPase definition 726, 847 functional roles 678 light-independent retinal degeneration and 732 membrane trafficking and mutation effects 679

Subject Index Rab6 678 Rab8 678–9 Rab11 678 rhodopsin targeting/trafficking 678, 682f SNARE interactions 679 Radial peripapillary capillaries (RPCs) 656, 657f Radiant power, definition 96 Radioimmunoassays, retinal neuropeptides 487 RALBP (retinal-binding protein) 585 Randomized controlled clinical trials (RCTs) definition 426 macular edema treatments 434 Ranibizumab therapy AMD 271, 834 choroidal neovascularization 92–3 macular edema 82 RANTES, secretion 767 Ras, circadian regulation of CNGCs 120 Rat, glaucoma models 39, 736 Rax 213 structural domains 213, 213f Rayleigh scattering 669–70, 669f definition 668 RDS, definition 847 Reactive oxygen species (ROS) apoptosis role 739, 739f mitochondrial dysfunction and 739, 739f Readily releasable pool, definition 661 Receptive field(s) (RFs) definition 290, 301, 309 Recessive optic atrophy 335 Recoverin (S-modulin) 629–30 definition 412 light adaptation in rods 600 light-driven translocation in photoreceptors 412, 413f, 614–15 active transport vs. diffusion 413–14 possible functions 413 Red–green color defects acquired optic neuritis and 137 RP and 136 definition 140 deutan defects 134, 143–4, 144f inherited 143 classification 143, 144f epidemiology 143 inheritance 143 L/M gene deletion/inactivation 143 structural basis 144, 145f perceptual consequences 143–4, 145f protan defects 134, 143, 144f tritan defects vs. 145–6 Refractive errors, retinitis pigmentosa 690 Refractory period definition 819 rod phototransduction 822 Refsum disease, retinitis pigmentosa and 705 Regression analysis, perimetry 556 Regulator of G protein signaling 5 (RGS5) 549–50 Regulator of G-protein signaling 9 (RGS9) cone inactivation 607, 628 definition 412, 605, 610 light-driven transducin translocation and 414 Relative afferent pupillary defect, ischemic vs. nonischemic CRVO 79, 79t Reprogramming definition 360 retinal remodeling 362 Research models 736 Resolution acuity 1, 2f measurement 1, 2f see also Visual acuity Resolution limit, definition 163 Retina anatomy/structure 470, 648 cell types 276 clock hour delineations 791, 793f goldfish 106, 106f membranes 708 vertebrate 105–6 cone distributions 101 definition 212, 830 drug delivery, BRB permeability and 49

evolutionary perspectives amphibians 847–8 pineal gland relationship 501 flat mounts 791, 793f flow of visual information 470 neurochemistry 228–34, 487 glutamate pathways 278, 503 melatonin synthesis 500 trauma vs. RP 692–3 visual acuity across 4, 4f Retina Implant AG, clinical trials 356 Retinal adaptation 105–6, 290, 318, 325–32 definitions 325 divergent rod/cone systems and 326, 841 horizontal cells and 315f DA and 315 dark vs. light adaptation 314–15 NO and 315 uncoupling via all-trans-retinoic acid 315 Limulus 622 study methods dichoptic stimulation protocols 325 ERG 325 periodic test stimuli 327–8 probe-flash paradigm 327 types/forms 325 chromatic adaptation 330, 330f contrast adaptation 329, 329f Retinal angiomatous proliferation (RAP) 93, 94, 94f Retinal-binding protein (RALBP) 585 Retinal cannabinoids 717–25 anatomy 719 biochemical assay 719 metabolizing enzyme distribution/localization 720, 721t receptor distribution/localization 719, 721t development and 724 functional role/physiology 720 bipolar cells and 719–20, 721 RGCs and 721 transmitter release and 720 neuroprotection and 724 Retinal cannabinoids, photoreceptor modulation 721 2-AG release from bipolar cells voltage-dependent 723 voltage-independent (mGluR1a-mediated) 723 presynaptic suppression role 724 voltage-gated currents and 721 2-AG release from bipolar cells 723 retrograde cone responses 722–3, 722f rod vs. cone responses 721–2 WIN 55,212-2 effects on cone light response 723f, 724 Retinal circadian clock circadian retinal responses 106 dopamine role 108 fly compound eye and 127–9 goldfish as model organism 106 illumination variation and compensation 105–6 rhythms associated 108 Retinal circadian clock, rod–cone regulation 105–11 circadian clock pathway 109, 110f DA/melatonin control 109–10 retinal adaptation vs. 110 clock vs. other retinal responses 108 conductance changes and 108, 109f dopamine release and 108–9 evidence against ambient/background light effects 109 day/night differences in outer retina responses 107, 107f cones and cone horizontal cells 106f, 107, 107f dopamine receptors and dopamine role 108–9 evidence for electrical coupling and 107 functional implications of 110 photoreceptor survival and 111 scotopic vs. photopic vision effects 105 synaptic switch 111 goldfish model 106, 106f rod/cone pathways and 106, 106f mammalian clock and control of rod–cone coupling 109 Retinal circuitry 22–4, 309 brain pathways 292–3 cone postreceptoral circuitry 22–4 signaling pathway 23f definition 22

901

902

Subject Index

Retinal circuitry (continued) evolutionary aspects 22 future studie 397–8 lateral communication networks 24 amacrine cells 24–5 horizontal cells 24 RGCs 25 rod pathways 25, 26f organization 389, 390f rewiring 363 rod postreceptoral circuitry 22, 25, 26f electrophysiological studies 25–6 primary pathway 26, 26f psychophysical studies 25 secondary pathway 26–7, 26f tertiary pathway 26f, 27 Retinal circuitry, development 389–98 lateral circuit assembly 395 inner retina (amacrine cells) 390f, 395 outer retina (horizontal cells) 390f, 396 spontaneous/light-evoked activity 396, 397f synaptic laminae 389 time-line 390f vertical pathway assembly 389 bipolar cells 392, 392f photoreceptors 393 retinal ganglion cells 391, 391f vertical pathway synaptogenesis 393, 393f, 394f, 395f Retinal degenerative diseases (RDDs) definition 354 imaging, adaptive optics (AO) 14f, 15, 15f Retinaldehyde (retinal) in visual cycle 649f clearance of all-trans-RAL from OS disks 648 oxidation of 11-cis-ROL to 11-cis-RAL 651 reduction to all-trans-ROL 649 regeneration of visual pigment from 11-cis-RAL 651 all-trans-Retinaldehyde (all-trans-RAL) 648, 649f clearance from OS disks 648 conversion to 11-cis-RAL 648 production from 11-cis-RAL 641, 642f, 648 reduction to all-trans-retinol 649 11-cis-Retinaldehyde (11-cis-RAL) 17, 648 competition between rods and cones for 19 cone opsins and 626 noncovalent interactions 629 covalent attachment to rhodopsin via Schiff base 638, 640–1, 640f visual cycle and 17, 648, 649f conversion of all-trans-retinaldehyde to 648 interaction with apo-opsin 651 photoisomerization to all-trans-retinaldehyde 641, 642f, 648 synthesis from 11-cis-ROL 651 Retinal densitometry, definition 7 Retinal detachment definition 790 PVR and 708, 712 retinopathy of prematurity and 792, 794f Stickler syndrome and 253, 256f treatment, ROP and 792 Retinal development assessment 791–2 dopamine role 498 oxygen role 795 Retinal dialyses 801, 803, 803f conservative management 804 laser demarcation 805 Retinal dystrophy definition 685, 699 Retinal eccentricity, chromatic function and 102 Retinal ganglion cells (RGCs) apoptosis 386, 734–44 in glaucoma 734 detection 734–5 axons degeneration in mouse glaucoma model (DBA/2J) 386, 387f elevated IOP-induced damage 382, 386 cell fate specification 750–1 circuitry 322 bipolar inputs 290–1, 319 dendritic stratification 391, 391f emergence of function 396, 397f lateral networks 25

classification/types 25, 290, 301 numbers 301 object motion sensitive 305–6 ON–OFF-cells 301, 305 clomeleon-labeled 454 definition 105, 381, 734 development 235–9 Islet1 role 238 Pou4f2/Brn3b role 237–8, 237f dynamic range and cone pathways and 846 rod bipolar cell pathway and 844 functional roles 25 imaging, adaptive optics 12f, 13f, 14 invertebrates 240 neuroprotection 734–44 reduction of misfolded proteins 741 optic neuritis 540 retinal disease and, proliferative vitreoretinopathy and 711 soma 386 Retinal ganglion cells, information processing 301–8 action potentials and 301 amacrine cells and 301, 306 bipolar cells and 301, 306 principles 301 spatial processing 302 center-surround receptive fields 302, 303f contrast sensitivity and 290 one cell–one pixel analogy 302–4 synchronous activity and 302–4 tiling and 302, 303f as spatiotemporal filters 305, 305f as specific feature detectors 305, 305f approach-sensitivity/looming and 305–6, 307 object motion sensitivity 306–7, 306 saccadic suppression and 307 stimulus features triggering RGC activity 305 temporal processing 301 cell-type-specific responses 301, 302f contrast sensitivity and 291 stratification and 301 ON vs. OFF responses to light 301, 302f Retinal ganglion cells, neurochemistry acetylcholine, directional selectivity 296, 299 dopamine and 497 endocannabinoids and 721 GABA receptors 233 neuropeptides and 490 Retinal gap junctions amacrine cells 447, 449–50 cone photoreceptors 159 rod photoreceptors 821 Retinal glial cells cell fate specification 750, 751f retinal remodeling and 360 scar formation 372, 372f proliferative vitreoretinopathy 710, 710f Retinal glutamate receptors amacrine cells 278 cone bipolar cells 23–4 Retinal G-protein receptor (RGR) 208, 651 functions 208 human 208 Retinal growth persistent neurogenesis in zebrafish 865, 867f, 870f, 871 Retinal guanylyl cyclase (RetGC) 606 cone inactivation and cGMP synthesis 627, 628 definition 605, 610 light adaptation 630 regulation 612–14 rod inactivation and cGMP synthesis 612 vertebrate isoforms 614 Retinal hemorrhage, in CRVO 80, 80f, 83f Retinal histogenesis 169–70, 170f, 745–52 birthdating 745 early vs. late cohorts 745, 746f procedure 745 cell autonomous vs. nonautonomous control 748 conflicting results 749 default progenitor state and 748 feedback density regulation and 749 inductive interactions and 748–9

Subject Index serial competence vs. progressive restriction model 749, 749f coordination 169–78 cyclin-dependent kinase (Cdk) role 172, 172f cyclin role 172, 172f E2f role 175 Ink4 role 172f, 174 p19Arf role 172f, 174 Rb family role 172f, 173–4, 174f environmental challenge and 748 lineage tracing 746, 747f, 747t embryonic timepoints 747–8 postnatal timepoints 746–7 stochastic cell fate determination 746–7, 748 timing of specification 748 progenitors 745 default state 748 multipotency 748 possible rod-only 748 symmetrical divisions 747–8 signaling pathways 263–9 future directions 269 hedgehog 265, 266f photoreceptor development 268, 268f, 570f, 572 transforming growth factor-b 267, 267f Wnt 264, 265 transcription factors and competence 750, 750f amacrine specification 750–1 Ascl1 (Mash1) 750 Atoh7 (Math5) 750–1 FoxN4 750 general requirement for Pax6 750 Math3 750–1 NeuroD1 750–1 neuronal vs. glial cell fate specification 750, 751f Notch signaling and 750 Ptf1a 750–1 RGC specification 750–1 Retinal holes 801, 802, 802f conservative management 804 laser demarcation 805 Retinal illuminance definition 325 light adaptation and 326, 326f Retinal imaging adaptive optics (AO) 9 cone mosaic and color vision 11 cones 11 eye motion compensation 9, 9f retinal disease 14f, 15, 15f retinal ganglion cells 12f, 13f, 14 retinal pigment epithelium 10f, 13 retinal vasculature 13f, 15 OCT see Retinal OCT see also specific techniques Retinal information processing 318–24 amplification 279–80, 280f mutual antagonism and 320 axonal cells 282 inner retina contrast gain 320 feature extraction 321 olfactory bulb vs. 277f organizational principles 320 concatenated lateral interactions 320 mutual antagonism as amplification 320 redundant feedforward/feedback interactions 320, 321f visual stream interactions 320, 321f, 322 outer retina (gain and level adjustment) 318, 320 ON vs. OFF streams 319 Retinal leakage analyzer, definition 44 Retinal mosaic, definition 447 Retinal neovascularization 541–50 inhibitors/antiangiogenic factors 541, 542t, 549 CD59 549 pigment epithelium-derived factor 549 Slit/Roundabout4 549 soluble VEGF receptor 1 549 as therapeutic agents 549 tryptophanyl-tRNA synthase fragment 549 VEGFxxxb isoforms 549 mechanism 541 promoters/proangiogenic factors 541, 542, 542t

903

angiopoietins 547, 548f complement system components 548 ephrins/Ephs 546, 547f erythropoietin 548 integrins 548 matrix metalloproteinases 548 Notch 545, 546f placental growth factor 544 platelet-derived growth factor 544, 544f, 545f tumor necrosis factor-a 546 vascular endothelial growth factor (VEGF) 542, 542t Retinal nerve fiber layer (RNFL), optic neuritis 539 Retinal neuropeptides 487–93, 488t distribution 487 GABA co-expression 490 historical aspects 487 peptide expression studies 487, 488t bioassays/radioimmunoassays 487 peptide localization studies 488 immunostaining 489, 489f, 490f mRNA 488 transgenic approach 488 ultrastructural studies 490 receptor expression studies 491 binding-sites/autoradiography 491 mRNA 491 pharmacological studies 491 receptor localization studies 491 immunostaining 491, 492f, 493f mRNA 491 signaling pathways 491 Retinal neuropeptides, function 477–86 cellular signaling 479 NPY 479, 480f somatostatin 479, 481f, 482f VIP 479, 482f electroretinogram results 482 somatostatin 483, 484f SP 482, 483f VIP 483 intracellular signaling 478 neurotransmitters and 479, 483, 485 overview 477, 484 receptors localization 478 variety 477, 478, 478t Retinal OCT 61, 61f, 525 adaptive optics OCT (AO OCT) 527–8, 529f, 530f clinical application 530–1, 531f axial resolution 525, 526f cellular resolution 527 intraretinal layers 528, 530f RPE 529–30, 530f in vivo 528, 529f data acquisition speed 525, 526f development 525, 526f functional imaging 531, 532f, 533f in vitro rabbit retina 531–2, 532f in vivo human retina 532–3, 533f future directions 534 RPE barrier assessment 61, 61f three-dimensional ultrahigh resolution 526, 527f Retinal pigment epithelial (RPE) cells 45, 47, 775 adaptive optics 10f, 13–14 culture 778–9, 778f human fetal model 761, 764f, 766 OCT tomograms 529–30, 530f Retinal pigment epithelial (RPE) tight junctions 45, 47, 762, 771–2, 771f, 774f, 775 assembly during differentiation 776f, 777 claudins 762, 775f, 776, 776f, 778 composition 775–6, 775f diseases and 779 modulation 47 occludin 775f, 776, 778 immunolocalization 60, 60f regulation 47 embryonic maturation studies 47 structure, assessment 60, 60f transepithelial electrical resistance (TER) 778–9 Retinal pigment epithelium (RPE) 375–6, 761–72 anatomy/structure 58, 59f, 755, 761, 762f asymmetry/epithelial polarity 761, 763f, 767

904

Subject Index

Retinal pigment epithelium (RPE) (continued) cell junctions 762, 771–2 dopaminergic neurons 496 hfRPE vs. 762, 764f morphology 762 SRS relationship 761 autologous grafts, RP treatment 697 choroid plexus epithelium vs. 774 definition 815, 830 development 200f, 753, 754f, 774, 774f, 777 early patterning 200t, 201f, 202–3 disease and AMD and 271, 761, 766 diabetic retinopathy 767–9 extracellular spaces and 761 leukocyte migration and disease 771, 771f oxidative stress and 766 PVR 710f, 711, 767–9, 770–1 RP 697 uveitis and 761, 770–1 disk membrane catabolism 817–18, 817f function 755 growth factor secretion 757, 758f immune system and cytokines/chemokines and 767, 768t, 769f innate immunity and 375 interactions around SRS 767, 768t, 769f optical coherence tomography 529–30, 530f retinal remodeling and 360, 364 regeneration in Xenopus retina 852 retinomotor movement of melanosomes 221–2 regulation 225 cAMP 222–3, 223f, 224f, 226 visual cycle stages 17, 648, 649f isomerization to cis form 650 11-cis-RAL synthesis 651 regulation 651 retinyl ester synthesis 650 transfer of all-trans-retinol from photoreceptors to 642, 649 Retinal pigment epithelium, physiology 761–72, 763f circadian effects (light/dark transitions) 502, 761, 763–4 dopaminergic neurons 496 GAT expression and 231 immune interactions 761, 767 cytokine/chemokine production by RPE 767 effects on proliferation/migration 767 IFNg and 769–70, 771f PDGF and 767–9, 770f polarized locations 767, 768t macromolecular movement across 182, 761, 763f leukocyte migration and disease 771, 771f tight junctions and 762, 771–2 melatonin receptors 502 metabolic load and fluid regulation 761, 763 CO2/HCO3 transport role 762, 764, 765f, 766f IFNg and 770 lactate transport and 765, 766f pHi and changes in fluid absorption 762 in vitro modeling 763–4 morphology, polarity and function 761, 762 oxidative stress and antioxidant role 761, 766 iron deposition and 767 monocyte invasion and 767 smoking relationship 766–7 Retinal pigment epithelium (RPE)–choroid complex 753–60 age-related changes 758 Bruch’s membrane 758 gene expression 758 development 179, 753, 754f function 755 structure 755 Retinal pigment epithelium (RPE)–choroid interactions 756, 759 in adults 757 growth factor secretion 757, 758f, 759 impact of choroidal change on RPE 757 isoform-specific VEGF mouse model 757 receptor expression 757 developmental 179, 756 Retinal plasticity, light damage, protection mechanisms 343 Retinal progenitor cells (RPCs) 169–70, 170f, 367 definition 169, 235, 263

differentiation into photoreceptor precursors 569, 570f, 571f retinal transition cells vs., cell cycle regulation 172, 172f Retinal regeneration 869 in mammals 872 in zebrafish 863–73 damage models 867 genes 869 mammals vs. 872 Mu¨ller glial cells involved in 867, 868f, 870f, 871–2 process of 870f, 871 Retinal remodeling 360–6 amacrine cells 283 definition 360 events/mechanisms 362 Mu¨ller cell remodeling 364 neuritogenesis 364 neuronal migration 363 receptor reprogramming 362 rewiring 361f, 363 RPE remodeling 364 self-signaling 363 synaptogenesis/microneuromas 363 vascular remodeling 364 light damage injury mechanisms 342 occurrence 360 overview 360 progression/phases 360, 361f cone effects on 366 factors affecting kinetics 360, 362f phase 0 361f phase 1 360, 361, 361f phase 2 360, 361, 361f phase 2+ 362 phase 3 360, 361f, 362, 363, 366 prosthetic devices and 355, 357–8 therapy and reversal 364 triggers 360 vascular 548 Retinal tears 802f conservative management 804 identification 802 laser demarcation 805 pathophysiology 801 Retinal topographic maps, starburst amacrine cells and 451 Retinal transition cells (RTCs) 169–70, 170f definition 169 retinal progenitor cells (RPCs) vs., cell cycle regulation 172, 172f Retinal transplantation 367–73 aim 373 early work 367 fetal donor material 369 growth factors and 373 inhibitory barriers and 371 limitations 367 retinal remodeling reversal/prevention 365 RPE transplants 367 Retinal vasculature 183, 653–60 adult 541 anatomy 29, 181f, 183 arterial blood supply 653 capillary-free zone around arteries 655, 656f dual nature (ophthalmic artery vs. choroidal) 653–4 perfusion pressure and 653–4 physiology 184 autoregulation 34, 659 blow flow rate 184 capillary bed 181f, 184, 655, 656f cotton wool spots and 656 foveal region 657, 657f layers 655, 657f macular region 655f, 657 physiology 184 radial peripapillary capillaries 656, 657f development 183, 541, 795 cell–cell interactions in 546 early 542 mechanisms 542 imaging, adaptive optics (AO) 13f, 15 nerve supply and 658, 659f pathology 185 causes 346–8

Subject Index diabetic retinopathy 185 infarction 183–4 retinal remodeling and 364 retinopathy of prematurity 185 physiology 184 retinal layers and 653, 654f, 655 vasoconstriction 34 venous drainage 183–4, 657 arteriovenous crossings 184, 655f, 656f, 657–8 central retinal vein 655f, 658, 658f, 659f physiology 184 postcapillary venules 656f, 657–8 superior branch veins 655f, 658, 658f Retinal vein occlusion 75 branch 75 hemi-central 75 Retinal vessel analyzer (RVA) 36 Retina Society Terminology Committee, PVR clinical classification 714 Retinitis pigmentosa (RP) 354, 470, 684–97, 854 acquired color blindness 136 allied disorders 684, 698 animal models Drosophila models 730–1 mouse (rd1) 857 Xenopus laevis models 849, 850 classification 687 age of onset 687 ERG and 692, 693f, 694f, 695f functional loss 690 inheritance pattern 687, 687t clinical features/diagnosis 690 anterior segment and cataracts 690, 695 OCT 692 refractive errors 690 visual symptoms 690, 857 definition 354, 637, 685, 699, 726, 847, 853 differential diagnosis 692 autoimmune retinopathy 693–4 diffuse unilateral neuroretinitis 693, 696f drug-induced disease 693 infectious disease 693, 695f inflammatory disease 693 trauma 692–3 gene mutations 650, 651, 685, 688t CHM gene 703 CRB1 gene 701–2 OAT gene 702–3 PDE6B gene 686, 857 RDH5 gene 702 rhodopsin mutations and 645–6, 686, 701, 730–1, 850 RLBP1 gene 702 RP1 gene 686 USH24 gene 686 histological features 690, 699 bone spicule pigmentation 685f, 690, 699, 700f bull’s eye maculopathy 699, 701f Coats-like response 700, 701f drusen 699, 700f optic nerve pallor 699, 700f historical aspects 685 inheritance 686, 687, 687t, 730–1 autosomal dominant 686, 690 autosomal recessive 686, 690 X-linked disorders 686, 701, 703 nonsyndromic 686 pathophysiology 690 apoptosis and 857 prevalence 686 prognosis 694, 857 retinal remodeling 360 RPE remodeling 364 self-signaling and 363 vascular remodeling 364 RPE barrier breakdown 66 syndromic forms 686, 703 abetalipoproteinemia 703 Alstro¨m syndrome 704 Bardet–Biedl syndrome 686, 687f CPEO 704 Drosophila models 730–1 Friedreich’s ataxia 704

incontinentia pigmenti 704 Joubert syndrome 704 mucopolysaccharidoses 704 neuronal ceroid lipofuscinosis 704 Refsum disease 705 Senior–Loken syndrome 705 spinocerebellar ataxia (type 7) 705 Usher syndrome 686, 730–1 vitamin E deficiency 704 terminology 698–707 treatment 694 autologous RPE grafts 697 CNTF/neuroprotection 696 docosahexanoic acid 696 gene therapy 696 microelectrode implants 697 optimizing remaining vision 695 patient resources/support 694 stem cell therapy 697 treatable vs. nontreatable forms 694 viral expression of photosensitive proteins 697 vitamin A 696 vitamin E 696 visual testing in 691, 705 dark adaptation 691 visual field 691, 691f, 692f Retinitis pigmentosa, fundus examination classification by pattern 689, 700 choroideremia 703, 704f classic pattern 685f, 700, 702f concentric RP 701 fundus albipunctata 702, 703f gyrate atrophy 702, 703f inverse RP 700 pigmented paravenous retinochoroidal atrophy 702, 703f retinitis punctata albescens 702 RP with preserved peri-arteriolar RPE 701 sector RP 701, 702f sine pigmento 701 tapetal-like reflex/sheen 701, 703f unilateral RP 689 diagnosis 685, 685f, 690, 692 Retinitis pigmentosa sine pigmento 701 Retinitis pigmentosa with preserved peri-arteriolar RPE (PPARPE) 701 Retinoblastoma gene/protein (Rb/Rb) in cell-cycle regulation 170, 171f retinal development role 172f, 173, 174f Retinochrome definition 582 squid 585 Retinohypothalamic tract 112, 113f Retinoic acid (RA) developmental role 268 optic cup patterning 203, 268 photoreceptors 268 pathway 268, 268f Retinoid isomerization 650 alternative in cone-dominant species 19, 19f all-trans-Retinoids horizontal cells and 315 isomerization by Rpe65-isomerase 650 Mu¨ller cells and 17–18 Retinol (ROL) 649f oxidation of 11-cis-ROL to 11-cis-RAL 651 transfer of all-trans-ROL from photoreceptors to RPE 649 all-trans-Retinol (all-trans-ROL) reduction of all-trans-RAL to form 17, 649 transfer from photoreceptors to RPE 642, 649 11-cis-Retinol (11-cis-ROL) 650 Mu¨ller cells and synthesis 18 all-trans-Retinol dehydrogenase (all-trans-RDH) 17 Retinol dehydrogenases (RDHs) 642 all-trans-RDH 17 RDH5 651 RDH8 649 RDH12 649 retinitis pigmentosa 702 Retinomotor movements in fish 219–27 circadian rhythms 219–20, 225 force production 221–2 elongation/contraction 220–1, 221f

905

906

Subject Index

Retinomotor movements in fish (continued) melanin granule movements 220, 222f function 226 overview 219, 220f regulation 219–20 adenosine 225 calcium 226 cAMP 222–3, 223f, 224f, 226 carbachol 226 dopamine 223–4, 225–6 Retinopathy of prematurity (ROP) 185, 790–800 animal models 796 mouse OIR 796, 796t, 797f rat 50/50 OIR 793f, 796, 796t stages modeled 796 classification 790 aggressive posterior 790, 796 flat mount vs. clock hour description 791, 793f peripheral severe 790, 796 plus disease 790, 791, 792f stage 790–1, 792f threshold/prethreshold 793t zone 790–1, 791f clinical background 790 epidemiology 790 genetic factors 795 pathophysiology 795 avascular retina and 790, 796 erythropoietin 798 HIF1a 798 IGF-1 and binding proteins 798 light role 799 oxygen and oxidative stress 795, 798 VEGF and 795, 798 retinal detachment and 792, 794f screening 791 treatment 792 acute neovascular changes 792 antioxidants 799 anti-VEGF antibodies 798 clinical studies 792 fibrovascular stages/retinal detachment 792 future considerations 799 surgery risks 793–5 type 1 prethreshold/threshold ROP 792, 793f, 793t visual rehabilitation 795 Retinopathy/retinal degeneration 698–707, 854 animal models 854 comparative merits 727 mice 854, 857 atrophy chronic rhegmatogenous retinal detachment 803 definition 685, 699 cilia and 680–1 cilia-associated proteins 681–2 definition 685, 699, 847 dopaminergic interplexiform cells and 475 dystrophy, definition 685, 699 inflammation role 341 glial scar formation 372, 372f light-dependent 731 light-independent 731 mixed mechanisms 732 primary definition 684, 698 with secondary photoreceptor degeneration 684, 698 therapy bionic implants 365 history 367 retinal remodeling reversal 364 trophic factors 365 Retinopexy, definition 801 Retinoschisis definition 118 due to mutation in retinoschisin 122 juvenile X-linked, mfERG results 510, 514f Retinotopy, definition 354 Retinotoxic drugs 837–9, 838t Retinyl ester(s) 649f definition 648 isomerization to cis form 650 synthesis 650

N-Retinylidene-phosphatidylethanolamine (N-ret-PE) 648–9 Retrobulbar space, definition 38 Retrobulbar vessels, definition 28 RGS5 (regulator of G protein signaling 5) 549–50 Rhabdom definition 416 Limulus photoreceptors 419, 616 eye-type-specific organization 419–20 shedding, circadian regulation 422, 422f structural changes, circadian regulation 420 Rhabdomeral lobes 583f definition 582 Rhabdomere 241f circadian changes in fly 127–9 definition 124, 205, 240, 567, 582, 726 Gq opsins 208 Rhabdomeric Gq opsins 208 Rhabdomeric photoreceptors 567–8 Rhegma, definition 801 Rhegmatogenous retinal detachment (RRD) 801–10 break types 802 retinal dialyses 801, 803, 803f retinal holes 801, 802, 802f tractional tears 801, 802, 802f clinical features 801 break localization 803 chronic RRD 803 signs 802 symptoms 801 definition 801 differential diagnosis 803, 805t historical aspects 801 management 804 complications 808 conservative 804 laser demarcation 804 new developments 809 outcomes 807 pneumatic retinopexy 805, 807, 809 primary aim 804 retinal dialysis 804, 805, 806, 807 retinal hole 804, 805, 806, 807 retinal tears 804, 805, 806 scleral bucking 806, 807f, 808, 809 treatment failure 809 vitrectomy 806, 808, 808f, 809 pathophysiology 801 Rhodopsin 610–11, 631–2, 634, 637–47, 648, 812–13 cone opsins vs. 18–19 cycling 17, 18f, 610, 611, 642f activation by light 610–11 bathorhodopsin 641 lumirhodopsin 641–2 metarhodopsin (I and II) 611, 641–2, 648 opsin 611 photorhodopsin 641–2 regeneration 642, 651 retinal isomerization 641, 648 vertebrate vs. invertebrate 642 definition 205, 206, 610, 819 desensitization 644 Drosophila 729 inactivation 611, 644, 644f, 645f light adaptation and 599–600, 601 intraflagellar transport 580–1 light damage role 338, 339 light protection role 343 mutation and disease 645, 646f autosomal dominant RP and 686, 731 Drosophila mutants 730–1 light-dependent retinal degeneration and 731 light-independent retinal degeneration and 731 sector retinitis pigmentosa and 701 transport/trafficking effects 678 Xenopus laevis models 849, 850 protein interactions 644, 644f rod cell structure and distribution 637, 638f, 676 signaling 412, 644 regulation by phosphorylation 644, 644f transducin activation 644 squid activation/deactivation 583f, 584–5, 584f

Subject Index arrestin and 587 structure 582–4 structure 638 chromophore binding region 639, 640, 640f conserved motifs 639 squid 582–4 three-dimensional 639, 640f two-dimensional 638, 639f vertebrate vs. invertebrate 643–4, 643 thermal stability 632 transport/metabolism retinal degeneration and 731 vertebrate vs. invertebrate 643 photoactivation 642 regeneration 642 structure 643–4, 643f Xenopus laevis models 849 Rhodopsin targeting/trafficking 676–83 disease associations 680 Bardet–Biedl syndrome 681 retinopathy 681–2 rhodopsin mutation effects 678 Usher syndrome 682 membrane trafficking apparatus 677 phospholipid co-transport 677 retinal cell-free assay 677–8 RTCs 677 sorting machinery 678 rod cell polarity and 676 small GTPases and 678, 682f Arfs 679, 680f Rabs 678 SNAREs and their regulators 679, 681f Rhodopsin transport carriers (RTCs) 677 Ribbon synapses 661–7 amacrine cells 278 bipolar cells 457f, 458 calcium and Ca2+ channels 664 Ca2+ currents 664–5, 664f intracellular 663, 665 role in vesicular release 665–6 definition 156, 661, 819, 841–2 proteins 663 Ca2+ -binding 663, 665 Ca2+ channel 664, 665 mutations 665, 666 ribbon function 663 rod photoreceptors 820–1, 820f, 822 structure 661, 662f vesicles 662, 662f numbers 661 release mechanism 665 Ribeye (protein) 663, 664 Riluzole 739 Ring perimetry 553, 554f RLBP1 gene/RLBP1 protein, retinitis pigmentosa 702 RNA helicases, definition 847 Rod bipolar cells 25, 285–6, 452, 454f, 455, 820–1, 820f AII amacrine cells and 323 definition 284 Rod bipolar pathway, dynamic range 842 adaptation to mean background light 845–6 evolutionary conservation 842 RGC sensitivity and 844 scotopic vision sensitivity and 842 signal transfer rod bipolar cell to AII amacrine cells 843 rod to bipolar cell 842, 843f, 844f Rod/cone (mixed) bipolar cells 284–5 Rod–cone dystrophy 853–4 bystander effect and 854 definition 685, 699 Rod–cone electrical coupling 161, 498, 823, 844 goldfish retina 106–7, 106f Rod–cone pathway 105 dynamic range and 105–6, 844 ERG responses 706 goldfish retina 106, 106f Rod dystrophies 853–4 Rodent(s) horizontal cell morphology 466–7, 466f

Rodent models, glaucoma 39 rat 39, 736 Rod-isolated ERG response 706 Rod–OFF pathway, dynamic range and 845 Rod outer segment (ROS) 637, 648 cytoskeleton 677 development from primary cilia 677 disk membrane proteins 676–7 trafficking 677 intraflagellar transport 677 as modified cilium 680 disease associations 680–1 structure 676 Rod photoreceptor(s) 17, 22, 558, 567, 568, 637, 819–24, 854 biophysical properties 821 absence of thresholds 615 cones vs. 18, 589 monochromacy (achromatopsia) 146 chromatic function and 102 degeneration Xenopus laevis models 849–50 zebrafish pde6 mutants 860 development 568, 569f from rod precursor 569f, 570f, 572 signaling molecules 268, 268f, 570f, 572 distribution 568 Drosophila photoreceptors vs. 729, 730f electrical coupling 821 rod–rod 823, 844 evolution 22, 841 function 841 goldfish retina 106, 106f light sensitivity 105–6, 596, 610, 611 saturation 589–90, 596–7, 597f, 598 Weber’s law and 326–7, 597 membrane trafficking 677 mouse retina 568 pathways amacrine cells and 280, 281f, 450, 843 critical fusion frequency and 25, 26f definition 105 photopigment cones vs. 626 stability 626 polarized light and 670–1 retinomotor movements 219, 220, 220f function 226 regulation by cAMP 222–3, 223f, 224f regulation by light 225 rod:cone densities 589 structure 637, 638f, 820f adherens junctions 677 axon 819 cilia 677, 680 cytoskeleton 677 gap junctions 821 inner segment 637, 677 invagination 821 ion channels 821 morphology/topology 819 outer limiting membrane 677 polarity 676 synapse 463, 820, 823–4 terminal 819 Rod phototransduction 22, 412, 610, 624–5, 625f, 631–6, 644, 821 activation 610–11 vertebrate rods 631–2, 633f adaptation 596–604 cyclic guanosine monophosphate (cGMP) role 635 gated channel 635 dark noise 633, 821 ‘dark thermal events’ 821–2 elimination mechanisms 842–3 sources 821 synaptic convergence and mitigation 821–2 dominant time constant 601 light-induced changes 602 electrical coupling and starlight 823 twilight 823 G-protein signaling pathway 631 melatonin effects 503–4

907

908

Subject Index

Rod phototransduction (continued) membrane hyperpolarization 631–2 graded 632 negative feedback 823 phosphodiesterase, second amplification step 634 quantum efficiency of photoactivation 632 refractory period 822 single-photon signal 631, 821, 822 suction electrode rod recording 631, 632f summation 821 synaptic transfer function high gain and temporal filter 822 nonlinear threshold 822, 842–3 vertebrate rods activation 631–2, 633f single photon detection 631 vesicle release rate 822, 842 Rod phototransduction inactivation 610–15, 613f lack of action potential and 615 light damage and 339–40 PDE inactivation 612 restoration of cGMP/Ca2+ levels 612 rhodopsin inactivation 611 transducin inactivation 612, 644 Rod spherule, definition 461, 841 Roll back definition 309 horizontal cells 310, 310f, 316 Rpe65-isomerase 650 Rpe65 definition 17 gene therapy in LCA 364–5 light damage role 339–40 in visual cycle 650, 651 RPE-retinal G-protein receptor (RGR) opsin 651 Rubella infection, RP vs. 693, 695f

S Saccade(s), suppression and RGCs 307 Saltatory nerve conduction, definition 506 SArr (squid arrestin) 584f, 587 Sattler’s layer 30 Saturation avoidance, definition 589 definition 589, 596 Scanning laser ophthalmoscope (SLO) 526 adaptive optics (AO) and 9–10 Schiff base 11-cis-RAL attachment to rhodopsin via 638, 640–1, 640f definition 637, 648 Sclera, posterior ciliary artery penetration 180, 180f, 181f Scleral buckle basic principle 806, 807f rhegmatogenous retinal detachment 806 complications 809 outcomes 808 retinal holes/dialysis 806 retinal tears 806 S-cones 134, 140, 159 blue cone bipolar cells 453 clomeleon-labeled 454 ‘genuine’ 452 L/M cones vs. 145–6 Parkinson’s disease and 137–8 photopigment gene 141, 142f species differences in horizontal cell selectivity 467 spectral sensitivity 141f tritan color defects and 144–6 Scotoma cecocentral, definition 333 mfERG results bull’s eye retinopathy 510, 512f, 513f central serous retinopathy 510, 511f small lesions 511, 515f Scotopic, definition 558 Scotopic vision 558, 559–60, 559f, 560f, 631 definition 22, 68, 105, 205, 705, 819, 841, 853 sensitivity 596, 842 trade-offs 596

Screening, diabetic retinopathy 788 Searching nystagmus definition 333 recessive optic atrophy 335–6 Second-order, first slice, definition 506 Second Sight Medical Products Inc. (SSMP) prostheses, clinical trials 356–7 Second-site modifier screens, definition 726–7 SECORD (severe early childhood-onset retinal dystrophy) 687–9 Secretory immunoglobulin A (sIgA) 377 Secretory phospholipid A2 (PLA2), innate immunity 376 Sector retinitis pigmentosa 701, 702f Self-signaling definition 360 retinal remodeling 363 Senior–Loken syndrome, retinitis pigmentosa and 705 Sensory cilia, definition 575 Sensory receptors Serial competence model definition 745 retinal histogenesis 749, 749f Serotonin (5-HT), fly circadian plasticity and 131 Severe early childhood-onset retinal dystrophy (SECORD) 687–9 Sexual dimorphism, color vision in butterflies 154 Shack–Hartmann wavefront sensor 8f definition 7 Shh (sonic hedgehog) signaling definition 863 in retinal development 864–5 Short posterior ciliary arteries (SPCAs) 179 anastomosis, circle of Zinn 180, 180f, 181f Short-wavelength automated perimetry 553, 553f Signaling pathways cell extrinsic, definition 263 cell intrinsic, definition 263 Drosophila–human similarities 727–8 retinal neuropeptides 491 Signal transduction, human–Drosophila similarity 727–8 Sign-conserving synapses, definition 276 Sign-inverting synapses, definition 276 Silent substitution 100–1, 101f Single nucleotide polymorphism (SNP) in AMD 272 definition 270 Single photon response, definition 631 Six3/Six3 212 mutations 215–16 structural domains 212, 213f Six6/Six6 212 mutations 215–16 structural domains 212–13, 213f Skylight, polarization 669–70, 669f navigation using 673 Slit/Roundabout4, antiangiogenic activity 549 Small-field amacrine cells 447, 448f Small GTPases definition 676 rhodopsin targeting/trafficking 678, 680f, 681f, 682f Smoking, AMD and 766–7, 833 Smooth endoplasmic reticulum (SER), IP3-mediated Ca2+ release in Limulus phototransduction 620 SNARE proteins definition 661, 676 membrane trafficking and 679 protein interactions 680 Rab interactions 679 rhodopsin targeting/trafficking 679, 681f, 682f ribbon synapses 663 structure 680 Snellen acuity 1–2, 2f acuity, definition 717 test–retest variability 3 Snellen chart, definition 536 Snowflake vitreoretinal degeneration 260 Socs3 gene, retinal regeneration 869–71 SOD-1 (superoxide dismutase 1), in mouse models of AMD 274 Sodium (Na) RPE transport and 764, 766f Sodium/bicarbonate co-transporter RPE and metabolic load reduction 764, 766f Sodium (Na+) channels, cone photoreceptors 158–9 Sodium pump, definition 58, 219

Subject Index Soluble VEGF receptor 1 (sVEGFR-1) antiangiogenic activity 549 corneal avascularity role 549 Soma definition 381 retinal ganglion cell 386 Somatostatin retinal cellular signaling 479, 481f, 482f circadian regulation of CNGCs in photoreceptors 121 excitation of ganglion cells 483, 484f intracellular signaling 478 neurotransmitter release 484 receptors 477–8 Somatotropin-release-inhibiting factor (SRIF) GABA co-localization 490 mRNA localization 488 peptide immunostaining studies 489, 490f radioimmunoassay 487–8 receptor autoradiography 491 receptor immunostaining 492, 492f receptor mRNA expression 491 Sorbitol pathway, pathogenesis of diabetic retinopathy 783 SPARC (secreted protein, acidic, and rich in cysteine), neovascularization and 351 Spatial contrast sensitivity, light level and 561 Spatial frequency channels 164, 165f, 166f definition 163 Spatially offset inhibition, definition 295 Spatial processing contrast adaptation 329 contrast sensitivity and 290 horizontal cells and 316 Spatiotemporal vision cone phototransduction and 162 definition 22 RGCs as spatiotemporal filters 304, 304f Spectral absorbance, definition 96 Spectral absorptance, definition 96 Spectral absorptivity, definition 96 Spectral luminous efficiency (SLE) definition 558 function applications 561 concept 560 definition 96 mesopic 559 Spectral power distribution (SPD), definition 558 Spectral radiance, definition 558 Spectral responsivity (SR), definition 558 Spectral sensitivity, definition 96 Spectral transmittance t(l), definition 96 Spectral tuning, definition 205 Specular reflection, definition 668 Spherule, definition 819 Spinocerebellar ataxia (type 7), retinitis pigmentosa and 705 Splinter hemorrhages, NA-AION 402, 403f Spondyloepiphyseal dysplasia 253, 258f, 260 SQRK (squid rhodopsin kinase) 586 Squid photoresponse 582–8 activation/inactivation 582, 583f, 584f, 585 arrestin 587 PLC 586 rhodopsin 584–5, 586 arrestin 587 calpain-like protease 586 eye structure 582, 583f Gq protein 585 light-activated ion channel 586 phospholipase C 586 rhodopsin 582, 586, 587 rhodopsin kinase 586, 588 polarized light, behavior based on 673–4 SSMP prostheses, clinical trials 356–7 Sst receptors, retinal 478 Standard achromatic perimetry (SAP) 553 definition 551 Starburst amacrine cells (SACs) 450 definition 318

morphology 276–8, 277f, 296–7, 297f, 450, 450f networks/information processing midget BC to GC pathway 281 mutual inhibition and 322, 322f rod pathway 280, 281f neurochemistry 451 OFF- vs. ON-types 451 populations of 450–1 postnatal development and 451 transretinal waves 451 Starburst amacrine cells, directional selectivity role 282, 282f, 298, 322, 322f, 451 ablation studies 297–8 cooperative integration and 299 geometrically asymmetric DSGC regulation 297, 298, 298f, 306 centrifugal excitation vs. centripetal inhibition 298–9 cholinergic excitation 296–7, 298–9 GABAergic inhibition 296–7, 306 mechanism of functional asymmetry 297f, 298 own DS properties 306 Stargardt’s disease 648–9 definition 847 Xenopus laevis model 851 stat3 gene, retinal regeneration 869–71 Static suprathreshold perimetry 552 test strategies 552t Static threshold perimetry 551 definition 551 Stell model of chromatic processing 316–17, 316f Stem cell(s), neovascularization and 351 Stem/progenitor cell therapy 367–73 aim 373 ethical issues 368 induced pluripotent stem cells 368 importance/advantages 368 production 368, 369f technical problems 368 inhibitory barriers and 371 glial scarring and 372, 372f myelin-associated ECM and 372 neural (brain-derived) progenitor cells 367 adult hippocampal 367 limitations 368 neuroprotection and 369 retinal progenitor cells 368–9 retinal remodeling reversal/prevention 365 retinitis pigmentosa treatment 697 total stem cell deficiency transplantation strategies 370 bolus cell injection techniques 370, 371f efficacy 370 MMP2 and ECM digestion 372–3 polymer scaffolds and 370 Stickler syndrome 252 arthropathy 253 cataract 253, 257f molecular genetics 260 myopia 253 ocular-only form 260–1 orofacial abnormalities 253, 257f retinal detachment 253, 256f type 1 vs. type 2 252, 256f Weissenbacher–Zweymuller syndrome 253 Stiles–Crawford effect 7, 326 Strabismus, retinopathy of prematurity and 795 Stroke, NA-AION vs. 401 Subjective day, definition 124 Subjective night, definition 124 Sublamina of the inner plexiform layer 470 Subretinal demarcation lines, chronic rhegmatogenous retinal detachment 803 Subretinal space (SRS) 761, 774, 774f chemical composition 761 blood–retinal barrier breakdown and 767 light to dark transition effects 763–4 definition 87, 761, 773 disease role 761 RPE relationship 761 immune interactions 767, 768t, 769f metabolic load and fluid regulation 763

909

910

Subject Index

Substance P (SP) retinal excitation of ganglion cells 482, 483f mRNA localization 488 neurotransmitter release 484 peptide immunostaining studies 490 radioimmunoassay 487 receptors 478 Suction-electrode recording 631, 632f definition 631 Superoxide dismutase (SOD) in glaucoma 739, 739f Superoxide dismutase 1 (SOD-1), in mouse models of AMD 274 Suprachiasmatic nuclei (SCN) as circadian master pacemaker 112 phase resetting and 112, 114f retinal input 112, 113f Surgery PION induction 408 management 410 Surgical decompression, central retinal vein 83 Swedish interactive threshold algorithm (SITA) 551–2 Synapse(s) cone photoreceptors 22–3, 158, 462 definition 105 dendrodendritic 276, 277f dyads 457f, 458 feedback definition 284 retinal remodeling 363 rod photoreceptors 463, 820, 823–4 tetrads, fly visual system 126f, 129 Synaptic ribbon, definition 661 Synaptogenesis inner plexiform layer 393–4, 393f, 394f, 395 outer plexiform layer 393–4, 393f, 394f, 396 retinal remodeling 363 vertical pathway 393, 393f, 394f, 395f Synaptotagmin 663 Synchronous neural activity, RGC spatial processing and 302–4 Synechia, definition 38 Systemic disease, central retinal vein occlusion and 75–6

T Tachykinin(s), retina immunostaining studies 490 receptor autoradiography 491 receptor mRNA expression 491 Tamponade definition 801 PVR management 714–15, 715f Tapetal-like reflex/sheen, retinitis pigmentosa 701, 703f Tapetoretinal, definition 333 Targeting-induced local lesions in genomes (TILLING), zebrafish 855 Tbx3 213, 213f T-cell(s) AMD role 272 optic neuritis 536–7 proliferative vitreoretinopathy and 711 T-cell receptor (TCR), T-cell activation and 126f Tear film/tears structure 374–5, 376f Tear film, defensive role 374 Telangiectatic, definition 87 Telodendria, definition 156, 819 Temporal frequency, definition 163 Temporal processing contrast sensitivity 166–8, 168f, 291 horizontal cells and 316 Tetrachromatic vision, definition 205 D9-Tetrahydrocannabinol (THC) 717 TGFb choroidal neovascularization role 91 choroidal neovascularization 91 signaling pathway 266, 267f retinal histogenesis 266–7 Threshold, definition 290, 325 Thrombospondin-1, CNV role 91 Tight junctions (TJs) blood–retinal barrier 51, 52f breach of 51–2, 52f inner blood–retinal barrier (iBRB) 44–5, 46

definition 44, 51, 58, 761, 773 diabetic retinopathy pathogenesis and 783 Tiling, RGC spatial processing and 302, 303f TILLING (targeting-induced local lesions in genomes), zebrafish 855 timeless (tim) gene/protein 127, 128f Tissue regeneration, retinal in mammals 872 in zebrafish 863–4, 865 damage models 867 genes 869 Mu¨ller glial cells involved in 867, 868f, 870f, 871–2 process of 870f, 871 Toll-like receptors (TLRs) adaptive immune response and 379–80 AMD role 273 eye locations 377, 378t innate–adaptive immunity interactions 379–80 innate immune response 377 initiation/amplification 379 mucin production and 374–5 ligand specificity 377, 378t neovascularization and 352 Tonometry, definition 38 Total deviation plots, perimetry 555 Total internal reflectance (TIRF) microscopy, definition 661 Toxins, acquired color blindness 138 TRAIL-R, pathogen clearance 379 Transcription factors early patterning 199f, 200–1 light damage injury mechanisms 341, 342 POU-domain, definition 235 ‘stemness’ 368 Transducin(s) achromatopsia and 146 activation during phototransduction cones 627 rods (rhodopsin interaction) 610–11, 631–2, 633, 644, 645f definition 412, 605, 610 guanosine diphosphate (GDP)-bound inactive state 634 guanosine triphosphate (GTP)-bound active state 634 inactivation after phototransduction cones 607, 628 rods 612, 613f, 644 light-driven translocation in photoreceptors 412, 413f, 614–15 active transport vs. diffusion 413–14 mechanisms 414, 414f possible functions 413 return to dark-adapted locations 415 threshold light intensity 412–13 rod vs. cone 627 subunit dissociation 414, 414f Transepithelial electrical potential (TEP), assessment of RPE barrier function 60 Transepithelial electrical resistance (TER) assessment of RPE barrier function 60 definition 773 RPE tight junctions 778–9 Transfer function definition 276 nonlinear in cone phototransduction 159, 160f Transgenic animals bipolar cell morphology 456f, 457, 459 retinal peptide localization and 488 Transgenic mouse, definition 567 trans-Golgi network (TGN), definition 676 Transient receptor potential (TRP), definition 438 Transient tritanopia 331–2 Transitional cell, definition 235 Transplantation, autologous RPE grafts, RP treatment 697 Transporter proteins, cone photoreceptors 158–9 Transretinal waves, starburst amacrine cells 451 Trauma head injury and acquired color blindness 137 inflammation and, RP vs. 692–3 Tree shrew (Tupaia belangeri ), horizontal cells 465–6, 466f Triad synapse(s) 309 cone photoreceptors 158 definition 156, 461 horizontal cells 461, 462f photoreceptors 309 Triamcinolone, intravitreal, macular edema treatment 82 Trichromacy/trichromatic vision cone subtypes and 140, 141f

Subject Index definition 134, 140, 205 horizontal cell cone selectivity and 467 Trophic factors bystander effect and 857–8, 858f definition 753 TRP (transient receptor potential), definition 438 TRP (transient receptor potential) channels definition 205, 240 Drosophila 240, 245 activation 246f, 248, 248f biophysical properties 245 discovery 245 future studies 251 phototransduction role 617, 622 structural features 247f Limulus phototransduction and 622 trp gene 245, 247f TRPL (transient receptor potential-like) channels comparison with squid photoreceptor channels 586 Drosophila 245 activation 246f, 248, 248f biophysical properties 245–6 inactivation no afterpotential D (INAD) protein and 249, 250f structural features 247f TRPV1 channels 717 arachidonoyl ethanolamide as ligand 720 Tryptophanyl-tRNA synthase fragment (T2-TrpRS), antiangiogenic activity 549 Tumor necrosis factor a (TNFa) blood–retinal barrier (BRB) breakdown and 54 proangiogenic actions 546 Tumors/mass lesions, PVR and 713, 713f, 714f TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) apoptosis role 868f definition 38, 863 Tupaia belangeri (tree shrew), horizontal cells 465–6, 466f Turtle(s), bipolar cells 459–60, 459f Two-flash apparent motion stimulation definition 295 starburst amacrine role in directional selectivity 298–9 Type properties, definition 452 Tyrosine kinase, role in circadian rhythm generation 120

U 3D Ultrahigh resolution retinal OCT (3D UHR OCT) 526, 527f Ultraviolet radiation (UVR), insect vision and 209–10 Unc119 (protein) 664 Unique hues, definition 96 United Kingdom Prospective Diabetes Study (UKPDS) definition 781 glycemic control as risk factor 782 U-shaped tears 801, 802, 802f Usherin, RP and 686, 730–1 Usher syndrome cilia involvement 682 retinitis pigmentosa and 686 Uveitis definition 44, 51, 541 RPE role 761 UV opsin, butterfly 150–1, 151t

V Vaccine, glaucoma 734 Vascular endothelial growth factor (VEGF) angiogenesis/neovascularization role 271, 350, 351f, 352, 542, 542t choroidal neovascularization 91 anti-VEGF therapy in AMD 271, 834 blood–retinal barrier (BRB) breakdown role 53 definition 270, 346, 781 isoform-specific mouse model 757 macular edema role 79, 83 neuroprotection and 798 retinal vasculature and development 183 diabetic retinopathy and 185 remodeling and 364 retinopathy of prematurity and 185, 795, 798 RPE secretion 757, 758f

Vascular endothelial growth factor receptor(s) (VEGFRs), neovascularization role 352 Vascular endothelial growth factor receptor-2 (VEGFR-2), choroid 757 Vascular remodeling, retinal 548 Vasculogenesis definition 179, 541 postnatal (adult), in choroidal neovascularization (CNV) 92 Vasoactive intestinal peptide (VIP), retinal excitation of ganglion cells 483 GABA co-localization 490 mRNA localization 488 peptide immunostaining 489–90 receptor autoradiography 491 receptor mRNA expression 491 signaling pathway 491 cellular 479, 482f intercellular 478–9 Vasoconstriction, retinal microvasculature 34 VEGF–TNF receptor-associated protein (TRAP) 93 in diabetic macular edema treatment 83 VEGFxxxb isoforms, antiangiogenic activity 549 Venules, retinal venous drainage 656f, 657–8 Vernier acuity 4–5, 5f definition 717 Versican, vitreoretinopathy 261 Vesicle release rate, rods 822 Vesicular inhibitory amino acid transporter (VIAAT) 228 retinal locations 228–9, 229t Vesicular transport blood–retinal barrier (BRB) 52, 53f definition 51 Viral vectors, retinitis pigmentosa and 697 Vision models 854 Visual acuity 1–6 across retina 4, 4f age-related changes 4, 5f dynamic 5 GCA and A-AION 405 hyperacuity 4, 5f light level and 561, 562f measurement 1–3 NA-AION 401–2 National Academy of Sciences recommendations 1 nonischemic CRVO ischemic CRVO vs. 79, 79t untreated 85, 85t opticak/neural limits 3 PION 409 reporting 3, 3t retinitis pigmentosa 690 standards/requirements 4 Visual adaptation, definition 22 Visual angle 165, 167f definition 1 Visual assessment, zebrafish models of retinal disease 856 Visual correction, adaptive optics (AO) 9 Visual cortex, acquired color blindness 137 Visual cycle 648–52 clearance of all-trans-RAL from OS disks 648 definition 624 overview 17, 648, 649f photopigment regeneration 642, 643f, 651 reduction of all-trans-RAL to all-trans-retinol 649 regulation 651 dark adaptation 651 RGR opsin and 651 retinoid isomerization 641, 642f, 650 retinyl ester synthesis 650 rhodopsin/opsin regeneration 651 RPE and 17 synthesis of 11-cis-RAL chromophore 651 transfer of all-trans-retinol from photoreceptors to RPE 642, 649 Visual-evoked potential definition 536 optic neuritis 536, 537–8, 538f Visual field, definition 551 Visual field defects NA-AION 401–2, 402f PION 409, 409f, 410f retinitis pigmentosa 690, 691, 691f, 692f

911

912

Subject Index

Visual loss, transient in GCA 405 Vitamin(s), protective against AMD 833 Vitamin A deficiency acquired color blindness 139 Xenopus laevis model 849 retinitis pigmentosa treatment 696 Vitamin E deficiency, retinitis pigmentosa and 704 treatment 696 Vitrectomy basic principle 806, 808f rhegmatogenous retinal detachment 806 complications 809 due to retinal holes/dialysis 807 due to retinal tears 806 outcomes 808 small-gauge sutureless 809 Vitreochoroidopathy, autosomal dominant (ADVIRC) 259 Vitreoretinal interface macular holes 825, 826, 827f, 828f Vitreoretinopathies, hereditary 252–62 abnormal retinal vasculature and 253t, 254t, 258 autosomal dominant vitreochoroidopathy 259 familial exudative vitreoretinopathy 259, 261, 261f classification 252, 253t clinical features 252, 254t corneal changes 253t, 254t snowflake vitreoretinal degeneration 260 molecular genetics 260, 262t collagen genes 260 fibrillin 261 inheritance patterns 261–2 phenotypic relationship 261 versican 261 Wnt signaling pathway 261 progressive retinal dysfunction and 253t, 254t, 257 Goldmann–Favre syndrome 258, 261 Wagner syndrome 257, 259f, 260f, 261, 261f skeletal abnormalities and 252, 253t, 254t Kniest dysplasia 253, 258f, 260 Knobloch syndrome 256 Marfan syndrome 256–7, 258f, 259f, 261 Marshall syndrome 253–4 spondyloepiphyseal dysplasia 253, 258f, 260 Vitreoschisis, definition 825 Vitreous anatomy, 825 biochemistry 825 definition 801 development 182 persistent hyperplastic primary vitreous 183 traction 825 Vitreous fluorometry blood–retinal barrier (BRB) assessment 54–5, 61 definition 44 Vitreous hemorrhage, in CRVO 80, 80f, 81f Voltage-gated calcium channels cone photoreceptors 158–9 rod photoreceptors 821 Voltage-gated currents horizontal cells and 311, 311f retinal cannabinoids and photoreceptor modulation 721 von Kries scaling 99 Vortex veins 179, 180–2, 180f

W Wagner syndrome 257–8, 259f, 260f, 261, 261f Wallerian degeneration 386 Water animal behavior relating to 673–4 generation of polarization 669–70, 670f Wavefront sensor, Shack-Hartmann 8f definition 7 Wavelets 164–5, 165f definition 163 Wavenumber, definition 96

Weber’s law 326–7, 591–2 cone adaptation and 591, 591f definition 589, 596 rod adaptation and 597 Weissenbacher–Zweymuller syndrome 253 White mutants 727, 728f Wide-field three-dimensional choroidal OCT 527, 528f Wnt/calcium pathway 264, 265f Wnt signaling pathway familial exudative vitreoretinopathy 261 noncanonical 264, 265f retinal histogenesis 264, 265f retinopathy of prematurity and 795 Wolfram’s syndrome 336

X Xanthopsia 137 Xenopus 500 Xenopus laevis eye development 851 retinal regeneration and 848, 852 utility as model organism 847–8 Xenopus laevis models of retinal disease 847–52 advantages 847–8, 848f, 852 developmental disorders 851 disadvantages 852 drug-inducible degeneration 851 electrophysiology and 848 expression of cone opsins in rod cells 851 glaucoma 849 historical aspects 848 retinitis pigmentosa rhodopsin P23H mutation 850 rhodopsin Q348ter mutation 850 rhodopsin transport and 849 transgenic animals 850 rhodopsin transport/metabolism and 849 Stargardt’s disease 851 vitamin A deficiency 849 X-linked disorders, juvenile retinoschisis, mfERG results 510, 514f

Z Zebrafish (Danio rerio) 853 cone outer segment axonemes 579f persistent neurogenesis of retina 865, 867f, 870f, 871 phototransduction in 856, 856f visual system 854–5 embryogenesis 854–5, 864, 864f retina 864 humans vs. 862 regenerative potential 860, 862 Zebrafish models 863, 867 glaucoma 42 Zebrafish models of retinal disease 853–62 advantages 854–6, 858, 862 high-throughput drug screening 856, 862 molecular techniques used 854 chimera production 856 DSBs 855–6 morpholinos 855 mutagenesis 855 observation of cells in intact animals 855, 855f TILLING 855 Pde6 mutants 857 bipolar cells 860 cone photoreceptors 860–1 missense mutant (els) 858 Mu¨ller cells 860 null mutant (pde6cw59) 858, 859f rod photoreceptors 860 structural effects 861, 861f vision evaluation in 856 Zeitgeber time, definition 68, 124 Zinc ions (Zn2+), GABA receptor modulation in retina 232–3

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