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THE PHOTOPIC ERG LUMINANCE-RESPONSE FUNCTION: DESCRIPTION, PHYSIOLOGICAL BASIS AND CLINICAL APPLICATION Marie-Lou Garon, O.D. Integrated Program in Neuroscience Montreal Children’s Hospital Research Institute McGill University Montreal October 2010 A thesis submitted to McGill University in partial fulfillment of the requirements for the Master’s degree (M.Sc.) in Neuroscience Marie-Lou Garon 2010 TABLE OF CONTENTS Acknowledgments ...................................................................................................................................iii Abstract....................................................................................................................................................... iv Résumé ........................................................................................................................................................V CHAPTER I INTRODUCTION.............................................................................................................. 1 REVIEW OF LITERATURE....................................................................................................... 3 1. THE RETINA ............................................................................................................................................ 3 1.1 General organization ................................................................................................. 3 1.2 Retinal cells............................................................................................................... 5 1.2.1. Photoreceptors................................................................................................... 5 1.2.1.2 Phototransduction ....................................................................................... 6 1.2.2 Horizontal cells .................................................................................................. 8 1.2.3 Bipolar cells ....................................................................................................... 8 1.2.4 Amacrine cells ................................................................................................... 9 1.2.5 Ganglion cells .................................................................................................... 9 1.3 Circuits of the cone pathways ................................................................................. 10 1.4 Circuits of the rod pathways ................................................................................... 12 2. THE ELECTRORETINOGRAM .............................................................................................................. 12 2.1 History..................................................................................................................... 12 2.2 Components of the flash ERG ................................................................................ 13 2.2.1 Short Flash full-field ERG ............................................................................... 14 2.2.2 Long Flash full-field ERG ............................................................................... 15 2.3 Origin of the ERG components............................................................................... 16 2.3.1 The a –wave ..................................................................................................... 16 2.3.2 The b-wave....................................................................................................... 17 2.3.3 The i-wave ....................................................................................................... 18 2.3.4 The oscillatory potentials ................................................................................. 18 2.4 Luminance-response functions of the Short Flash full-field ERG.......................... 19 2.4.1 Scotopic b-wave luminance-response function: the Naka-Rushton equation.. 20 2.4.2 Photopic b-wave luminance-response function: the Photopic Hill.................. 21 3. RETINAL DEGENERATIONS ................................................................................................................ 22 3.1 Retinitis pigmentosa................................................................................................ 22 3.2 Congenital stationary night blindness (CSNB)....................................................... 23 3.3 Congenital postreceptoral cone pathway anomaly (CPCPA) ................................. 24 PURPOSE OF MY RESEARCH PROJECT .................................................................................................. 25 i CHAPTER II STUDY 1 - SPECTRAL ELECTRORETINOGRAPHY: WHAT DOES THE LUMINANCE-RESPONSE FUNCTION REVEAL? .................................................................................. 27 1 Abstract ............................................................................................................................................. 29 2 Introduction ...................................................................................................................................... 30 3 Methods ............................................................................................................................................. 31 4 Results ............................................................................................................................................... 32 5 Discussion......................................................................................................................................... 34 6 References ........................................................................................................................................ 38 7 Captions to Figures and Tables .................................................................................................. 41 CHAPTER III STUDY 2 – ASYMETRICAL GROWTH OF THE PHOTOPIC HILL DURING THE LIGHT ADAPTATION EFFECT ........................................................................................................ 54 1 Abstract ............................................................................................................................................. 56 2 Introduction ...................................................................................................................................... 57 3 Methods ............................................................................................................................................. 59 4 Results ............................................................................................................................................... 61 5 Discussion......................................................................................................................................... 63 6 References ........................................................................................................................................ 68 7 Captions to Figures and Tables .................................................................................................. 72 CHAPTER IV STUDY 3– ESTIMATING THE ON AND OFF RETINAL PATHWAYS FUNCTION WITH THE GLASGOW EQUATION: NORMATIVE DATA AND CLINICAL APPLICATION 83 1 Abstract ............................................................................................................................................. 85 2 Introduction ...................................................................................................................................... 86 3 Methods ............................................................................................................................................. 87 4 Results ............................................................................................................................................... 89 4 Discussion......................................................................................................................................... 91 4 References ........................................................................................................................................ 94 4 Captions to Figures and Tables .................................................................................................. 97 CHAPTER V GENERAL DISCUSSION & CONCLUSION ........................................... 103 REFERENCES ..................................................................................................................................... 106 APPENDIX ........................................................................................................................................... 115 ii ACKNOWLEDGMENTS First of all, my most sincere thanks to Dr. Pierre Lachapelle, for his help, time and support throughout this project. Merci Pierre de m’avoir accueillie dans ton laboratoire, d’abord comme étudiante d’été, puis comme étudiante graduée, et d’avoir partagé avec moi ton expertise et tes connaissances en électrophysiologie. Merci aussi pour tous tes précieux conseils, qu’ils soient d’ordre scientifique, culinaire, sportif ou sur la vie en général. I also want to thank my lab colleagues for their help and support, but mostly for creating such a great atmosphere in the laboratory. Merci Marianne de m’avoir montré les bases de l’électrophysiologie humaine à mes débuts au laboratoire. Merci Julie R. pour ta grande disponibilité et ton infinie patience. On peut toujours compter sur toi. Thank you Allison for your encouragements late at night when I felt like going to bed instead of working on this thesis. Merci Catherine pour ta bonne humeur, ton enthousiasme et ta franchise. Thank you Malgosia for your computer skills (you saved me a few times!) and for making me laugh. Merci Colin pour tes conseils mathématiques. My biggest thanks go to Anna for all your help at the lab, but mostly for your friendship. The last three years would not have been the same without you. Arigato! To Mikheil, Wenwen, Julie L., Sandrine, Jarred, Stéphanie, Mathieu, Marie-Maxime, and all the other students I did not mention, it was a pleasure working with you all. I would also like to thank the members of my Advisory committee, Dr. Marc Hébert, Dr. Michelle McKerral and Dr. Yong Rao, as well as the Graduate Program in Neuroscience, the Réseau Vision, and the Montreal Children’s Hospital Research Institute for studentship awards and travel support. Un gros merci à mes amis et à ma famille pour leur support, tout particulièrement à mes parents, Louis et Louise, qui sont toujours là pour m’appuyer. Finally, I would like to thank Dan, for his love and his support these past three years. iii ABSTRACT In order to further define the photopic ERG luminance-response function and explore its potential value in the investigation of normal and pathological phenomena involving the cone visual pathways of the human retina, we recorded photopic ERGs on a broad range of intensities: 1) in response to very dim flashes of red, blue and white light; 2) during changes in the retinal light adaptation level; and 3) in patients affected with selected retinal diseases. Our experimental approach revealed that the photopic ERG luminanceresponse function is a useful tool to understand the basis of retinal physiology and can be used to some extent to facilitate segregation/dissociation of retinopathies involving specifically the ON or OFF-retinal pathways. ERG specialists would thus benefit from adding luminance-response recordings to their standard protocols. iv RÉSUMÉ Afin de mieux définir la fonction luminance-réponse de l’ERG photopique et de déterminer son potentiel pour étudier les phénomènes normaux et pathologiques des voies visuelles liées aux cônes, nous avons enregistré des ERGs photopiques sur une large étendue d’intensités de stimulation dans les 3 trois conditions suivantes : 1) en réponse à de faibles stimuli de lumière rouge, bleue et blanche; 2) pendant l’adaptation de la rétine à la lumière; 3) chez des patients atteints de maladies rétiniennes choisies. Notre approche expérimentale a montré que la fonction luminance-réponse de l’ERG photopique est un outil utile pour comprendre les bases de la physiologie rétinienne et qu’elle permet également de catégoriser les rétinopathies affectant spécifiquement la voie ON ou la voie OFF de la rétine. Il serait donc indiqué d’ajouter l’enregistrement d’une courbe luminance-réponse aux protocoles cliniques standards. v Chapter I INTRODUCTION When stimulated by light, retinal cells produce a transient electric response that allows for transmission of visual information via the visual pathways to higher visual structures located in the brain. This signal, although produced at the retinal level, can be recorded by a non-invasive electrode positioned on the cornea, the eye acting as a dipole. The signal thus obtained is called the electroretinogram (ERG). The ERG waveform is usually composed of a negative deflection produced by the photoreceptors’ activation, the awave, followed by a positive peak, the b-wave, which results from the interaction between second order neurons, namely the ON and OFF bipolar cells, and the glial Muller cells. The ERG has been used clinically for decades, mainly to diagnose and/or follow progression of various retinal disorders and to investigate retinal function in pediatric and uncooperative patients, such as non-verbal or mentally challenged individuals. Standard ERG protocols require that light and dark-adapted ERG be obtained in order to assess cone and rod functions, respectively. Usually, a single flash intensity is used and diagnosis is based on a- and b-wave amplitude and peak time measurements in each adaptation state. At the Montreal Children’s Hospital Electrophysiology Clinic, however, we have been recording ERGs on a broad range of flash intensities for most of our patients over the past 15 years or so, with our main interest being the photopic ERG luminance-response function. Indeed, previous studies suggested that the photopic bwave luminance-response function could be useful to differentiate retinal pathologies (Rufiange & al., 2003), and that it could allow for possible segregation of ON and OFF retinal responses (Hamilton & al., 2007). The photopic luminance-response function thus represents an interesting mean of studying retinal function. In the following pages I present a brief review of literature on the structure and function of the normal and diseased retina. The first section focuses on the retina, specifically on its organization and the pathways involved in the transmission of visual information. The second section describes the ERG in details, with emphasis on the photopic luminance- 1 response function of the short flash ERG, the main subject of this thesis. Finally, the last section provides an overview of selected retinal diseases that were studied during the course of this project. 2 REVIEW OF LITERATURE 1. THE RETINA 1.1 General organization The retina is a thin transparent layer that lies against the back of the eye ball. Rays of light refracted by the cornea and the lens and entering the eye through the pupil must pass through the entire retinal thickness before reaching the photoreceptors, the main lightsensitive cells of the retina. The human retina, as all vertebrate retina, is organized into three distinct neuronal layers, separated by two synaptic layers. The photoreceptor cell layer, which is directly in contact with the pigmented epithelium, includes rod and cone outer and inner segments, as well as their cell bodies (the outer nuclear layer). In the second cell layer, called the inner nuclear layer, are the bipolar, horizontal and amacrine cells. The last neuron layer is composed of the ganglion cells whose axons eventually form the optic nerve that carries the visual information to higher visual structures. The space where the photoreceptors synapse with bipolar and horizontal cell dendrites is called the outer plexiform layer; the bipolar and amacrine cells, on the other hand, connect to the ganglion cells at the level of the inner plexiform layer. The retina also contains interplexiform cells that connect the inner and outer plexiform layers as well as Müller glial cells which serve as support cells and potassium buffer (figure 1). In summary, the retina is usually divided into 10 distinctive layers (figure 1), from the outermost to innermost: the retinal pigmented epithelium (RPE), the photoreceptor layer (PR) with outer and inner segments, the outer limiting membrane (OLM), the outer nuclear layer (ONL), the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), the ganglion cell layer (GCL), the nerve fiber layer (NFL) and the inner limiting membrane (ILM). (Dowling, 1987; Rodieck, 1998; Kolb, 1994, 2003). 3 Figure 1: Simple organization of the retina (from Anna Polosa, with permission) [1=RPE cell; 2=cone; 3=rod; 4=Müller cell; 5=bipolar cell; 6=horizontal cell; 7=amacrine cell; 8=ganglion cell] 4 1.2 Retinal cells Six of the above mentioned retinal cells are of the neuron type and thus participate in visual information processing: the photoreceptors, the bipolar cells, the ganglion cells, the horizontal cells, the amacrine cells and the interplexiform cells. The Müller cells, as mentioned above, are glial cells and do not take part in the visual transmission. The most direct pathway for visual information transmission is from photoreceptors, to bipolar cells, to ganglion cells. The horizontal and amacrine cells, on the other hand, influence retinal processing by modifying respectively the responses of bipolar and ganglion cells via lateral connections. The interplexiform cells, as opposed to the photoreceptor-bipolar-ganglion cells pathway, would transmit the visual information following a centrifugal flow from the ganglion cells to the photoreceptors. Their exact role, however, is not fully understood. (Dowling, 1987; Rodieck, 1998; Kolb, 1994, 2003) 1.2.1. Photoreceptors There are two types of photoreceptors in the human retina: the rods which are used in dim conditions (scotopic vision), and the cones which are active in bright-colored environments (photopic vision). Several publications report a count of 120 million rods and 6 million cones in the human retina, based on a 1935 study by Ostenberg. However, recent studies using more advanced techniques and performed on a larger number of eyes showed lower cell counts, with averages ranging from 60 to 92 millions rods and 3.2 to 4.6 millions cones (Cursio & al, 1990; Jonas & al., 1992). The same studies showed that cone density is maximal at the fovea and falls abruptly over the first 1 to 1.5 mm of eccentricity, beyond what it decreases more slowly towards the periphery (Curcio & al., 1990). Rods, on the other hand, are absent from the central 0.35 mm of the fovea (Curcio & al., 1990). They gradually increase in number to reach their highest density in an ellipse-like area located 5 to 7 mm from the center of the fovea. Their density then declines slowly from this rod-rich area to the far periphery (Curcio & al, 1990; Jonas & al., 1992). 5 All photoreceptors present roughly with the same general structure: they have an outer segment that contains the visual pigment connected by a cilium to an inner segment that contains the metabolic machinery, a perikaryal region containing the cell nucleus, and a synaptic terminal (Rodieck, 1998; Dowling, 1987). However, there are morphologic differences in some of these structures that distinguish cones from rods. First, cone synaptic terminals, termed pedicles, are larger and contain a number of invaginations where horizontal and bipolar cells connect with the cone. Rod terminals, called spherules, are smaller and present a single invagination of the membrane (Dowling & Boycott, 1966). Also, the cone outer segment is composed of infoldings of the surface membrane whereas the rod outer segment contains a set of stacked membranous discs derived, but detached, from the surface membrane (Sjöstrand, 1953). Rod outer segment discs contain only one type of visual pigment capable of capturing photons, the rhodopsin. On the other hand, there are three types of cones- short (S), medium (M) and long (L) - based on the spectral sensitivity peaks of their photopigments, called cone opsins, which are respectively in the blue (410-430 nm), green (530/535 nm) and yellow (556/562 nm) regions of the visual spectrum (Jacobs, 1996). L- and M-cones, which account together for about 90% of the cone population, both transmit the visual information through the parvo (P) and magnocellular (M) pathways. S-cones differ from the other two types of cones in many aspects. Firstly, they only represent 10% of all the cones and are absent from the center of the fovea. Secondly, they pass on the visual signal via the koniocellular (K) pathway (Calkins, 2001; Rigaudière & Legargasson, 2007). We will examine in detail later the retinal circuits underlying color vision. 1.2.1.2 Phototransduction The photoreceptor’s main function is to convert light energy into an electrical signal. The cell membrane potential depends upon the distribution of ions on either side of the membrane. This is determined by the selective permeability of the membrane to different ions, especially Na+ and K+. The concentration of K+ is higher on the inside, whereas that of Na+ is higher on the outside of the cell. This equilibrium is maintained by the ATP-dependent Na-K pump. At rest (i.e. in the dark), the photoreceptor membrane is 6 depolarized compared to a typical neuron. This depolarization is due to Na+-cGMP-gated channels located in the outer segment membrane. In the dark, these channels are open, thus permitting Na+ to move into the cell according to its electrochemical gradient. At the level of the inner segment, K+ ions leak out of the cell through another type of channels, the K+-selective non-gated channels. This movement of positive ions across the photoreceptor membrane is called the dark current. In its depolarized state, the photoreceptor continuously releases the neurotransmitter glutamate. When the photoreceptor is exposed to light, the Na+-cGMP-gated channels close, resulting in a decrease of the Na+ current into the cell. On the other hand, K+ ions continue to leak out. Consequently, there is hyperpolarization of the photoreceptor membrane and interruption of glutamate release. (Bear, Connors & Paradiso, 1996; Djamgoz, Archer & Vallerga, 1995) The closure of the Na+-cGMP-gated channels is the result of the phototransduction, the process through which a photon is transformed into an electrical signal in the retina. This takes place in the outer segment of the photoreceptors, more precisely at the level of the membranous discs containing the photosensitive pigment. Although the phototransduction has been studied for over a century, a coherent scheme of the process was first described by Yau and colleagues in 1986. They have since published several extensive reviews on the subject (Koutalos & Yau, 1993; Yau, 1994; Luo & al., 2008). Briefly, upon light absorption, the conformation of the photosensitive pigment molecule changes. The activated photopigment interacts with several molecules of the G-protein transducin. This interaction catalyzes the replacement of a molecule of guanosine diphosphate (GDP) bound to the transducin by a molecule of guanosine triphosphate (GTP). The transducin-GTP complex then activates a phosphodiesterase (PDE). Each PDE molecule hydrolyzes more than 2000 molecules of cGMP into 5’-GMP. The resulting reduction in cGMP concentration then leads to the closure of the Na+-cGMPgated channels. The activated components of the phototransduction cascade are inactivated at light cessation. The photopigment is inactivated through two mechanisms. The first one 7 involves its phosphorylation by a specific opsin kinase. Once the photopigment is phosphorylated, a protein called arrestin binds to it, and prevents its interaction with transducin molecules. Inactivation of transducin results from its intrinsic GTPase activity. Finally, the recovery of the intracellular cGMP concentration is achieved through the increased activity of guanylate cyclase (Djamgoz, Archer & Vallerga, 1995). 1.2.2 Horizontal cells The horizontal cells receive from and transmit information to photoreceptors and other horizontal cells. They also send messages to bipolar cells. Each photoreceptor invagination receives two horizontal cell processes, each from a different horizontal cell. Two types of horizontal cells are found throughout the human retina, named HI and HII. One end of the HI cells forms a small dendritic arbor that synapses with both L- and Mcones while the other end consists of a long axon with a widespread telodendritic tree sharing synaptic contacts with approximately 700 rods. Instead of a long axon, HII cells have a shorter process with few short dendritic extensions that connect with all three types of cones. The S-cones exclusively synapse with HII cells (Rodieck, 1998; Dowling, 1987). Both types of horizontal cells are involved in a mechanism called lateral inhibition that will be explained in detail later (Rodieck, 1998; Kolb, 1994). 1.2.3 Bipolar cells As mention above, the bipolar cells are the second neurons involved in the direct transmission of visual information, receiving direct input from the photoreceptors. They also receive lateral input from the horizontal cells and transmit information to the ganglion cells and amacrine cells (Kolb, 1994; Rodieck, 1998). Their structure consists of dendritic terminals, a cell body containing the nucleus as well as the cell metabolic material, and the axon terminals (Dowling, 1987; Rodieck, 1998). The dendritic terminals of a bipolar cell connect exclusively to cones or exclusively to rods, never both. Consequently, bipolar cells can be divided in two categories: rod bipolar cells and cone bipolar cells. While there is only one type of rod bipolar cells, cone bipolar cells can be of 8 several types depending mainly on three factors: the number of cones that contact the bipolar cell (one or several), the type of contact made (on- or off-) and the type(s) of cones that contact the bipolar cell (L-, M- or S-) (Rodieck, 1998). We will examine in more detail pathways involving some of these bipolar cells later in this review of literature. 1.2.4 Amacrines cells Amacrine cells receive input from bipolar cells as well as other amacrine cells. On the other hand, they send outputs to bipolar cells, other amacrine cells and ganglion cells. There is a wide variety of amacrine cells based on their size, morphology and function (Rodieck, 1998). However, most of the amacrine cells can be classified either as diffuse or stratified. Diffuse amacrine cells extend their processes throughout the thickness of the IPL. They can be of the narrow or wide-field type depending on how spread out their dendritic tree is. Stratified amacrine cells, in contrast, extend their processes on one or a few strata of the IPL and are called mono, bi or multi-stratified accordingly so (Dowling, 1987). 1.2.5 Ganglion cells Ganglion cells receive direct input from the bipolar cells and lateral input from the amacrine cells. They are the last neurons to relay the visual information within the retina, as they send their messages out of the eye via their axons which form the optic nerve. They transmit their signal to the brain via the lateral geniculate nucleus (LGN) by generating action potentials at different rates. This is different from the photoreceptors and bipolar cells which pass on visual information through graded changes in their membrane potential (Rodieck, 1998). The ganglion cells synapse in different regions of the LGN according to the specific aspect of vision they code for, such as contrast, color, movement or direction, to name a few (Rodieck, 1998). In that matter, most ganglion cells in primates can be classified according to the subdivision of the LGN where they 9 synapse, i.e. parvocellular (P), magnocellular (M) or koniocellular (K) (Martin & al., 1997). 1.3 Circuits of the cone pathways Cone pathways in humans are organized into two parallel streams of information. The cone bipolar cells receive visual input from the cone photoreceptors via glutamate receptors, either metabotropic or ionotropic. Metabotropic receptors close the ionic channels in the cell membrane in response to glutamate (inhibitory receptors) and thereby hyperpolarize the bipolar cell. Since photoreceptors constantly release glutamate unless exposed to light, bipolar cells with metabotropic receptors do not fire in the dark. When the photoreceptors stop releasing glutamate during light exposure, the ionic channels open, the cell depolarizes and starts firing. These bipolar cells, called ON-bipolar cells, are the start of the ON retinal pathway which provides information concerning brighterthan-background stimuli. In contrast, ionotropic glutamate receptors open the ionic membrane channels in presence of glutamate resulting in the bipolar cell depolarization and firing. These OFF-bipolar cells are thus activated when the light is reduced and are part of the OFF retinal pathway that allows for detection of darker images against a lighter background. ON- and OFF-bipolar cells then transmit information directly to ONand OFF-ganglion cells, respectively. This organization into ON and OFF pathways is the basis of successive contrast in visual perception (Kolb, 2003). However, the retina needs other circuits to improve the image resolution. This is achieved by lateral inhibition, which allows for discrimination of simultaneous contrast. The main cells involved in this process are the horizontal cells. As described in the previous section, one end of HI cells, the dendritic arbor, connects to L- and M-cones and bipolar cells. The dendrites of adjacent HI cells are tightly coupled together by small gap junctions, allowing for communication between them (Rodieck, 1998; Kolb, 2003). By this mean, HI cells accumulate information from a wide group of cones and add an antagonistic surround signal to the bipolar cells’ receptive fields, either directly and/or by feeding back information to the cones (Kolb, 1994, 2003; Rodieck, 1998). The bipolar cells are thus 10 said to have a center-surround receptive field: OFF-surround for the ON-center bipolar cells and ON-surround for the OFF-center bipolar cells. The overall effect of this opponent center-surround neural circuitry is to produce a relative enhancement of the bipolar cells response to differences in light intensity and help sharpen the boundaries of images. This opponent center-surround organization is maintained at the level of ganglion cells. Similarly, HII cells are connected together by gap junctions and influence the bipolar cells that receive from S-cones (Rodieck, 1998). The circuitries involving the HI dendritic system and the HII cells are also of utmost importance for color vision, which is also based on center-surround antagonism (red/green or blue/yellow). Small-field amacrine cells are also thought to take part in the spatial and spectral center-surround organization of the ganglion cells’ receptive fields. The organization of red/green channels can thus be of 4 types: red ON-center/green OFFsurround, red OFF-center/ green ON-surround, green ON-center/red OFF-surround and green OFF-center/red ON-surround (Dacey, 2000; Kolb, 1994). The only well documented blue/yellow channel, on the other hand, is associated with a spatially coextensive S-cone excitatory/(L+M) cone inhibitory receptive field (Dacey, 2000). Differences between red/green and blue/yellow channels are to be expected given that information arising from L- and M-cones travels through the same pathways (M or P), whereas that coming from S-cones is carried separately (K pathway). In the P or midget pathway (responsible for color vision and details), each L- or M- cone connects with two midget bipolar cells, one ON-center and the other OFF-center. In turn, they connect respectively to an ON-center and an OFF-center midget ganglion cell (Kolb, 1994; Dacey, 2000). In the M pathway (dedicated to gross shape discrimination and movement perception), information from the L- and M-cones is processed as a whole. Briefly, several L- and M-cones connect to a diffuse ON- or OFF-bipolar cell. Each type of diffuse bipolar cell then converges respectively to ON- and OFF-parasol ganglion cells (Kolb, 1994). In contrast, the K pathway receives direct signals from the S-cones through specific S-cone bipolar cells and indirect signals from the L- and M-cones via diffuse OFF-bipolar cells. This information is then relayed to the bi-stratified ganglion cells (Dacey & Lee, 1994; Martin & al., 1997; Dacey, 2000). 11 1.4 Circuits of the rod pathways Our knowledge of the rod pathways so far, suggests that scotopic signals use two or three distinct pathways to transmit the visual information from the outer to the inner retina (Sharpe & Stockman, 1999). In contrast to the cones, rods synapse with only one type of bipolar cell, which is of the ON-type. These ON-center rod bipolar cells do not connect directly to ganglion cells: instead, they transmit information to ON- and OFF-midget ganglion cells through different types of amacrine cells, the most important being the AII. The AII amacrine cells transmit rod signals to OFF-center ganglion cells via glutamatergic chemical synapses. They also send electrical input to cone ON-center bipolar cells through gap junctions, resulting in transmission of the rod signal to ONcenter ganglion cells. The second rod pathway involves transmission of information through small gap-junctions between cone pedicles and rod spherules. The rod signals then reach the ON- and OFF-cone bipolar cells, and consequently, the ON- and OFFganglion cells (Kolb, 1994; Sharpe & Stockman, 1999; Bloomfield & Dacheux, 2001). Finally, studies done on transgenic mice suggest a possible third pathway connecting the rods to OFF-cone bipolar cells (Sharpe & Stockman, 1999; Bloomfield & Dacheux, 2001). 2. THE ELECTRORETINOGRAM 2.1 History The first ERG recordings were reported in 1865 when Holmgren discovered that a light stimulus causes a change in the electrical potential of the frog eye. Less than a decade later, in 1877, Dewar recorded the first human ERG, which was also the first human biopotential ever recorded (de Rouck, 2006). However, the actual waveforms were never published. We will have to wait until the 1920’s to see the first published human ERGs (Khan & Lowerstein, 1924; Hartline, 1925; Sachs, 1929) and to the 1940’s for the emergence of recording techniques suitable for clinical use (Riggs, 1941; Karpe, 1948). In the mean time, our understanding of the ERG waveform continued to evolve with studies 12 conducted on different animal species. Gotch (1903), using a capillary electrometer on the frog eye, reported that the ERG consists of two major waves: a negative deflection followed by a positive peak of larger amplitude. A few years later, Einthoven and Jolly (1908) separated the ERG response into three waves (A, B and C), which they attributed to transient chemical processes triggered by the light stimulus. They also identified an "off-effect" at light cessation, which is now called the d-wave in contemporary long-flash electroretinography. Piper (1911) also divided the ERG waveform into three components: I, II and III. In contrast to Einthoven and Jolly, however, he suggested that all three components lasted for the duration of the light stimulus, the a-, b- and c-waves resulting from their summation. The later hypothesis was confirmed by Granit (1933), whose classic study of the cat ERG under ether intoxication contributed the most to our understanding of the ERG. In this study, Granit showed that the ERG was basically composed of three processes, named PI, PII and PIII, according to their order of disappearance as the level of anesthesia deepened. These processes were shown to correspond respectively to the c-, b- and a-waves of the modern ERG. A few modifications to Granit’s model have been added over the years; of importance, it has been shown that P-III can be divided into a fast P-III and a slow P-III (Sillman & al., 1969). Also, other waves have been added, such as the i-wave (Nagata, 1963) and the oscillatory potentials (OPs) (Cobb & Morton, 1954) to name a few. In modern electroretinography, however, diagnosis is usually based on amplitude and peak time measurements of the two major waves of the ERG, the a- and b-waves, as well as the OPs (Hébert & Lachapelle, 2003). 2.2 Components of the flash ERG The ERG can be evoked to a diffuse (flash ERG) or structured (pattern and multifocal ERG) light stimulus, which results in different ERG waveforms. This thesis concentrated on the flash ERG, most commonly used for both clinical and research purposes. The components of the flash ERG waveform vary according to a number of factors, amongst them the duration, intensity, frequency and color of the flash, as well as the background color and illumination. 13 2.2.1 Short Flash full-field ERG In humans, the photopic short flash ERG is usually composed of a negative trough, the awave, followed by a larger positive peak, the b-wave, on which smaller wavelets called oscillatory potentials (OPs) are often seen (Lachapelle & Hébert, 2003). In order to isolate the OPs, one must raise the recording bandwidth low frequency cut off to 100 Hz. Three main OPs are usually easily identifiable: the short latency OP2 and OP3 as well as the long latency OP4, labeled as 2, 3 and 4 in figure 3. In the case of a retina fully adapted to light (photopic condition), the cones alone contribute to the genesis of the a-wave (Armington, 1974). When the retina is dark adapted (scotopic condition), the a-wave is recordable only for flash luminances in the photopic range; this is what is called the scotopic mixed response because both cones and rods then generate the response. The pure scotopic response, evoked to dimmer flashes, does not include an a-wave. Figure 2 shows typical photopic, scotopic mixed and pure scotopic short flash ERG b OPs 50 V Flash i a b 25 ms 5 0 V a b 50 m s 5 0 V 50 ms Figure 2: Typical photopic (top, scotopic mixed (middle) and pure scotopic (bottom) short flash ERGs 14 4 2 3 10 V 20 ms Figure 3: Typical isolated photopic oscillatory potentials 2.2.2 Long Flash full-field ERG The retina responds to changes in luminance level in the environment; consequently, if a flash of light is presented, it will generate a potential at both light onset and cessation. This is what is called the ON response and the OFF response, respectively. With a short flash stimulus (<5 ms), both pathways are combined to yield a single response. With a longer duration flash (usually 100 ms), a second positive wave, called the d-wave, appears distinctively at the stimulus offset. In photopic conditions, the waveform resulting from the long flash ERG thus presents with a negative a-wave, followed by a positive b-wave identified as the ON response and finally a positive d-wave representing the OFF response (Sieving, 1993; Sustar & al., 2006; Miyake, 2006) (Figure 3). Although a long flash ERG is not commonly used in clinical electrophysiology, because it is not available with all stimulation systems and requires more cooperation from the patient, it is useful in the diagnosis of certain retinal disorders affecting specifically the ON or OFF pathways such as congenital stationary night blindness (CSNB). On Off 10 V 50 ms Figure 4: Typical long flash full-field ERG 15 2.3 Origin of the ERG components For years, electrophysiologists have been investigating the origin of the different components of the ERG waveform using multiple techniques. This section summarizes our knowledge on the a-wave, b-wave, i-wave and OPs generators to date. These waves were studied at some point in the manuscripts included in this thesis. 2.3.1 The a –wave It has been accepted for decades now that the a-wave comes from the hyperpolarization of the photoreceptors in response to light stimulation (Armington, 1974). Several studies using intra-retinal microelectrodes suggested the photoreceptor layer as the origin of the fast P-III component (Tomita, 1950; Brown & Wiesel, 1961a, 1961b; Brown & Murakami, 1964). Since then, Hood & Birch (1990, 1995) were able to demonstrate a quantitative association between the electrical activity of the photoreceptors and the leading edge of the a-wave. 2.3.2 The b-wave It is well accepted now that the ERG b-wave is produced by the bipolar/Müller cells complex (Dowling, 1970; Newman & Frishman, 1991). Briefly, light-evoked depolarization of the second order neurons, the bipolar cells, increases the extracellular concentration of K+ in the distal retina, leading to an influx of K+ into Müller cells. It results in depolarization of the Müller cells and outflow of K+ at the more proximal regions of the cells. The return current thus created in the extracellular space generates a transretinal potential recorded as the b-wave. The role of the Müller cells was first revealed by current source density analysis in the rabbit retina (Faber, 1969, unpublished, reported in Dowling, 1970; Newman & Frishman, 1991). Similar findings were later reported in the monkey (Heynen & Van Norren, 1985). Based on these studies, it appeared that the Müller cells, which extend from the outer to the inner limiting membrane, were the only retinal elements compatible with the distribution of the b-wave sources and sinks 16 within the retina. A different approach, used by Miller and Dowling (1970) also pointed to the Müller cells as the generators of the b-wave. Working on the mudpuppy retina, they compared the intracellular light-evoked voltage response of Müller cells to the b-wave and found that they shared many similarities. Based on the above, the b-wave can then be considered as a glial response. However, as many studies have shown, it reflects the neural activity of the bipolar cells. In that matter, there are differing views as which bipolar cells contribute to the building of the b-wave. The first view is that only ON-bipolar cells are involved. Supportive of this idea are studies on both amphibian (Dick& Miller, 1985) and rabbit (Dick, Miller & Bloomfield, 1985) retinas that identified the ON-bipolar cells as being at the origin of the K+ increase in the OPL. In addition, injection of 2-Amino-4-phosphonobutyric acid (APB), a specific blocker of the ON-bipolar cell glutamate receptors, eliminates the b-wave in the vertebrate retina (Gurevich & Slaughter, 1993). Based on their study of the primate retina, Sieving and colleagues (1994) came up with the conclusion that the OFF-bipolar cells, although playing a smaller role, were also involved in shaping the b-wave. They introduced the push-pull concept, with the ON-bipolar cells pushing the ascending phase of the b-wave and the OFF-bipolar cells limiting the b-wave amplitude by competing with the Muller cells for the K+ released by the ON-bipolar cells. 2.3.3 The i-wave As represented in figure 2, the i-wave of the photopic short flash ERG is a low-voltage positive component that often follows the b-wave (Nagata, 1963; Rousseau & al., 1996; Rosolen & al., 2004). Nagata (1963) initially presented the i-wave as a remnant of the OFF response normally evoked when using a flash of longer duration. However, Seiple and Holopigian (1994) challenged this hypothesis with results suggesting that there is not a significant recordable OFF ERG response elicited by brief flash stimuli. Later work from Rousseau & al. (1996) indeed proposed a more distal site for the i-wave generators: these investigators linked the i-wave with the P50 component of the pattern ERG which is allegedly produced by the retinal ganglion cells (Maffei & al., 1985; Berardi & al., 1990). 17 . 2.3.4 The oscillatory potentials Despite several studies conducted on the subject, there is currently no consensus regarding the origin of the OPs, the high-frequency, low-amplitude wavelets rising on the ERG b-wave. However, certain retinal cells have been excluded as generators of the OPs. Effectively, Brown (1968) showed that central retinal artery occlusion abolishes both the b-wave and the OPs. The OPs generators are thus dependent on the retinal circulation, excluding the outer retinal structures, i.e the photoreceptors and horizontal cells, as OPs generators. Confirming the postreceptoral origin of the OPs, results of a microelectrode depth study from Ogden (1973) pointed to membranes of the IPL as generators of the OPs in the primate retina. In the same study, Ogden excluded the Müller cells as it was shown that they are incapable of rapid membrane potential fluctuations. However, Ogden identified three structures of the IPL able to do so: the axon terminals of the bipolar cells, the processes of the amacrine cells and the dendrites of ganglion cells. There are evidences both for and against the contribution of the ganglion cells to the genesis of the OPs. For example, Gur & al. (1987) and Veagan & al. (1995) showed that OPs were reduced in groups of patients affected with glaucoma or optic nerve diseases. However, it was not always the case in individual patients. In contrast, a few studies showed that the OPs are not affected in patients with optic nerve atrophy (Wanger & Persson, 1983; Wachtmeister & el Azazi, 1985). In a study on the rabbit visual system, Molotchnikoff & al. (1989) showed that the amplitudes of the long latency OPs were significantly increased after optic nerve blockade, with no change in the shorter latency OPs. Along the same line of thought, discrepancies between the short and long latency OPs were observed in numerous pharmacological studies (Lachapelle & al. 1990; Guité & Lachapelle, 1990) and with pathology (Lachapelle, 1994; Lachapelle & al., 1998). For example, in patients affected with CSNB-1, a condition known to result from an ON retinal pathway anomaly (Houchin & al., 1991; Khan & al., 2005), OP2 and OP3 are specifically abolished, whereas OP4 is of normal amplitude (Lachapelle & al., 1998). This is in accordance with previous publications by Wachtmeister (1980, 1981) suggesting that 18 the early OPs are associated with the ON retinal pathway, and the late OPs with the OFF retinal pathway. In a review on the subject, Wachtmeister (1998) indeed identified the bipolar cells as the most probable generators of the OPs, given that they carry the ON and OFF pathways of the retina. However, Wachtmeister could not exclude the involvement of the amacrine and interplexiform cells. In summary, although some questions remain regarding the origin of the OPs, these results strongly suggest that each OP is probably generated by different retinal elements. 2.4 Luminance-response functions of the Short Flash full-field ERG The morphology as well as amplitude and peaktime measurements of the short flash ERG vary with the intensity of the flash stimulus. The amplitude change in relation to flash luminance is particularly interesting to study. The a-wave, in both photopic and scotopic conditions, increases almost linearly as the retina is stimulated with progressively brighter flashes (Hébert & Lachapelle, 2003; Rufiange & al., 2002). In contrast, the b-wave amplitude varies in a unique way with increases in flash luminance depending on the adaptation state of the retina, as explained in sections 2.4.1 and 2.4.2. 2.4.1 Scotopic b-wave luminance-response function: the Naka-Rushton equation In scotopic condition, with increasing flash luminance, the amplitude of the b-wave grows rapidly following a sigmoidal trend which can be described with the Naka-Rushton equation (Naka & Rushton, 1966): V/Vmax=In/(In + Kn) where V is the amplitude of the bwave evoked to a stimulus of intensity I, n the slope of the function and K the flash intensity that produces a b-wave half the maximal amplitude (Vmax). The plateau of the sigmoidal function (Vmax) usually occurs at the intensity where the a-wave is first seen, suggesting that for brighter flashes, cones contribute to the response (mixed scotopic response). 19 Figure 5: Naka-Rushton equation fit to the scotopic b-wave luminance-response function (from: Rufiange & al., 2002b, with permission) 2.4.2 Photopic b-wave luminance-response function: the Photopic Hill With progressively brighter flash stimuli, the luminance-response function of the photopic ERG b-wave adopts a unique shape where the amplitude of the b-wave first increases, reaches a maximal value (Vmax) and then decreases gradually to finally form a plateau (Wali & Leguire, 1992; Rufiange & al., 2002, 2003, 2005), a function called the Photopic Hill by Wali and Leguire (1992) because of its particular shape. The most recent explanation regarding the fall of the photopic b-wave amplitude with brighter flashes consists of the combination of a gradually reduced ON component amplitude with a gradually delayed OFF component, both effects being enhanced with brighter intensities of stimulation (Ueno & al., 2004). While the scotopic b-wave luminance-response function can be easily fitted with the Naka-Rushton equation, as mentioned above, the particular shape of the Photopic Hill 20 complicates its analysis. A first method was proposed by Rufiange & al. (2003) who showed that the function can be analyzed using 7 easily identifiable and reproducible parameters (figure 6): the maximal b-wave amplitude (Vmax), the amplitude of the a-wave at the Vmax intensity (amax), the flash intensity needed to generate the Vmax response (Imax), the stimulus intensities generating a b-wave half the amplitude of Vmax on the ascending (Ka) or descending (Kd) limb of the photopic hill, the ratio of the amplitude of the b-wave over that of the a-wave measured at Vmax intensity (b/amax) and the intensity of stimulation needed to generate an ERG where the amplitude of the b-wave equals that of the a-wave (Ka=b). This analysis method proved the usefulness of the Photopic Hill in studying various retinal disorders (CSNB-1, cone anomaly and pigmentary retinopathy). More recently, Hamilton & al. (2007) presented a mathematical equation combining a Gaussian and a logistic growth functions to fit the Photopic Hill curve (Glasgow equation). Based on the analysis of CSNB-1 patients (known to have an abolished ON pathway), their work suggested that the Gaussian and the logistic growth functions would reflect respectively the OFF and ON retinal responses. Amplitude (V) 2.84 a-wave 150 2.39 b-wave 1.90 1.63 100 1.40 1.13 50 0.90 0.64 0.39 0 -2 -1 0 1 2 3 -2 Flash luminance (log cd.s.m ) 0.17 -0.02 -0.23 -0.41 -0.62 Figure 6: Photopic a- and b-waves luminance-response functions (Photopic Hill) -0.80 -1.00 -1.21 -1.62 -1.81 100 -2.04 50 21 3. RETINAL DEGENERATIONS As mentioned earlier, the ERG is the only objective tool available to assess retinal function. Dark-adapted ERGs provide information about the function of the rod pathways whereas light-adapted ERGs assess the function of the cone pathways. Depending on the retinopathy, the cone or the rod pathways, or both, will be affected. Analysis of rod and cone ERGs, together with other clinical findings, such as fundus appearance, visual complaints and visual fields to name a few, will lead to the appropriate diagnosis. 3.1 Retinitis pigmentosa With a prevalence of 1/3000 to 1/4000 worldwide (Sharma & Ehinger, 1999), retinitis pigmentosa (RP) is by far the most common inherited retinal degeneration. RP is a group of hereditary progressive retinal degenerations caused by gene abnormalities on different chromosomes. A survey-based study performed in Maine (Bunker, 1984) estimated the frequency of transmission modes as follows in the US population: 19 % autosomal dominant (AD), 8 % X-linked, 19 % autosomal recessive (AR), 46 % isolated and 8% undetermined. The first descriptions of the disease go back to the 1850’s with Van Trigt, Ruete, and Donder (Weleber & Gregory Evans, 2006). RP indeed presents with characteristic features, the major being night blindness, progressive visual field constriction with relative preservation of the macular function and bone spicule-like pigmentation within the posterior pole (Birch, 2006). Attenuated retinal vessels and waxy pallor of the optic nerve head are also common findings as the disease progresses (Benson, 1993). Depending on the stage of the disease, ERG responses range from normal to undetectable. Scotopic ERG responses are usually severely attenuated early in the disease process, sometimes years before signs and symptoms arise. A delay in the cone b-wave implicit time is also noted. As the condition progresses, the photopic ERG amplitude is also significantly reduced. Eventually, both responses are abolished. (Benson, 1993; Birch, 2006) 22 3.2 Congenital stationary night blindness (CSNB) In contrast to RP, CSNB is a non progressive retinal disorder characterized by night blindness, slightly decreased visual acuity and a normal fundus appearance. There are two major types of CSNB: complete (CSNB-1) and incomplete (CSNB-2). Patients with CSNB-1 show moderate to severe myopia, undetectable rod function and a normal cone response, whereas patients with CSNB-2 show moderate myopia to hyperopia and subnormal but measurable rod and cone function (Bech-Hansen & al., 1998). The condition is transmitted through a X-linked or an AR mode. X-linked CSNB-2 has been shown to result from a mutation in the CACNA1F gene (Hoda & al, 2005) whereas the AR form is due to a mutation in the CABP4 gene (Zeitz & al., 2006). These mutations would result in a defect in channels located in the photoreceptor terminals, thus affecting transmission from the photoreceptors to the ON- and OFF-bipolar cells (Zeitz & al., 2006). Using the long flash ERG technique, several investigators suggested that CSNB-1 results from a postreceptoral anomaly at the synapse between the photoreceptors and the ON-depolarizing bipolar cells (ON-DBC) (Miyake & al., 1987; Quigley & al., 19961997; Langrovà & al., 2002). Supporting of the later, recent literature revealed that mutations on the NYX (Pusch & al., 2000; Bech-Hansen & al., 2000) and GRM6 (Dryja & al, 2005; Zeitz & al., 2005) genes were respectively responsible for the X-linked and AR forms of CSNB-1. NYX and GRM6 encode respectively a small leucine-rich repeat protein called nyctalopin and the glutamate receptor mGluR6, which have both been linked to synapses between cone and rod photoreceptors and ON-depolarizing bipolar cells (Morgans & al., 2006; Nakajima & al., 1993; Slaughter & Miller, 1985; Gregg & al., 2007). Furthermore, studies have shown that these molecules are required for normal synaptic transmission between retinal photoreceptors and ON-depolarizing bipolar cells in mice and zebrafish models of CSNB-1 (Bahadori & al., 2006; Pinto & al., 2007). As mentioned above, in CSNB-1 the rod ERG is completely abolished and the cone response, i.e. the a-wave, is of normal amplitude. However, the photopic short flash ERG is not totally normal. Effectively, it has a characteristic square-like morphology and a truncated b-wave as a result of the specific abolition of OP2 and OP3 (Lachapelle & al., 1998). 23 3.3 Congenital postreceptoral cone pathway anomaly (CPCPA) In a previous publication (Lachapelle & al., 1998), our laboratory introduced a new clinical entity with clinical signs suggestive of a form of cone pathway anomaly. In this paper, the condition was referred to as a form of cone dystrophy, although ERG analysis revealed a postreceptoral defect, with normal cone function. The disease was later renamed CPCPA in order to describe more adequately the location of the anomaly (Garon & al., 2008).add reference Our patients with CPCPA are from 2 different families, unlinked to each other. Their main complaint is usually decreased vision (VA ranging from 20/30 to 20/100). They also present with a red/green color defect as tested with the FM-100. No genetic testing has been performed on this group of patients. However, their ERG waveforms have been studied thoroughly. Their photopic ERG demonstrates unique features never reported before. The a-wave is of normal amplitude while the b-wave is markedly attenuated with a truncated appearance. Analysis of the OPs revealed normal amplitude values for OP2 and OP3 while OP4 is severely attenuated, a pattern which complements that seen in CSNB-1. The scotopic mixed oscillatory potentials revealed a marked enhancement of OP2, another finding never reported previously (Lachapelle & al., 1998). CPCPA thus seems to represent a distinct retinal disease with electrophysiological features complementary to CSNB-1. 24 PURPOSE OF MY RESEARCH PROJECT Based on the above, the purpose of the project that will be presented in this thesis was to further define the photopic ERG luminance-response function and test its usefulness to investigate normal and pathological phenomenon involving the cone visual pathways of the human retina. Results of my investigations are reported in the following three studies: 1- Study 1(Chapter 2): Spectral electroretinography: What does the luminanceresponse function reveal? In this study I compared the morphology of ERGs evoked to progressively brighter blue, red and white flashes in order to better understand the origins of the alleged S-cone and L,M-cone ERGs in normal subjects. 2- Study 2 (Chapter 3): Asymmetrical growth of the photopic hill during the light adaptation effect. In this study I examined how the photopic luminance-response function curve varies as the retina is adapting to a photopic background light following a prolonged period of dark-adaptation (the so-called light adaptation effect: LAE). In doing so, I hoped to bring light on the still unknown retinal mechanisms underlying the light adaptation effect (LAE). 3- Study 3 (Chapter 4): Estimating the ON and OFF retinal pathways function with the Glasgow equation: Normative data and clinical application. Finally, in the last study, I put to the test the recently published mathematical fitting of the b-wave luminance-response function (Hamilton & al., 2007) in differentiating ON and OFF retinal responses and studied its clinical relevance in more accurately segregating patients affected with selected retinal diseases. There are many reasons to focus our investigations on the photopic ERG. First, many of the most severe and debilitating retinal diseases initially involve the gradual loss of rod function with significant deterioration of cone vision occurring later during the course of the disease. At the time of diagnosis, the cone component of the ERG response is thus often the only one remaining. This is frequently the case with patients affected with 25 retinitis pigmentosa (RP). Secondly, one would agree that functionally, cone vision is more important than rod vision, given that most of our daily activities are done in a photopic environment. Finally, as opposed to the scotopic ERG, which has been studied thoroughly (origin, morphology, equation fitting) and used widely to assess normal retinal physiology and pathology, the photopic ERG has somehow been neglected. It is therefore important to improve our understanding of the cone function and refine the techniques used to assess it, in order to better classify and stage retinopathies and possibly offer better insights on prognosis to patients affected with progressive retinal degenerations ultimately leading to blindness. Such information will be of utmost significance should a cure become available in the future. 26 Chapter II Study 1 Spectral electroretinography: What does the luminance-response function reveal? In this first study, we used luminance-response ERG recordings to evaluate the changes in morphology of ERGs evoked to progressively increasing dim red and blue flashes in comparison to ERGs evoked to white flashes, in order to further describe and understand a feature of the normal retina, namely the spectral ERG. 27 Spectral electroretinography: What does the luminance-response function reveal? Marie-Lou Garon, O.D.1, Marianne Rufiange, Ph.D.1,, Pierre Lachapelle, Ph.D.1 1 Department of Ophthalmology & Neurology-Neurosurgery, McGill University – Montreal Children’s Hospital Research Institute Short title: Spectral electroretinography, S-cone response This manuscript contains 26 pages, 1 table and 9 figures. Last revision: September 25th, 2010 Address for correspondence: Pierre Lachapelle, Ph.D. Department of Ophthalmology (D-164) McGill University, Montreal Children’s Hospital Research Institute 2300 Tupper Street Montreal, Quebec, Canada, H3H 1P3 Tel : 514-412-4400 ext 23890 Fax : 514-412-4331 E-mail : [email protected] 28 ABSTRACT Many ERG laboratories are using very dim short-wavelength flashes against a bright white or yellow background to separate the short-wavelength (S-) cone response from the long- (L-) and middle-wavelength (M-) cone response. We examined if the typical biphasic ERG waveform, suggested to result from the summation of the L-, M- (early component) and S- (late component) cones, is unique to short-wavelength stimuli or if it can be obtained with other types of stimuli. Photopic ERGs in response to blue, red and white flashes of different intensities were recorded against a white background of 50 or 200 cd.m-2 from 8 normal subjects. Our results suggest that the typical S-cone ERG waveform can also be generated with long-wavelength stimuli as well as with white flashes in some patients. Furthermore, analysis of the white-flash luminance-response function reveals that what is identified as the S-cone contribution is most probably the iwave. Finally, our results also shows that to separate the alleged L, M- and S-cone contribution, it is not necessary to use a bright photopic environment, as a 0.6 log unit increase in background luminance simply shifted the resulting luminance-response curve by 0.5-0.6 log unit. In fact, when the retina was adapted to the 200 cd.m-2 background, the S-cone typical response was lost, indicating that very bright background should be avoided when recording spectral ERGs. Keywords: human electroretinogram (ERG); spectral ERG; S-cone response; luminanceresponse function. 29 INTRODUCTION It is well accepted that color vision in humans is due to comparison of photons absorption by 3 types of photoreceptor cells: the short (S-), medium (M-) and long (L-) wavelength sensitive cones, each presenting with a distinct spectral sensitivity with maximal absorption at 430, 535 and 570 nm (Calkins, 2001), respectively. Several ERG laboratories are using a selective adaptation technique (Arden & al., 1999; Gouras & MacKay, 1990; Gouras, MacKay & Yamamoto, 1993; Gouras, 2003; Simonson & Rosenberg, 1996; Yamamoto & al., 1997, 1999; Hayashi & Yamamoto, 2001) , which consists in using very dim short-wavelength (blue) flashes against a bright background (yellow or white), to separate the S-cone response from the L- and M-cone response. The typical ERG response obtained using this technique is composed of an early small positive b-wave, suggested to result from the L- and M-cones, followed by another larger and delayed b-wave that would be produced by the S-cones activity. This second positive peak is absent when red flashes are used. Furthermore, a S-cone achromat patient placed in the same conditions produced a single b-wave matching the late b-wave obtain in normal subjects (Gouras & MacKay, 1990). These findings point to the S-cones as generators of this biphasic ERG waveform in response to dim blue flashes of light on a bright background. The ISCEV standards for clinical electroretinography recommend using a 3.0 log cd.s.m-2 stimulus on a retina previously light-adapted to a 30 cd.m-2 white background light in order to evaluate the cone function (Marmor & al., 2009). The role of retinal light adaptation is to desensitize rods and is essential to avoid rod contamination to the response. In our clinic, we routinely record cone responses on a broader range of flashes (-0.8 to 2.84 log cd.s.m-2) in order to generate a photopic ERG luminance-response function or Photopic Hill, which has proved to be useful in both clinical and experimental studies (Rufiange & al., 2002a, 2002b, 2003, 2005; Brûlé & al., 2007; Beaulieu & al., 2009). In an attempt to study the changes in the ERG waveform morphology in response to dimmer white flashes as low as -2.04 log cd.s.m-2, we noticed that the ERG responses obtained resembled the typical S-cone ERG waveform. 30 Based on the above, the purpose of this study was to compare ERGs evoked to dim white, red and blue flashes of light in order to examine if the typical biphasic ERG waveform suggested to result from the summation of the L-,M- (early component) and S- (late component) cones, is indeed unique to short-wavelength stimuli or if it can be obtained with other light stimuli/background combination. METHODS Preparation of subjects A total of 8 normal subjects (age: 20-27, mean: 22.5; 7 women, 1 man) were tested for the purpose of the study. They all signed an informed consent approved by the Institutional Review Board of the Montreal Children’s Hospital attesting of their voluntary participation and received a symbolic financial compensation. ERGs were recorded on both eyes with DTL fiber electrodes in double thickness (27/7 X-Static silver coated conductive nylon yarn: Sauquoit Industries, Scranton, PA, USA) according to a method previously described (Rufiange et al., 2002a). Briefly, pupils were maximally dilated (8-9 mm) with 1 % tropicamide drops. DTL electrodes were positioned deep into the inferior conjunctival bags and secured at external and internal canthi of each eye with doublesided adhesive tape. Reference and ground electrodes (Grass gold cup electrodes filled with Grass EC2 electrode cream) were pasted at the external canthi and forehead respectively. ERG recordings The subjects were facing a Ganzfeld of 30 cm in diameter that provided both the white background light and color flash stimuli needed for this study. Subjects were first lightadapted to a background of 50 cd.m-2 for 10 minutes. Then, photopic ERGs (bandwidth: 0.3-500 Hz) in response to 9 intensities of blue (410 nm, -2.40 to 0.30 log cd.sec.m-2), red (640 nm, -2.23 to 0.08 log cd.sec.m-2) and white (-2.23 to 0.64 log.cd.sec.m-2) flashes were recorded simultaneously on both eyes using a LKC UTAS-E-3000 system (LKC Systems Inc., Gaitherburg, MD, USA). Photopic ERG responses to blue and red flashes were also obtained against a 200 cd.m-2 white background light in order to compare the effect of background luminance on the morphology of the waves. The flash duration, the 31 interstimulus interval and the pre-stimulus baseline were fixed respectively at 20 ms, 0.33 s and 40 ms. 50 to 300 responses were averaged for each flash luminance. Background and flash luminance were measured with a research photometer (IL 1700; International Light, Newburyport, MA, USA). Data analysis Analysis of the ERGs included peak time and amplitude measurements of a-, b- and iwaves for all stimulus intensities against both backgrounds used (a-wave not showed in this paper). Data from both eyes were averaged to yield a single data point. The amplitude of the a-wave was measured from baseline to the most negative trough, that of the b-wave from the later trough to the most positive peak and that of the i-wave from the trough following the b-wave to the next positive peak. Finally, peak times were measured from baseline to the peak of each wave. RESULTS Figure 1 shows the averaged typical photopic b-wave luminance-response function in response to white flashes of -2.04 to 2.84 log cd.s.m-2. Representative waveforms from one of the subjects are illustrated to the left. As shown on the plot graph, increase in flash luminance resulted in a gradual increase of the b-wave amplitude until it reached a maximal value (Vmax); from there, the amplitude of the b-wave decreased with further increase in flash luminance to the point where it plateaued. This is the typical “Photopic Hill” morphology as reported previously (Wali & Leguire, 1992; Rufiange & al., 2002, 2003, 2005; Kondo & al. 2000; Hamilton & al., 2007). Responses evoked to flashes dimmer than -0.8 log cd.s.m-2 appear to be flat on this figure; however, when shown on an appropriate scale, as in figure 2, the amplitude and peaktime of their components can easily be measured. The ERG tracings from two representative individuals in response to blue, red and white flashes of increasing luminance are shown in figures 2 and 3. In figure 2, the typical Scone ERG was elicited in response to blue flashes of -2.22 and -2.01 log cd.s.m-2, where the early positive b-wave was followed by a late larger one. For higher flash luminance, 32 the early positive peak grew larger than the second which became very similar to what is usually call the i-wave in standard white full-field ERGs. This same subject also produced waveforms similar to S-cone responses with a red flash of -1.84 log cd.s.m-2 and a white flash of -1.62 log cd.s.m-2. Results obtained from the second subject (figure 3) were different. In response to blue flashes, the waveform showed a third higher positive peak following the biphasic S-cone response (present for flashes from -2.22 to -1.83 log cd.s.m-2). This third positive peak, identified as k, was also noted for responses to red and white flashes, although for these flash stimuli, the second positive peak was always lower than the first, showing a morphology closer to the L-,M-cone response. Figure 4 shows the composite ERG waveforms from the 8 subjects to progressively brighter blue, red and white flash stimuli. Blue flashes equal or dimmer than -1.83 log cd.s.m-2 elicited the typical morphology of the short-wavelength ERG, i.e. the second positive component peaked at an amplitude equal or above that reached by the first one. In contrast, brighter blue flashes and all red and white flashes produced a larger early positive peak followed by one or two positive peaks of smaller amplitude. This resulted in the typical long-wavelength ERG morphology given that only the L-,M-cone component was readily obvious. Comparison of these typical responses is eased at figure 5, where typical waveforms in response to blue and red (top) and blue and white (bottom) flashes are superposed. In some subjects, however, there were intensities of white and red which elicited ERG responses similar to those obtained in response to blue flashes, as exemplified at figure 6. Illustrated at figure 7 are photopic b- (top left) and i-wave (top right) luminance-response functions (mean ± 1 SD) obtained to white, red and blue stimuli against a 50 cd.m-2 white background. Irrespective of the wavelength of the stimulus, the amplitude of the b-wave always increased, although it is hardly detectable for the lowest flash intensities due to the scaling used. However, the b-wave grew faster in response to blue stimuli compared to white and red (approximately 0.4 and 0.5 log unit difference compared to blue, respectively). This is in accordance with Rufiange & al. (2005) who showed a shift of sensitivity between luminance-response functions evoked to white and color stimuli, the 33 retina being most sensitive to blue followed by green, white and red. The i-wave amplitude followed a similar path, except for dimmer flashes where its amplitude plateaued before it started increasing. Subjects were approximately 0.5 and 0.6 log unit less sensitive to white and red, respectively, compared to blue. The bottom two graphs of figure 7 show the corresponding b-wave (left) and i-wave (right) luminance-peak time functions. With progressively brighter flashes, the peak time of the b-wave first shortened gradually and then lengthened. These results are in accordance with those obtained by Rufiange & al. (2005). In contrast, the i-wave peak time lengthened progressively as the flash intensity increased. In all instances, there was no marked difference in peak time between the three wavelength stimuli. The average peak times for b- and i-waves evoked to a blue flash of -2.01 log cd.s.m-2 (the flash luminance that produced the most representative S-cone ERG) were respectively 27.1 ms ±1.5 (SD) and 43.9 ms ±1.3 (SD). They correspond approximately to peak times reported previously for S- and L-,M-cones responses (Gouras & MacKay, 1990; Gouras & al., 1993; Tsuruoka & al. 2004). Refer to table 1 for values comparison. The effect of the background luminance on the ERG waveforms obtained in response to blue (figure 8) and red (figure 9) flashes was also investigated in one of the subjects. Increasing the background by 0.6 log unit (from 50 to 200 cd.m-2) simply shifted the resulting b-wave luminance-response curve by approximately 0.6 log unit for the blue and 0.5 log unit for the red stimuli. Of interest, when examining the waveforms obtained against the 200 cd.m-2 background in response to blue flashes, their morphology appeared closer to the typical L-,M-cone response, as the second positive component always peaked at an amplitude significantly below that reached by the first positive component. DISCUSSION In this study, we were able to reproduce typical S-cone responses (Gouras & MacKay, 1990, 1993, 2003) using dim blue flashes against a moderately bright white background. Indeed, the waveform obtained first showed a negative deflection, followed by two positive peaks, the second one, claimed to originate from the S-cone activation, being 34 larger than the first one, presumably produced by the L,M-cone activation. In contrast, in response to red and white flashes, the second positive peak was of significantly smaller amplitude compared to the first one, resulting in the typical L,M-cone ERG. In some subjects, however, we noticed that ERGs evoked to red and white flashes presented morphologies almost identical to ERGs generated in response to blue flashes; only the flash intensity varied slightly (figure 6). Could these results suggest that the typical, biphasic, short-wavelength ERG can also be generated with a long-wavelength stimulus as well as with white flashes? Based on the present results, we cannot apply this conclusion to all of our subjects, as it was not a finding obtain in all cases. It appears, however, that red and white flashes produce similar ERG waveforms, which is not a surprising findings given that L,M-cones account for 90% of all cones (Rodieck, 1998). The most interesting result of our study is probably the waveform progression with increasing flash intensities. Previous studies which investigated the changes in the S-cone response with progressively brighter flashes (Gouras, 1990; Tsuruoka & al., 2004) were limited to very dim flashes. Further increases in flash intensity revealed that the second positive component of the response eventually peaked at an amplitude significantly below that reached by the first positive component, as reported in figure 4 for blue flashes brighter than -1.83 log cd.s.m-2. When compared to the white flash luminance-response function, it appears that what is identified as the S-cone contribution in the blue flash ERG is most probably the i-wave. Indeed, we identified the L,M-cone as the b-wave and the S-cone as the i-wave on the figures. Supportive of that claim, b- and i-wave peak times presented in the present study were comparable to L,M- and S-cone peak times reported in previous studies (Table 1). There were small variations in the values; however, they could be explained by variation in background and flash luminance, type of electrode, age of the subjects (Gouras & al., 1993), bandwith, as well as differences in the wavelength transmission of the filters used in each study. The i-wave being a component of the photopic ERG suggests that it is dependent on cone stimulation. The S-cones transmit the visual signal exclusively through the koniocellular 35 pathway. This pathway receives direct signals from the S-cones through specific S-cone bipolar cells and indirect signals from the L- and M-cones via diffuse OFF-bipolar cells. This information is then relayed to the bistratified ganglion cells (Dacey & Lee, 1994). Interestingly, Rousseau & al. (1996) presented experimental evidence that associated the flash ERG i-wave to the pattern ERG P50 wave, the later long used to assess the ganglion cells function (Maffei & Fiorentini, 1981; Veagan & al., 1995; Parisi & al., 1997). This could suggest that the S-cone/i-wave response originates from the bistratified ganglion cells. Of interest, one of the earliest signs of primary open angle glaucoma (POAG), a disease known to affect ganglion cells, is a subjective reduction in blue sensitivity as showed with color vision testing (Greenstein & al., 1989) and blue on yellow automated perimetry (Heron & al., 1988; Johnson, 1996). Drasco & al. (2001) indeed reported significantly attenuated amplitude for the S-cone b-wave in early POAG. In a study on the minipig, Rosolen & al. (2003) observed a significant reduction in the i-wave amplitude 2 months and 6 months following induced ocular hypertension, providing further support to the claim that the i-wave is generated at the level of the retinal ganglion cells. Based on the above, the i-wave could represent the response of the bistratified ganglion cells involved in the koniocellular pathway which carries information from the S-cones. The last part of our study showed that it is not necessary to use a bright photopic environment to separate the alleged L,M- and S-cone contribution, as a 0.6 log unit increase in background luminance simply shifted the resulting luminance-response curve by 0.5-0.6 log unit (figures 8 and 9). In fact, when the retina was adapted to the 200 cd.m-2 background, the S-cone typical response was lost, indicating that very bright background should be avoided when recording spectral ERGs. Our study presents valuable evidence challenging the concept of spectral ERG, more specifically the S-cone response. However, more research is needed before we can conclude that the i-wave of the photopic full-field ERG can be used to assess the function of the S-cones pathway instead of the spectral ERG technique. Specifically, comparison 36 of i-wave and traditional spectral ERG waveforms in blue-cone achromats and blue-cone monochromats would be of additional value. 37 References Arden G, Wolf J, Berninger T, Hogg CR, Tzekov R, Holder GE (1999) S-cone ERGs elicited by a simple technique in normals and in tritanopes. Vision Res 39: 641-650. Beaulieu C, Rufiange M, Dumont M, Lachapelle P (2009) Modulation of ERG retinal sensitivity parameters with light environment and photoperiod. Doc Ophthalmol 118(2): 89-99. Brûlé J, Lavoie MP, Casanova C, Lachapelle P, Hébert M (2007) Evidence of a possible impact of the menstrual cycle on the reproducibility of scotopic ERGs in women. 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Rufiange M, Dumont M, Lachapelle P (2002b) Correlating retinal function with melatonin secretion in subjects with an early or late circadian phase. Invest Ophthalmol Vis Sci 43:2491-2499. Rufiange M, Dumont M, Lachapelle P (2005) Modulation of the human photopic ERG luminance-response function with the use of chromatic stimuli. Vision Res 45:2321-2330. Rufiange M, Rousseau S, Dembinska O, Lachapelle P (2002a) Cone-dominated ERG luminance-response function : the Photopic Hill revisited. Doc Ophthalmol 104(3):231248. Simonsen SE, Rosenberg T (1996) Reappraisal of a short-wavelength-sensitive (S-cone) recording technique in routine clinical electroretinography. Doc Ophthalmol 91:323-332. Tsuruoka M, Yamamoto S, Ogata K, Hayashi M (2004) Built-in contact lens electrode for S-cone electroretinographic recordings. Doc Ophthalmol 108:61-66. Vaegan, Graham SL, Golberg I, Buckland L, Hollows FC (1995) Flash and pattern electroretinogram changes with optic atrophy and glaucoma. Exp Eye Res. 60:697-706. 39 Wali N, Leguire LE (1992) The photopic hill: a new phenomenon of the light adapted electroretinogram. Doc Ophthalmol 80(4):335-345. Yamamoto S, Hayashi M, Takeuchi S (1999) S-cone electroretinogram to Ganzfeld stimuli in patients with retinitis pigmentosa. Doc Ophthalmol 99:183-189. Yamamoto S, Nitta K, Kamiyama M (1997) Cone electroretinogram to chromatic stimuli in myopic eyes. Vision Res 37:2157-2159. 40 Captions to illustrations FIGURE 1 Mean b-wave luminance-response function (right) obtained from 10 normal subjects for white flash intensities of -2.04 to 2.84 log cd.s.m-2. Representative corresponding ERG waveforms from one of the subjects are presented to the left. Amplitude is expressed in V and luminance in log cd.s.m-2. FIGURE 2 Representative photopic ERG waveforms recorded from a normal subject in response to blue, red and white flashes of light of progressively brighter intensities (from top to bottom, values indicated at the left of each tracing in log cd.s.m-2). All waveforms were elicited against a 50 cd.m-2 rod-desensitizing white background light. a-, b-, i- and kwaves are indicated as a, b, i and k respectively. Horizontal calibration: 20 ms; vertical calibration: 5 to 30 V. FIGURE 3 Representative photopic ERG waveforms recorded from another normal subject in response to blue, red and white flashes of light of progressively brighter intensities (from top to bottom, values indicated at the left of each tracing in log cd.s.m-2). All waveforms were elicited against a 50 cd.m-2 rod-desensitizing white background light. a-, b-, i- and kwaves are indicated as a, b, i and k respectively. Horizontal calibration: 20 ms; vertical calibration: 5 to 30 V. FIGURE 4 Mean photopic ERG waveforms (n=8) elicited to progressively increasing flashes (top to bottom) of blue, red and white light against a 50 cd.m-2 white background. Flash intensities are indicated at the left of each tracing in log cd.s.m-2. a-, b-, i- and k-waves are identified as a, b, i and k, respectively. Horizontal calibration: 40 ms; vertical calibration: 1 to 10 V. 41 FIGURE 5 Superposition of mean ERG waveforms for typical S- and L,M-cone response to blue and red (top) and blue and white (bottom) stimuli. Flash intensity and color are indicated at the left of the tracings. Vertical calibration:1.5 to 2 V. FIGURE 6 Comparison of similar waveform morphologies obtained from normal subjects in response to flashes of different colors. Color and intensity (in log cd.s.m-2) of stimulus are indicated at the left of the tracings. Horizontal calibration: 40 ms; vertical calibration: 1 to 2 V. FIGURE 7 Mean (±1 SD) values for amplitude (top left: b-wave; top right: i-wave, ordinate in V) and peak time (bottom left: b-wave; bottom right: i-wave, ordinate ms) as a function of flash intensity (abscissa in log cd.s.m-2) for blue, red and white stimuli presented against a 50 cd.m-2 rod-desensitizing background. FIGURE 8 Comparison of representative ERG waveforms in response to blue flashes obtained from a normal subject against background lights of 50 and 200 cd.m-2, as indicated at the bottom of each column of tracings. Flash intensities (in log cd.s.m-2) are indicated at the left of each tracing. Horizontal calibration: 40 ms; vertical calibration: 1 to 16 V. The two graphs at the right represent respectively amplitude (top) and peak time (bottom) as a function of flash intensity for the two backgrounds tested. Amplitude is expressed in V and peak time in ms. FIGURE 9 Comparison of representative ERG waveforms in response to red flashes obtained from a normal subject against background lights of 50 and 200 cd.m-2, as indicated at the bottom of each column of tracings. Flash intensities (in log cd.s.m-2) are indicated at the left of each tracing. Horizontal calibration: 40 ms; vertical calibration: 1 to 16 V. The two 42 graphs at the right represent respectively amplitude (top) and peak time (bottom) as a function of flash intensity for the two backgrounds tested. Amplitude is expressed in V and peak time in ms. 43 2.84 2.39 1.90 1.63 150 Amplitude (V) 1.40 1.13 0.90 0.64 0.39 0.17 100 50 -0.02 0 -0.23 -2 -0.41 -1 0 1 2 3 Flash luminance (log cd.s.m-2) -0.62 -0.80 -1.00 -1.21 -1.62 -1.81 100 Figure 1 -2.04 50 44 -2.40 -2.23 -2.23 -2.22 -2.01 -2.05 -1.84 -2.04 b i -1.83 -1.62 -1.65 a 10 -1.43 -1.18 -0.96 -0.47 5 -1.43 b i -0.92 -0.41 -0.69 -0.02 10 b i i 0.08 30 b i a 10 -1.00 a b 0.30 -1.81 -1.62 -1.21 30 a a 20 b i 0.64 k a 20 Blue 30 20 Red White Figure 2 45 -2.40 -2.22 -2.01 -1.83 b i k -2.23 -2.23 -2.05 -2.04 -1.84 -1.81 -1.65 -1.62 -1.43 10 b -1.43 -1.62 k i 5 -1.18 -0.96 0.30 i 30 a 10 b i 0.08 k 10 -0.02 b 30 a 20 i 0.64 k 30 a 20 Blue k i -0.41 -0.69 b -1.21 -1.00 -0.92 -0.47 b Red White 20 Figure 3 46 -2.40 1 1 -2.22 L,M -1.65 1.5 1.5 i -1.83 1.5 -1.62 b b i -1.18 k a 1 1 -2.01 b -2.04 -1.81 -1.84 1 S 1 -2.05 2 i k 2 -1.21 k 2 a a 2 -1.62 -0.92 2 2 -1.00 b b b i -0.96 k 6 a a Blue 40 i -0.41 i 0.08 k Red 10 40 6 k a White 40 Figure 4 47 L,M S 1.5 blue -2.01 2 red -1.18 L,M 1.5 S blue -2.01 1.5 white -1.62 Figure 5 48 1 white -2.04 blue -2.22 white -1.81 blue -2.22 1.5 1 40 red -1.65 2 blue -2.01 40 red -1.65 blue -2.01 1 1 40 40 Figure 6 49 25 125 i-wave amplitude (µV) b-wave amplitude (µV) 150 100 75 50 25 20 15 10 5 0 0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 -2.5 1.0 flash intensity (log cd.s.m -2) -1.5 -1.0 -0.5 0.0 0.5 1.0 55 i-wave peak time (ms) b-wave peak time (ms) -2.0 flash intensity (log cd.s.m -2) 40 35 30 25 20 15 -2.5 blue red white -2.0 -1.5 -1.0 -0.5 0.0 0.5 -2 flash intensity (log cd.s.m ) 1.0 50 45 40 35 30 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 -2 flash intensity (log cd.s.m ) Figure 7 50 -1.43 1 2 2 -1.83 2 -0.96 3 -1.62 b-wave i-wave 200 cd.m -2 b-wave i-wave 100 -1.21 amplitude (µV) -2.01 50 cd.m -2 75 50 25 0 2 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 -2 flash intensity (log cd.s.m ) -1.43 -0.70 2 70 -0.96 peak time (ms) 4 -0.47 6 4 40 40 30 20 -2.0 -1.5 -1.0 -0.5 0.0 0.5 -2 10 50 cd.m-2 50 10 -2.5 0.30 -0.47 60 16 200 cd.m-2 flash intensity (log cd.s.m ) 40 Figure 8 51 -1.65 -1.18 60 2 1 -1.18 amplitude (µV) 50 -0.69 50 cd.m -2 b-wave i-wave 200 cd.m -2 b-wave i-wave 40 30 20 10 2 0 2 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 -2 flash intensity (log cd.s.m ) -0.19 -0.69 3 60 4 peak time (ms) 0.33 0.08 50 40 30 20 10 12 0 40 -2.5 10 -2 50 cd.m -2 200 cd.m -2.0 -1.5 -1.0 -0.5 0.0 0.5 flash intensity (log cd.s.m -2) 40 Figure 9 52 Table 1: Comparison of S- and L-,M-cones peak time values previously published Study Tsuruoka & al. (2004) Blue filter (nm) Age of subjects (years) Recording electrode Bandwith (Hz) Background Flash luminance (log cd.s.m-2) S-cone implicit time (ms) L-,M-cone implicit time 433 Not mentioned Build-in LED contact lens electrode 1-1000 2 log cd/m2= 100 cd/m2 -2.0 to -1.3 43-48 24-28 11-29 Gouras & al. (1993) 450 30-59 60-83 Gouras & MacKay (1990) 430 11-66 BurianAllen bipolar contact lens electrode BurianAllen bipolar contact lens electrode 38.0 (SD=3.9) 5-1500 17000 photopic trolands≈ 85 cd/m2 Units not provided 39.6 (SD=3.2) 44.8 (SD=4.9) Not provided 5-1500 9000 photopic trolands≈ 45 cd/m2 Units not provided 38.2 (SD=2.5) 23.0 (SD=1.1) 53 Chapter III Study 2 Asymmetrical growth of the photopic hill during the light adaptation effect* In this second study, in continuity with the first one, we examined another feature of the normal retinal physiology, namely the light adaptation effect, by recording ERG luminance-response functions as the retina was light adapting. Previous studies on the light adaptation effect never looked at the changes in the Photopic Hill morphology, as most of these studies were restricted to dimmer flashes. The study helped us further define the properties of the Photopic Hill and identify the possible mechanisms at the origin of the light adaptation effect. *With kind permission from Springer Science + Business Media: Doc Ophthalmol, Asymetrical growth of the photopic hill during light adaptation effect, 2010 Aug 15. [Epub ahead of print] Garon ML, Rufiange M, Hamilton R, McCullogh DL, Lachapelle P, original copyright notice displayed with material. Asymmetrical Growth of the photopic hill during the light adaptation effect Marie-Lou Garon, O.D.1, Marianne Rufiange, Ph.D.1, Ruth Hamilton, Ph.D.2, Daphne L. McCulloch, O.D., Ph.D3., Pierre Lachapelle, Ph.D.1 1 Department of Ophthalmology and Neurology-Neurosurgery, McGill University – Montreal Children’s Hospital Research Institute; 2 Department of Clinical Physics, Yorkhill Hospital and University of Glasgow, UK; 3 Vision Sciences, Glasgow Caledonian University, Glasgow, UK; Short title: LAE and Photopic Hill This manuscript contains 28 pages, 3 tables and 6 figures. Status: accepted for publication in Documenta Ophthalmologica, July 26th, 2010 Address for correspondence: Pierre Lachapelle, Ph.D. Department of Ophthalmology (D-164) McGill University - Montreal Children’s Hospital Research Institute 2300 Tupper Street Montréal, Québec, Canada, H3H 1P3 Tel : 514-412-4400 ext 23890 Fax : 514-412-4331 E-mail : [email protected] 55 ABSTRACT Purpose: In response to progressively stronger flashes delivered against a rod saturating background light, the amplitude of the photopic ERG b-wave first increases, reaches a maximal value (Vmax) and then decreases gradually to a plateau where the amplitude of the b-wave equals that of the a-wave, a phenomenon known as the photopic hill (PH). The purpose of this study was to investigate how the PH grew during the course of the light adaptation (LA) process that follows a period of dark adaptation (DA): the so-called light adaptation effect (LAE). Methods: Photopic ERG luminance-response (LR) functions were obtained prior to (control-fully light adapted) and at 0, 5 and 10 minutes of LA following a 30-minute period of DA. A mathematical model combining a Gaussian and a logistic growth function, suggested to reflect the OFF and ON retinal contribution to the PH respectively, was fitted to the LR functions thus obtained. Results: Our results indicate that the magnitude of the cone ERG LAE is modulated by the stimulus luminance, with b-wave enhancements being maximal for luminance levels that result in the descent of the PH. The Gaussian function grew significantly with LA while the logistic growth function remained basically unchanged. Conclusion: Our findings would therefore suggest that the LAE reflects primarily an increase in the retinal OFF response during LA. Keywords: human electroretinogram (ERG); light adaptation effect (LAE); photopic hill; luminance-response function; b-wave. 56 INTRODUCTION The photopic, cone mediated, electroretinogram (ERG) represents the electrical potential evoked from the retina in response to a flash stimulus delivered against a rod desensitizing background light. The photopic ERG response thus reaches maximal amplitude when the retina is fully adapted to the predetermined photopic environment. This is best exemplified with the Light Adaptation Effect (LAE) that characterizes the gradual enhancement of the cone ERG amplitude observed during the course of light adaptation (LA) that follows a period of dark adaptation (DA). Nearly all ERG components [i.e. a-, b-, d- waves and oscillatory potentials (OPs)] are enhanced as a result of this process [1-5], with the b-wave demonstrating the most pronounced effect as it nearly doubles in amplitude within the first 15 minutes or so of LA [1, 3]. The magnitude of the LAE was also suggested to be stimulus dependent, the effect increasing with brighter flashes [2, 3, 6-8]. Although the LAE was first documented more than half a century ago [6, 9], the retinal mechanisms at its origin are not fully understood. Previously advanced explanations include: change in the standing potential of the retina [6], repolarization of the cones following their hyperpolarization by the adapting light [2] and change in the volume of retinal extracellular space [5]. Furthermore, given that nearly all the ERG components are affected, more than one retinal mechanism is likely to be involved in generating the LAE, a concept that was previously advanced by Murayama and Sieving [5]. In line with the above, Bui and Fortune [10] showed, with the use of selective blockers of postreceptoral transmission in rats, that most of the LAE originated in the pre-inner retinal layers, suggesting that the growth in b-wave amplitude observed during the LAE process simply reflected the concomitant enhancement of photoreceptor activity. However, contrasting with the latter view, Alexander et al. [11] reported a clear dissociation between a- and bwave intensity-response parameters in humans, suggesting that the cone redepolarization contributed minimally to the b-wave LAE. Moreover, although a period of DA must precede the recording of the light-adapted (cone dominated) ERG to reveal the LAE, fully dark-adapted rods do not appear to be required since a maximal LAE was reached after less than 5 minutes of DA [12]. Of interest, the cone ERG is also enhanced following a 57 rapid decrease in the intensity of the adapting background level but this effect is more transient than the LAE [13]. It would thus appear that growth of the photopic ERG is the normal response of the cone-mediated signal in reaction to an abrupt change in the level of retinal adaptation. However, the retinal mechanisms and/or pathways at the origin of this effect remain to be determined. The distinctiveness of the cone-mediated photopic ERG is also demonstrable with its unique luminance-response function. It is now well documented that with progressively brighter flash stimuli, the luminance-response (LR) function of the photopic ERG b-wave adopts a unique shape where the amplitude of the b-wave first increases, reaches a maximal value (Vmax) and then decreases gradually to finally form a plateau, explaining the term photopic hill originally coined by Wali and Leguire [14] to describe this unique LR function that was since then reproduced by several investigators [15-20]. Contrasting with the above, the amplitude of the a-wave grows gradually (and almost linearly) in response to the same increment in stimulus luminance [15-18, 20]. With the use of long duration stimuli, to separate ERG ON and OFF responses, Kondo et al. [15] showed that, compared to the ON response, the OFF response (d-wave) markedly decreased with brighter flashes, suggesting that a gradual inhibition of the OFF retinal pathway could be at the origin of the descent of the photopic hill. A subsequent study by the same group, using pharmacological agents to manipulate the retinal ON and OFF pathways of the primate ERG, provided evidence suggesting that the descent of the photopic hill could be explained by a combination of a gradually reduced ON component amplitude with a gradually delayed OFF component, both effects being enhanced with brighter intensities of stimulation [19]. More recently, Hamilton et al. [21] showed that the human photopic hill could be fitted to a mathematical equation that combined a Gaussian and a logistic growth function. Their results also suggested that the Gaussian and logistic growth functions reflected the respective contribution of the retinal OFF and ON pathways to the genesis of the photopic hill, thus offering a new means to test Ueno et al’s [19] claim. Given the above, the purpose of this study was to examine how the photopic hill grew during the course of the light adaptation effect (LAE) in order to determine if our 58 knowledge of the mechanisms underlying the photopic hill phenomena could help us identify those involved in generating the LAE. METHODS Preparation of subjects Experiments were performed on a total of 10 normal subjects (age: 17-26, mean: 20.6 ± 2.3 years; 7 women, 3 men) who signed an informed consent form, approved by the Institutional Review Board of the Montreal Children’s Hospital, attesting to their voluntary participation. All procedures followed the tenets of the Declaration of Helsinki. ERGs were recorded from both eyes with DTL fiber electrodes (27/7 X-Static silver coated conductive nylon yarn: Sauquoit Industries, Scranton, PA, USA) according to a method previously described by us [16]. Briefly, the pupils were maximally dilated with 1 % tropicamide after application of 0.5 % proparacaine hydrochloride drops. DTL electrodes were positioned deep into the inferior conjunctival bags and secured at the external and internal canthi of each eye with double-sided adhesive tape. Reference and ground electrodes (Grass gold cup electrodes filled with Grass EC2 electrode cream) were pasted at the external canthi and forehead respectively. ERG recordings The subjects faced a Ganzfeld of 30 cm in diameter that provided a white light rod desensitizing background of 30 cd.m-2 [in accordance with the 17-34 cd.m-2 recommended by the International Society for Clinical Electrophysiology of Vision (ISCEV) [22]. Subjects were first light-adapted to this background for 10 minutes in order to standardize the level of retinal adaptation following which photopic ERGs (bandwidth: 1-500 Hz; amplification: 20000 X; attenuation 6 dB) and OPs (bandwidth: 75-500 Hz; amplification: 50000 X; attenuation 6 dB) were recorded [LKC UTAS-E-3000 system (LKC Systems Inc., Gaitherburg, MD, USA)] simultaneously from both eyes prior to (control) and at 0, 5 and 10 minutes following a 30 minute period of DA (t=0, t=5 and t=10, respectively). In the control condition, ten responses were averaged for each of the 14 time-integrated [23] flash luminance levels presented (ranging from –0.8 to 2.84 log.cd.s.m-2 in 0.3 log-unit steps; flash duration: 20 ųs; interstimulus interval: 1.5 s.; prestimulus baseline: 20 ms). 59 Given the time necessary (approximately 2.5 minutes) to record a complete LR function and the anticipated growth in ERG amplitudes during LA, LR functions obtained during the LA process (i.e. t=0, t=5 and t=10) were generated with only 9 time-integrated flash luminance levels instead of 14 (-0.80, -0.41, -0.02, 0.17, 0.39, 0.64, 1.13, 1.63, 2.39). These flashes were presented in a random order to minimize the contribution of the LAE within each of the three LR functions. Background and flash luminances were calibrated with a research photometer (IL 1700; International Light, Newburyport, MA, USA). Data analysis Data analysis included peak time and amplitude measurements of the a- and b-waves and of the three major OPs (OP2, OP3 and OP4). The amplitude of the a-wave was measured from baseline to the most negative trough while that of the b-wave was measured from the trough of the a-wave to the most positive peak. Values from both eyes were averaged to yield a single data point and then normalized to account for normal interindividual variations in ERG amplitude. Data from the 10 subjects obtained at the three LA time intervals (t=0, 5 and 10 minutes respectively) were averaged to maximize our chance of demonstrating significant changes in photopic hill parameters during the LA process. OPs were measured only for the flash that evoked maximal OP amplitudes in the control photopic LR function, usually 0.90 log cd.s.m-2. The amplitude of each OP was measured from the preceding trough to the peak except for OP2 which was measured from baseline to the peak. OPs were reported both individually and collectively (SOPs= OP2 + OP3 + OP4). Peak times were measured from flash onset to the peak of each wave. Statistical analyses were performed using one-way within subject ANOVAs followed by Tukey’s post hoc test. Two separate methods were used to analyze the LR functions of the photopic ERG. In the first one [18], the following photopic hill parameters were measured: the maximal b-wave amplitude (Vmax), the time-integrated luminance of the flash needed to generate the Vmax response (Imax), the amplitude of the a-wave at Imax (amax), and the time-integrated luminance of the flash generating a b-wave equal to half the amplitude of Vmax on the ascending (Ka) and descending (Kd) limb of the photopic hill. 60 In the second method of analysis, photopic LR functions were fitted, by least squares regression, to a mathematical equation combining a Gaussian (G) and a logistic growth (LG) function [21]: R ln I B 2I V b max I Vb Gb R I b (Equation 1) where Vb is the b-wave amplitude (% of Vmax) for the corresponding time-integrated flash luminance I (cd.s.m-2), Gb is the maximal amplitude of the Gaussian function (% of Vmax), B is the width of the Gaussian (cd.s.m-2), R is the flash strength corresponding to Gb, Vbmax is the height of the logistic growth function (% of Vmax) and b is the flash strength (cd.s.m-2) corresponding to half the maximal amplitude of the logistic growth function. Data obtained from 28 normal subjects [18 from a previous study [18] and the control photopic hills obtained from the subjects reported in the present study] were averaged to yield the normal (mean ± S.D.) values for each of the photopic hill parameters mentioned above. The latter constituted the normative data that described the fully light-adapted photopic hill and against which the representative (averages taken from the 10 normal subjects, see above for details) photopic hills generated during the light adaptation process (i.e. at t=0, t=5 and t=10) were compared using z-score statistics. RESULTS Representative a- (top left) and b-wave (top right) photopic LR functions obtained from one subject prior to (control) and at 0 (t=0), 5 (t=5) and 10 (t=10) minutes following a 30 minute period of DA are shown in figure 1. Amplitude is expressed as a percentage of the maximal amplitude measured in control recordings. Representative ERG responses (from the same subject) are also illustrated at the bottom of figure 1. For all subjects, irrespective of the moment at which the LR function was sampled (control, t=0, t=5 and t=10), the awave always increased with brighter flashes while the b-wave always demonstrated the photopic hill phenomenon. Of note, the amplitude of the a-wave was minimally affected 61 by the light-adaptation process, with only 3 flash intensities yielding small but significant amplitude increments (0.39, 0.64 and 2.39 log cd.s.m-2, p<0.05). In contrast, the b-wave was significantly attenuated at t=0, especially for flashes stronger than Imax. Of interest, Vmax increased markedly as the retina light adapted. At figure 2 are illustrated mean a- and b-wave LR functions averaged from the amplitude values obtained from the 10 experimental subjects. Note that the overall morphology of the LR functions is comparable to the individual ones shown in figure 1 for both a- and bwaves. The photopic hill parameters obtained using Rufiange et al., [18] method (e.g. Vmax, amax, Imax, Ka and Kd) are reported in Table 1. The increase in Vmax with light adaptation was significant (p<0.0001), the amplitude being maximal for the control condition and followed, in order, by the t=10, t=5 and t=0 values. Post Hoc analyses revealed that t=0, t=5 and t=10 Vmax values were significantly different from control. Similarly, a significant (p<0.01) difference in Imax values was measured between control and t=0. Finally, there were no significant (p>0.05) light adaptation effect measured for amax, Ka and Kd parameters (table 1). In all instances, the peak time of the a-wave shortened with increasing time-integrated flash luminance (figure 3). However, for a given flash strength , the a-wave peak time remained the same despite progressive changes in the level of retinal adaptation (control, t=0, t=5 or t=10). In contrast, the peak time of the b-wave first increased with progressively brighter flashes to reach a maximal value and then decreased to finally form a plateau (figure 3, bottom). It is of interest to note that the morphology of this peak time LR curve is similar to that of the amplitude LR curve (i.e. the photopic hill). Of interest, the intensity needed to reach maximum values do not coincide, peaking at 0.39 log cd.s.m2 for the b-wave amplitude LR function compared to 1.13 log cd.s.m-2 for the peak time LR one. It is also at 1.13 log cd.s.m-2 that the LAE amplitude increment [{(amplitude at control - amplitude at t0) ÷ amplitude at t0} × 100] was maximal (figure 4). As shown at figure 4, the amplitude enhancement associated with the LAE varied with the timeintegrated luminance of the flash, suggesting that the kinetics of the cone ERG LAE is modulated by the strength of the flash. In fact, the increase in b-wave amplitude was 62 maximal for flashes slightly stronger than those required to yield Vmax and returned to baseline for ERGs found on the plateau of the photopic hill. It is of interest to note that the ERG that showed the largest increase in b-wave amplitude with light adaptation was that with the longest b-wave implicit time. Representative OPs obtained from one of the subjects in the four conditions of LA (control, t=0, t=5 and t=10) are shown in figure 5 and group data are reported in table 2. Post hoc analyses revealed that OP4 was significantly reduced at t=0, t=5 and t=10 compared to control, t=0 presenting the largest reduction followed by t=5 and t=10. Similarly, SOPs amplitudes at t=0 and t=5 were also significantly reduced from control condition (p<0.05), but not at t=10 (p>0.05). Finally it is only at t=0 that OP2 and OP3 were significantly smaller than control (p<0.05). Curves obtained by fitting equation 1 to our experimental data are shown at Figure 6, while the values of the equation parameters (Gb, B, R, Vb max and b) are reported in table 3. The peak amplitude of Gaussian curve (Gb,) was significantly lower than control at t=0 (z=-3.46; p=0.0003) and t=5 (z=-2.05; p=0.02), but not at t=10 (z= -1.20; p=0.11). No significant differences were found for any of the parameters defining the logistic growth function. Thus, our results indicate that the LAE had a significant impact on the Gaussian but not on the logistic growth component of Equation 1. DISCUSSION The purpose of this study was to explore the retinal mechanisms at the origin of the gradual increase in amplitude of the ERG that occurs when the retina is exposed to a photopic background following a period of dark-adaptation, a phenomenon known as the light adaptation effect (LAE). To do so, we recorded photopic ERG luminance-response functions (i.e. photopic hills) at different time intervals following the onset of a rod desensitizing background light (0, 5 and 10 minutes) and compared them to the photopic hill recorded prior to the dark-adaptation period (control photopic hill). Our results strongly suggest that the LAE is mostly a post-receptoral phenomenon: the measured LAE effect being significantly more pronounced when assessed with post-receptoral ERG 63 components (b-wave and OP4) compared to the alleged photoreceptor component, the awave, where only 3 flash intensities yield small but significant amplitude increments (0.39, 0.64 and 2.39 log cd.s.m-2, p<0.05). Of interest, although it is generally well accepted that the corneal ERG a-wave reflects the hyperpolarization of the photoreceptors, previous reports also suggested that, under specific stimulating conditions, the corneally recorded a-wave can also conceal a second, cornea-negative potential, that would be generated at a more proximal site, most probably by the OFFhyperpolarizing bipolar cells (OFF-HBC) [24,25]. This postreceptoral contribution is said to be most evident in ERGs evoked to stimuli near the photopic ERG threshold, that is at intensities that are well within the clinical ERG range, such as those found on the ascending limb of the photopic hill and to which the 0.39 and 0.64 log cd.s.m-2 intensities referred to above belong. In contrast, the a-waves of ERGs evoked to brighter stimuli, such as those found on the descending limb of the photopic hill, would mostly (if not solely) reflect photoreceptor activation. Given the lack of a reproducible a-wave LAE, irrespective of the flash intensity used (e.g. only 3 intensities out of the 9 used yielded a small but significant a-wave LAE effect: 2 on the ascending and 1 on the descending phase of the photopic hill), our results would suggest that neither the “photoreceptor” awave nor it’s OFF-HBC contribution were reliably solicited with our LAE paradigm. In contrast, our results obtained with b-wave measurements strongly suggest that the b-wave LAE is essentially a retinal OFF pathway phenomenon, to which the OFF-HBCs are important contributors. At this point, it is difficult reconcile how the LAE could, on the one hand activate the OFF-HBC in such a way to yield a significant growth in the corneal b-wave and, on the other hand, not interfere with the genesis of the corneal ERG a-wave. Irrespective of the time during the LAE at which the photopic LR function was generated, the resulting function always exhibited a photopic hill-like morphology. However, as detailed in table 1, the Vmax and Imax were significantly reduced at t=0 compared to control and their values grew progressively as the retina light adapted, while Ka and Kd did not show significant changes. Previous studies have shown that light and dark-adapted LR functions can be similarly interpreted with measures of retinal sensitivity [K] and responsiveness [Vmax] [16-18, 20, 26, 27]. Our results would thus suggest that the amplitude growth obtained with the LAE results from a gain in retinal responsiveness 64 rather than a gain in retinal sensitivity, control Vmax amplitude (that obtained prior to the DA period) being reached only once the retina is fully light adapted. However, of interest, the magnitude of the amplitude gain between the t=0 and control values was found to be stimulus dependent. As exemplified at figure 4, there is a gradual increase in the magnitude of the amplitude gain with flash intensity to reach a maximum with ERG responses evoked by flashes of 1.13 log cd.s.m-2 in intensity, a value that is normally found on the descent of the photopic hill. Previous studies have also reported that the LAE was stimulus dependent [1, 2, 6-8]. However, in our hands a near linear LAE enhancement of the b-wave amplitude was only observed with ERGs evoked by weak and moderate flashes (i.e. the rising and descending phases of the photopic hill), while ERGs evoked by the strongest intensities (in the plateau region of the curve) did not yield a measurable ERG amplitude gain with LAE. The LR function for OP4 amplitude also has a ‘photopic hill’ morphology [16]. The current study demonstrates that OP4 also has a stronger LAE than the other OPs, its amplitude doubling between t=0 and control compared to approximately 30% increase for OP2 and OP3 (Table 2). Similarly, while after less than 5 minutes of light adaptation OP2 and OP3 have nearly recovered their control amplitudes, OP4 only reaches 70% of control (p<0.05). These results agree with previous findings that showed a more pronounced LAE for OP4 compared to OP2 and OP3 [1, 4, 5, 12]. Of interest, the timing of photopic OP4 was previously shown to be concomitant with the peak of the photopic b-wave [1, 28,29], and time-locked to a segment of the photopic ERG response that included remnants of the OFF response evoked by longer flashes [30,31], thus suggesting that the retinal pathways leading to the OFF ERG response could explain most of the LAE. Supportive of the latter claim, Hamilton et al. [21] showed that the photopic LR function from a patient affected with complete congenital stationary night blindness (CSNB-1) could be fitted with the Gaussian function only, with no significant contribution from the logistic growth function component of Equation 1. Given the abundant (anatomical, functional and molecular) literature that identified a synaptic malfunction between the photoreceptors and the ONdepolarizing bipolar cells (ON-DBC) to be at the origin of the CSNB-1 anomaly [32-40], Hamilton et al’s results [21] suggest that the logistic growth function of Equation 1 65 predominantly reflect the ON pathway contribution to the photopic hill. Consequently, given that only the Gaussian component of Equation 1 was significantly modified with light adaptation (Table 3 and Figure 6), our data suggests that the LAE is predominantly an OFF pathway effect. This claim would find support with Miyake et al’s [41] results which showed a normal LAE in CSNB-1 patients (e.g. ON retinal pathway anomaly with normal OFF). Along the same lines of thought, Ueno et al. [19] reported that the descent of the photopic hill resulted from a reduction in the contribution of the ON component to the ERG genesis along with a delay in the contribution of the OFF component for flashes with high luminance levels. It is of interest to note that OP4, the alleged remnant of the OFF response in short flash ERGs, is the only OP that sees its peak time increase significantly with brighter flashes [29]. Also, the present study showed that compared to control, OP4 is the OP which shows the most significant amplitude decrease at t=0 of the LAE. When examining Ueno et al’s [19] results, we see that the b-wave of ERGs evoked to a short duration stimulus represents a summation of ON and OFF-ERG components. Of interest, in responses evoked by the brighter flashes, the positive peak of the OFF response is delayed to such an extent that it can no longer summate with the positive ON response. This could explain the nearly inexistent LAE at the plateau phase of the Photopic hill: the OFF pathway contributing less to the building of the photopic ERG b-wave evoked by the brightest stimuli. The later is in accord with Alexander et al. [11] who noticed that the amplitude growth was significantly less for ERGs evoked by brighter flashes. Of interest, the morphologies of their dim and bright flash ERGs are, in the present study, similar to those found at the peak and at the plateau of the photopic hill respectively, thus giving further support to our claim. Our findings would thus suggest that the increase in b-wave amplitude with LAE is governed by the same retinal pathways as those involved in the descent of the photopic hill or the descent of the photopic b-wave, namely the OFF retinal pathway and how it contributes to the genesis of the photopic ERG b-wave. 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Zeitz C, van Genderen M, Neidhardt J, Luhmann UF, Hoeben F, Forster U, Wycisk K, Matyas G, Hoyng CB, Riemslag F, Meire F, Cremers FP, Berger W (2005) Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram. Invest Ophthalmol Vis Sci 46:4328-4335. 40. Morgans CW, Ren G, Akileswaran L (2006) Localization of nyctalopin in the mammalian retina. Eur J Neurosci 23:1163-1171. 41. Miyake Y, Horiguchi M, Ota I, Shiroyama N (1987b) Characteristic ERG flicker anomaly in incomplete congenital stationary night blindness. Invest Ophthalmol Vis Sci 28:1816-1823. 71 Captions to illustrations FIGURE 1 Representative LR functions for a-waves (upper left) and b-waves (upper right) are illustrated for a normal subject prior to (control) and at 0 (t=0), 5 (t=5) and 10 (t=10) minutes following a 30-minute period of DA. The four lower panels illustrate the corresponding ERG waveforms. On the left side of the control ERG waveforms, the timeintegrated luminance levels of the flashes are indicated in log cd.s.m-2. Background luminance was 30 cd.m-2. The a- and b-waves are indicated as a and b, respectively. The vertical arrow corresponds to the flash onset. Horizontal calibration: 20 ms; vertical calibration: 50 V. FIGURE 2 Mean SR functions for a- and b-wave amplitudes recorded prior to (control) and at 0 (t=0), 5 (t=5) and 10 (t=10) minutes following 30 minutes of DA. Values are means of the two eyes for 10 healthy adult volunteers. Error bars are not shown to ease comparisons among curves. FIGURE 3 LR functions for a- and b-wave peak times recorded prior to (control) and at 0 (t=0), 5 (t=5) and 10 (t=10) minutes following 30 minutes of DA. Values are means of the two eyes for 10 healthy adult volunteers. Error bars are not shown to ease comparisons among curves. FIGURE 4 LR functions for LAE amplitude enhancement of the photopic ERG are illustrated for awaves (○) and for b-waves (■) (mean 1 SD, n=10 subjects). Enhancement is measured 72 as the difference in amplitude between control and t=0, expressed as relative amplitude (% of t=0). Photopic hill phases are also indicated on the graph. FIGURE 5 Representative OPs obtained prior to (control) and at 0 (t=0), 5 (t=5) and 10 (t=10) minutes following a 30-minute period of DA. OPs were analyzed for the flash luminance giving the maximal amplitude of the three major OPs under the control condition (see text), which was 0.64 log cd.s.m-2 for the subject shown. OP2, OP3 and OP4 are indicated with the numbers 2, 3 and 4, respectively. Horizontal calibration: 25 ms; vertical calibration: 50 V. FIGURE 6 Curve-fitting of b-wave LR functions obtained prior to (control) and at 0 (t=0), 5 (t=5) and 10 (t=10) minutes following a 30 minute period of DA. Upper graph: lines are best fit of equation 1 to the data points (symbols). Lower graphs: decomposition of equation 1 into the Gaussian and the logistic growth function curves. 73 a-wave b-wave 120 100 80 60 control t=0 t=5 t=10 40 20 0 -1.0 -0.5 Amplitude (% of max) Amplitude (% of max) 120 control t=0 t=5 t=10 100 80 60 40 20 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 -0.6 -0.1 Time-integrated flash luminance (log cd.s.m-2) control t=0 0.4 0.9 1.4 1.9 2.4 2.9 Time-integrated flash luminance (log cd.s.m-2) t=5 t=10 2.39 1.63 1.13 0.64 0.39 b 0.17 a -0.02 -0.41 -0.80 50 20 Figure1 74 a-wave Amplitude (% of max) 120 control t=0 t=5 t=10 100 80 60 40 20 0 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time-integrated flash luminance (log cd.s.m-2) b-wave Amplitude (% of max) 120 control t=0 t=5 t=10 100 80 60 40 20 0 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time-integrated flash luminance (log cd.s.m-2) Figure 2 75 a-wave Peak time (msec) 20 control t=0 t=5 t=10 15 10 5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time-integrated flash luminance (log cd.s.m-2) b-wave Peak time (msec) 40 control t=0 t=5 t=10 35 30 25 20 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time-integrated flash luminance (log cd.s.m-2) Figure 3 76 amplitude increment Increment (% of t=0) 150 descending ascending phase phase a-wave b-wave 100 plateau 50 0 -50 -100 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time-integrated flash luminance (log cd.s.m-2) Figure 4 77 23 4 control t=0 t=5 50 t=10 25 Figure 5 78 Figure 6 79 Table 1: Main ERG Photopic Hill Parameters (Method 1) Light adaptation duration Control t=0 t=5 t=10 ANOVAs Vmax 100.0 (0.0)1,2,3 65.4 (8.8)2,3,4 82.0 (7.0)1,4 86.7 (8.2)1,4 p<0.0001 amax 60.1 (5.8) 47.8 (19.5) 47.6 (8.1) 54.8 (8.4) p>0.05 Imax 0.52 (0.13)1 0.29 (0.19)4 0.42 (0.14) 0.42 (0.14) P<0.01 Ka -0.17 (0.10) -0.27 (0.21) -0.22 (0.13) -0.21 (0.11) p>0.05 Kd* 1.29 (0.05) 1.07 (0.21) 1.31 (0.15) 1.23 (0.17) p>0.05 Values are means ( 1 SD) for the 10 subjects tested. Refer to the text for definition of the parameters presented. Vmax and amax are expressed in % of max whereas Imax, Ka and Kd are in log cd.s.m-2. Post Hoc analyses (Tukey): 1=different from t=0; 2=different from t=5; 3=different from t=10; 4=different from control; p<0.01. * Only 5 subjects presented with a measurable Kd at all 4 levels of light adaptation; therefore, the mean as well as the paired one-way ANOVA were performed for n=5 for this parameter. The other subjects were dropped for the analysis. 80 Table 2: Photopic OP amplitudes during LA Light adaptation duration Control t=0 t=5 t=10 ANOVAs OP2 14.0 (2.3)1 11.1 (2.8)2,3,4 14.0 (3.4)1 13.7 (3.5)1 p<0.05 OP3 16.9 (5.0)1 12.7 (4.8)3,4 16.1 (5.1) 16.9 (4.7)1 p<0.05 OP4 30.8 (7.5)1,2,3 14.9 (2.9)2,3,4 22.1 (5.1)1,4 23.7 (6.8)1,4 p<0.0001 Sum of OPs 61.7 (11.2)1,2 38.6 (8.2)2,3,4 52.1 (9.8)1,4 54.3 (11.1)1 p<0.0001 Values are means ( 1 SD) for the 10 subjects after different light adaptation durations. Amplitudes are given in V and light adaptation duration in minutes. OPs were measured for the time-integrated flash luminance giving the maximal amplitude of the three major OPs under the control condition (see text). Post Hoc analyses (Tukey) : 1=different than t=0; 2=different than t=5; 3=different than t=10; 4=different than control; p<0.05 81 Table 3: Curve fitting parameters for the photopic b-wave LR functions during LA (Method 2, Equation 1) Light adaptation duration Control T=0 T=5 T=10 Gb 63.008 (10.372) 27.12841 41.72842 50.5192 B 1.555 (0.224) 1.4564 1.6328 1.4692 R 3.222 (0.834) 2.0670 2.7941 2.6357 Vbmax 42.589 (8.049) 33.1156 40.1781 38.7707 b 0.279 (0.106) 0.2214 0.3557 0.3172 In the control condition, parameters are mean (SD) obtained from a representative sample of 28 normal subjects. At t=0, t=5 and t=10, ten subjects were tested. Refer to the text for definition of the parameters presented. Gb, and Vbmax are expressed in % of max whereas B, R and b are in cd.s.m-2 z-score: 1 different from control, p<0.0005; 2 different from control, p<0.05. 82 Chapter IV Study 3 Estimating the ON and OFF retinal pathways function with the Glasgow equation: Normative data and clinical application The previous study provided evidence supporting that the model proposed by Hamilton & al. (2007) to fit the Photopic Hill function can be used to segregate the ON and OFF retinal responses. In the third study presented in this thesis, we further investigated this claim through analysis of the photopic b-wave luminance-response function in the diseased retina. This manuscript is still in preparation. However, I thought this thesis could still benefit from the incomplete version. 83 Estimating the ON and OFF retinal pathways function with the Glasgow equation: Normative data and clinical application Marie-Lou Garon, O.D.1, Pierre Lachapelle, Ph.D.1 1 Department of Ophthalmology & Neurology-Neurosurgery, McGill University – Montreal Children’s Hospital Research Institute. Short title: Photopic Hill Glasgow equation This manuscript contains 18 pages, 4 figures and 1 table. Last revision: September 25th, 2010 Grant information: This study was supported by McGill university-Montreal Children’s Hospital Research Institute, Canadian Institutes of Health Research (MOP-14639) and FRSQ-Réseau-Vision. Address for correspondence: Pierre Lachapelle, Ph.D. Department of Ophthalmology (D-164) McGill University - Montreal Children’s Hospital Research Institute 2300 Tupper Street Montreal, Quebec, Canada, H3H 1P3 Tel : 514-412-4400 ext 23890 Fax : 514-412-4331 E-mail : [email protected] 84 ABSTRACT With progressively brighter stimuli, the b-wave of the photopic ERG first increases in amplitude to a maximal value and then decreases to finally form a plateau, a phenomenon known as the Photopic Hill (PH). A mathematical model combining a Gaussian function and a logistic growth function was developed by a team from Glasgow (Hamilton & al., 2007) to fit this unusual luminance-response curve. The same study suggested that the logistic growth function represents the ON response, and the Gaussian the OFF response of the retina. In the present paper, we tested this claim by comparing luminance-response functions of normal subjects and patients affected with selected retinal disorders (CSNB1, CPCPA and RP) using this new method of analysis. PHs obtained from CSNB-1 patients showed a normal Gaussian, but a significantly reduced logistic growth function; the opposite was observed for the CPCPA patients. Patients affected with RP, however, showed variable contributions from each function. Given that CSNB-1 presents with selective dysfunction of the ON pathway while CPCPA is thought to have a defect of the OFF pathway, our results support Hamilton’s claim. The model could thus be useful to discriminate between ON and OFF pathway anomalies. However, further studies will be needed to determine its utility in studying progressive retinal diseases like RP. Keywords: human electroretinogram (ERG); Photopic Hill; luminance-response function; b-wave; modelization. 85 INTRODUCTION In response to progressively brighter flash stimuli, the amplitude of the short flash ERG photopic b-wave first increases to a maximal value, then decreases with further increments in flash luminance until it reaches a plateau (Wali & Leguire, 1992, 1993; Kondo & al. 2000, Rufiange & al. 2002, 2003, 2005; Ueno, 2004). This unique luminance-response function was named Photopic Hill by Wali and Leguire (1992) due to its particular shape reminiscent of a hill. The most probable explanation for the decrease of the photopic b-wave amplitude at higher flash intensities came from Ueno & al. (2004) who showed a decreased ON-component amplitude as well as a delayed OFFcomponent with brighter flashes in primates treated with NMDA+TTX or APB. While the scotopic b-wave luminance-response function can easily be fit using a sigmoidal curve known as the Naka–Rushton equation (Naka & Rushton, 1966), the particular shape of the Photopic Hill makes it much more difficult to analyze. A first method was proposed by Rufiange & al. (2003) who showed that the function can be analyzed using 7 easily identifiable and reproducible parameters: the maximal b-wave amplitude (Vmax), the amplitude of the a-wave at the Vmax intensity (amax), the flash intensity needed to generate the Vmax response (Imax), the stimuli intensities generating a b-wave half the amplitude of Vmax on the ascending (Ka) or descending (Kd) limb of the photopic hill, the ratio of the amplitude of the b-wave over that of the a-wave measured at Vmax intensity (b/amax) and the intensity of stimulation needed to generate an ERG where the amplitude of the b-wave equals that of the a-wave (Ka=b). Using this method of analysis, the investigators demonstrated the clinical usefulness of the Photopic Hill in various retinal disorders such as congenital stationary night blindness (CSNB), cone anomaly and pigmentary retinopathy. More recently, Hamilton & al. (2007) proposed a mathematical model combining a Gaussian and a logistic growth functions to fit the Photopic Hill luminance responsefunction. This model is defined with five independent parameters (Gb, B, R, Vbmax, b) as illustrated and described in Figure 1. The study showed accuracy in fitting of normal subjects. Furthermore, application of the model to a complete congenital stationary night 86 blindness (CSNB-1) patient’s luminance-response function revealed an almost nonexistent logistic growth component. Based on both electrophysiologic (Miyake, 1987; Quigley, 1996; Langrova, 2002) and molecular (Pusch, 2000; Bech-Hansen, 2000; Dryja, 2005; Zeitz 2005; Morgans, 2006; Slaughter, 1985; Nakajima, 1993; Gregg, 2007; Bahadori, 2006) evidences, it is well accepted that CSNB-1 is a disorder affecting the ON retinal pathway, with a normal OFF pathway. Indeed, reduced b-waves (ON responses) but normal d-waves (OFF responses) were reported in these patients using long flash photopic ERGs (Miyake, 1987; Quigley, 1996; Langrova, 2002). Hamilton & al. (2007) thus suggested that the logistic growth component of the equation could be associated to the ON retinal response, while the Gaussian could represent the OFF retinal reponse. If their assumption is correct, the Glasgow Photopic Hill equation could be a useful clinical tool to diagnose ON and OFF pathway disorders and to better understand the contribution of the ON and OFF pathways in progressive retinal diseases. The purpose of this paper was to determine if the modelization of the photopic short flash b-wave luminance-response function using the Glasgow equation could effectively allow for discrimination between the ON and OFF retinal pathways function. To do so, we used this new method of analysis to compare luminance-response functions of patients affected with various retinal disorders. METHODS Subjects/Patients ERGs from a total of 21 patients [3 congenital stationary night blindness type 1 (CSNB1), 4 congenital postreceptoral cone pathway anomaly (CPCPA) (Lachapelle & al., 1998; Garon & al., 2008) and 14 retinitis pigmentosa (RP)] were obtained from the Montreal Children’s Hospital Electrophysiology Clinic. All patients or their parents signed an informed consent form, approved by the Institutional Review Board of the Montreal Children’s Hospital. A complete ophthalmological examination was performed by experienced ophthalmologists prior to electrophysiological testing. All procedures followed the tenets of the Declaration of Helsinki. ERGs were recorded from both eyes with DTL fiber electrodes (27/7 X-Static silver coated conductive nylon yarn: Sauquoit 87 Industries, Scranton, PA, USA) according to a method previously described by us (Rufiange & al., 2003) and used routinely at the Montreal Children’s Hospital Electrophysiology Clinic. Briefly, the pupils were maximally dilated with 1 % tropicamide after application of 0.5 % proparacaine hydrochloride drops. DTL electrodes were positioned deep into the inferior conjunctival fornix and secured at external and internal canthi of each eye with double-sided adhesive tape. Reference and ground electrodes (Grass gold cup electrodes filled with Grass EC2 electrode cream) were pasted at the external canthi and forehead respectively. ERG recordings The subjects faced a Ganzfeld of 30 cm in diameter that provided a white light rod desensitizing background of 30 cd.m-2 as recommended by the International Society for Clinical Electrophysiology of Vision (Marmor & al., 2009). Subjects were first lightadapted to the background for 10 minutes in order to standardize the level of retinal adaptation. Photopic ERGs (bandwidth: 1-500 Hz; amplification: 20000 X; attenuation 6 dB) were evoked to flashes of white light using the LKC UTAS-E-3000 system (LKC Systems Inc., Gaitherburg, MD, USA)]. Five to fifteen responses were averaged for each of the 11 flash luminance levels presented (ranging from –0.8 to 2.84 log.cd.s.m-2 in 0.3 log-unit steps; flash duration: 20 μs; interstimulus interval: 1.5 s.; prestimulus baseline: 20 ms). Background and flash luminance were calibrated with a research photometer (IL 1700; International Light, Newburyport, MA, USA). Data analysis Data analysis included peak time and amplitude measurements of the a- (not shown) and b-waves for each photopic ERG response. The amplitude of the a-wave was measured from baseline to the most negative trough while that of the b-wave was measured from the trough of the a-wave to the most positive peak. For all subjects, except for RP patients, responses from both eyes were averaged to yield a single data point. For RP, since the disease is progressive and often asymmetric, eyes were analyzed separately. Values obtained were then normalized to ease Photopic Hill morphology comparison between diseased and normal retina. The photopic ERG b-wave luminance-response functions of 88 these patients were then fit using the mathematical model proposed by Hamilton & al. (2007) and parameters thus obtained were compared to those of a group of 28 healthy individuals (age: 17-43, mean: 26.7; 15 F, 13 M) tested in the same conditions (data from study 2). Statistical analysis consisted of one-way ANOVAs, followed by Tukey’s post hoc tests. In order to compare the relative contribution of the ON and OFF components to the ERG response, we also calculated the GL ratio as previously described: [GL=Gb/(Gb+Vbmax)] (Garon & al., 2007, 2008). RESULTS Figure 1 illustrates the Glasgow Photopic Hill equation applied to the photopic b-wave luminance-response function of a representative normal subject. The Photopic Hill curve is represented in green, whereas its decomposed components, the Gaussian and the logistic growth functions, are traced respectively in blue and purple. This color scheme is also used in figure 4. The parameters of the equation, namely Gb, R, B, Vbmax and σb, are described on the right end side of the figure. In figure 2, representative ERG and OPs waveforms obtained from two of our patients affected respectively with CSNB-1 (left) and CPCPA (right) are compared to waveforms from a normal subject (center). For each patient, the waves are presented in the following order from top to bottom: photopic ERG, photopic OPs, scotopic ERG and scotopic OPs. The photopic ERG of the CSNB-1 patient exhibits the characteristic “square” a-wave (Heckenlively, Martin & Rosenbaum, 1983; Lachapelle, Little & Polomeno, 1983) due to the absence of OP2 and OP3 as revealed by the OPs waveform. The scotopic ERG is somehow unusual, showing a rather small b-wave. Furthermore, while OP3 is missing as expected, OP2 does not seem attenuated as one would expect. The signal, however, is contaminated by significant noise, which could explain the presence of an apparent early OP. The photopic ERG of the CPCPA patient also differs from the normal ERG in that the signal lacks OP4 in both photopic and scotopic conditions, while OP2 and OP3 are easily identifiable, characteristic features previously reported (Lachapelle & al, 1998). Waveforms of RP patients are not shown, but were characteristic of the disease, with 89 severely reduced scotopic ERGs and moderate to markedly reduced photopic ERGs (bwave ranging from 15 to 70 V), according to the stage of the disease. Application of the Glasgow model to representative patients and normal subjects is shown in figure 3. In accordance with previous data published by Hamilton & al. (2007), CSNB1 patients presented with a PH composed of a large Gaussian and a significantly reduced logistic growth function whereas patients affected with CPCPA showed opposite results. Effectively, the decomposition of their PH revealed a larger than normal logistic growth function and a significantly reduced Gaussian function. This is reflected in GL ratios significantly higher than normal (0.60±0.08) for CSNB-1 (0.80±0.07) and significantly lower for CPCPA (0.33±0.06). Note the small standard deviation for both groups of patients, indicating that all patients within each group showed fairly similar results. In fact, although there were only 3 CSNB-1 patients and 4 CPCPA patients, their SD values were comparable to that of the group of 28 normal subjects, reinforcing the accuracy of the trends presented in this study. On the other hand, RP patients showed a broad range of PH morphologies and consequently, of GL ratios, as illustrated at the bottom of figure 3. As reported in Table 1, GL ratio values varied from 0 to 0.77 for RP patients. No correlation was observed between the maximal b-wave amplitude and the GL ratio. Furthermore, we noted significant variations in PH morphology and GL ratio value between OD and OS of individual patients despite minimal variations in maximal b-wave amplitude between both eyes. Figure 4 better exemplified the discrepancy between the GL ratio distributions of the different groups examined in this study. Normal subjects presented with a distribution close to a normal curve, with ratios distributed almost equally on each side of the mean. In contrast, the CSNB-1 ratios were concentrated at the far right of the graph while the CPCPA ratios occupied the left side. The RP ratios were spread throughout the whole range of values. 90 Discussion The purpose of this study was to evaluate the clinical usefulness of the Glasgow Photopic Hill equation proposed by Hamilton & al (2007). To do so, we applied the proposed mathematical model to patients affected with stationary retinal ON/OFF pathways disorders and retinitis pigmentosa (RP), a well-known progressive retinal disorder. Our results clearly demonstrate that retinal pathologies affecting specifically the ON or OFF retinal pathway can be distinguished using the Glasgow model. Effectively, all 3 patients affected with CSNB-1 presented with a PH mainly composed of a Gaussian function, while the 4 patients with CPCPA showed a large logistic growth function and a significantly reduced Gaussian function. These findings support Hamilton’s claim that the Gaussian component of the model reflect the OFF retinal response whereas the logistic growth function reveal the ON retinal response. Rufiange & al. (2003) analyzed patients affected with similar diseases with their PH analysis method. Of interest, they reported normal parameters (Ka, Vmax and Imax) for the ascending phase of the PH in CSNB-1. However, the descending phase of the PH was in the lower limit of normal values, and the plateau was significantly lower than normal. This correlates with a decreased logistic growth function, i.e. an abnormal ON response. Furthermore, they reported an abnormal PH ascension for the "suspected cone anomaly" patients, a finding consistent with an abnormal Gaussian or, in other words, an abnormal OFF response. In two studies, Rufiange & al, (2002, 2003) suggested that the "push-pull" concept presented by Sieving, Murayama and Naarendorp (1994) to explain the amplitude of the photopic b-wave could also applied to the Photopic Hill descent. Briefly, this concept stipules that with light activation, the ON-depolarizing bipolar cells push the amplitude of the b-wave towards its maximal amplitude, while the OFF-hyperpolarizing bipolar cells pull it back to its baseline value. Rufiange & al. proposed this push-pull interaction to be dependent upon the stimulus intensity, the pull effect being stronger in the descending phase of the PH. However, according to this theory, as they acknowledge themselves in one of the studies (Rufiange & al., 2003), we would expect to observe an abnormal ascending phase in CSNB-1 and an abnormal descending phase in cone anomaly or CPCPA, when we actually observe the opposite. Since this study was published, Ueno & al. (2004) published a convincing study attributing the descent and plateau of the PH to the 91 combination of progressively decreasing ON responses and delayed OFF responses with brighter flashes. The Gaussian is compatible with Ueno’s hypothesis. Effectively, the descending phase of the Gaussian function could very well correspond to the gradual decreased contribution of the OFF response to the b-wave peak as the OFF response peak time increases. The logistic growth function, on the other hand, shows increasing ONresponse amplitudes with progressively brighter flashes, a finding that goes against Ueno’s demonstration. As of now, we do not have a justification for this discrepancy. The results obtained for RP patients, on the other hand, did not show a characteristic pattern for the ON/OFF contribution, as highlighted by the broad range of GL ratios obtained for this group of patients. As shown in figure 2, PHs of RP patients can be of the CSNB-1 type, normal type of CPCPA type. This suggests the idea that RP could progress in three different ways: 1- early loss of ON response; 2-equal loss of ON and OFF responses; 3-early loss of OFF response. Given the fact that rods are affected early in the course of the disease compared to cones and that rods connect only to ON-bipolar cells, one may have expected to see a PH of the CSNB-1 type in patients with relatively preserved b-wave amplitudes. However, it did not seem to be necessarily the case with our patients. That being said, a limited number of patients were involved in the study, making it difficult to show a trend and most patients showed a maximal b-wave already significantly reduced, suggesting that cones are already affected. A prospective study following a large group of patients from the early stages of the disease up to when their photopic ERG is almost extinguished would certainly be the best way to determined if the stage of the disease is determinant for the GL ratio. However, this type of study is not easy to carry and it would take years to obtain conclusions. Comparison of large groups of patients at different stage of the disease could be another option, but given the fact that RP can be caused my mutations on several genes (Rivolta & al., 2002), that would add another factor susceptible to impact how the ON and OFF pathways are affected. Genetic testing would also be necessary in that case, which can imply major costs. 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Graph on the left shows the decomposition of the function (green) into the logistic growth (purple) and the Gaussian (blue) components. The 5 parameters describing the function are also represented with their definition to the right. FIGURE 2 Representative CSNB-1, normal and CPCPA flash ERG and OPs waveforms. From top to bottom: photopic ERG, photopic Ops, scotopic ERG, scotopic Ops. Flash luminance (log cd.c.m-2) is indicated at the left of each tracing. Amplitude is expressed in μV and time in ms. OP2, OP3 and OP4 are indicated as 2, 3 and 4, respectively. FIGURE 3 Application of the Glasgow model to representative patients and normal subjects. PH: green; LG: purple; G: blue. Mean GL ratios ±SD for normal subjects, as well as CSNB-1 and CPCPA patients are reported under their respective graphs. Chosen RP PHs are illustrated on the bottom row with their corresponding GL ratios. FIGURE 4 GL ratio distribution for normal subjects and patients (CSNB-1, CPCPA, RP). Abscissa: ratio; ordinate: number of patients (normal subjects, CSNB-1 and CPCPA) or number of eyes (RP). 97 R ln I B 2I Vb max I Vb Gb I b R Gaussian Gb Vbmax ½Vbmax b R Logistic Growth Gb : maximal Gaussian amplitude R : flash luminance at which Gb occurs B : width of the Gaussian where G is its SD Vbmax : maximal saturated logistic growth amplitude b : flash luminance which evokes ½Vbmax I : flash luminance Figure 1 98 CSNB-1 Normal CPCPA 25 V +0.64 +0.64 +0.64 4 25 ms 25 V 2 4 2 25 ms 25 ms 3 +0.64 3 10 V 10 V +0.64 25 V 25 ms 25 ms 25 ms +0.17 +0.17 25 V +0.17 10 V +0.64 25 V 25 V 3 25 ms 2 2 4 25 ms 3 4 10 V +0.17 10 V +0.17 25 ms 25 ms 2 25 ms 10 V +0.17 25 ms Figure 2 99 Figure 3 100 Normal subjects CSNB patients 18 2 14 Number of patients Number of patients 16 12 10 8 6 4 1 2 0 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0 0,9 0 0,1 0,2 0,3 Ratio 0,4 0,5 0,6 0,7 0,8 0,9 0,6 0,7 0,8 0,9 Ratio CPCPA patients RP patients 3 8 Number of eyes Number of patients 7 2 1 6 5 4 3 2 1 0 0 0 0,1 0,2 0,3 0,4 0,5 Ratio 0,6 0,7 0,8 0,9 0 0,1 0,2 0,3 0,4 0,5 Ratio Figure 4 101 Table 1: Patients demographics and individual GL ratios Patient Gender Age Diagnosis Max b-wave amplitude ID OD OU OS 01 M 14 CSNB-1 189 02 M 14 CSNB-1 62 03 M 17 CSNB-1 83 04 F 9 CPCPA 72 05 F 21 CPCPA 38 06 F 16 CPCPA 59 07 M 14 CPCPA 36 08 M 29 Sporadic RP 33 38 09 F 35 AD RP 15 19 10 F 26 RP 29 24 11 F 56 RP sine pigment 69 63 *12a F 44 RP sine pigment 31 48 *12b F 41 RP sine pigment 59 35 13 M 13 Usher syndrome 21 49 14 F 54 RP 43 47 15 F 71 RP 35 55 16 M 66 RP 50 60 17 M 55 RP 15 18 18 M 30 RP 32 27 19 F 34 RP 8 26 20 F 52 RP 16 19 31 44 21 F 37 RP *The luminance-response function of patient 12 was analyzed at 3 year interval OD 0.46 0 0.28 0.69 0 0.67 0.48 0.52 0.21 0.38 0.30 0.29 0.47 0.27 0.54 GL ratio OU 0.83 0.85 0.72 0.29 0.23 0.41 0.35 OS 0.56 0 0 0.51 0.77 0.72 0.50 0.67 0.30 0.45 0.57 0.44 0.50 1.0 0.41 102 Chapter V General discussion and conclusion The three manuscripts included in this thesis added to the previous descriptions of the luminance-response function of the photopic ERG, in both normal and pathological retinae. We were the first, to our knowledge, to describe in detail the progressive changes in waveform and compare spectral and white flash ERG for very dim intensities as presented in the first study. Tsuruoka & al. (2004) did look at spectral ERGs evoked to blue and red over a 3-log intensity range and obtained results similar to ours, but they did not compared the waveforms to white flash ERGs, nor did they make a link between the S-cone response and the i-wave. Miyake & al. (1985) previously showed an absence of Scone responses in patients with a homozygous defect in the S-cone opsin gene. It would be of interest to record white flash ERGs on a patient with such a defect in order to validate our claim that the i-wave and the S-cone response are from the same origin. If it is indeed the case, a patient lacking S-cones should also lack an i-wave. However, such patients are rare, and we did not find reports of a patient with a similar defect in our patient database. On the other hand, patients affected with glaucoma are common. As reported in our first study, Drasco & al. (2001) showed a significant attenuation of the Scone in patients affected with early POAG. Again, if our i-wave/S-cone claim is valid, we should observe a reduction of the i-wave in those patients who present with a significantly reduced S-cone. Testing of such patients with the protocol used in our first study would certainly be an interesting venue to explore in the future. Of interest, Rufiange & al. (2002) showed that the i-wave also exhibited a Photopic Hill-like luminance-response function. Could the Glasgow equation also be used to analyze the i-wave luminanceresponse function and add to our knowledge of the retinal interactions behind the i-wave as it did with the LAE? Indeed, the modelization of the Photopic Hill function with the Glasgow equation has provided additional information regarding the origin of the light adaptation effect (study 103 2). Changes in the Photopic Hill equation parameters as the retina light adapts corroborated with oscillatory potentials findings. Effectively, in both cases, components associated with the OFF response (Gb, OP4) in previous publications (Hamilton & al. 2007; Lachapelle & al, 1998) were markedly affected at background onset and slowly grew back to normal amplitude with light adaptation. Similarly, OP2 and OP3, as well as Vbmax (although not significantly), elements thought to represent the ON response, showed a decrease in their amplitude at t=0, but quickly recovered to their normal amplitude after less than 5 minutes. These findings are in line with previous studies that suggested two distinct processes (Peachey & al, 1992; Benoit & Lachapelle, 1995) at the origin of the LAE, the OFF pathway process explaining, however, most of the LAE observed with the PH. This discrepancy in the ON and OFF pathways during the LAE provided additional clues pointing to the logistic growth and the Gaussian functions as representing respectively the activity of the ON and OFF pathways. The last study was intended to look at this last point. The modelization of the Photopic Hills obtained from patients with retinopathies involving specifically the ON and OFF retinal pathways, namely CSNB-1 and CPCPA, confirmed Hamilton’s claim. However, if CSNB-1 presents with a complete abolition of the ON pathway, one would think that the logistic growth function would be completely absent from the PH. Nonetheless, a reduced, but present logistic growth function was obtained for all three subjects analyzed. Similarly, the Gaussian function was not completely abolished in CPCPA patients. This could suggest that the Gaussian and the logistic growth functions may not represent pure OFF and ON responses, although the results suggest a strong association between each function and the OFF or ON pathways, respectively. One of the weaknesses of our investigations was the lack of molecular evidence to support our hypothesis. In that regard, it would be interesting to apply the Glasgow model to the PH of animals treated with chemicals blocking selectively the ON or OFF pathways. We are limited, however, in the animals to be used as only primates (Ueno & al, 2004) and guinea pigs (Racine & al., 2005) were reported to have a PH. Finally, our analysis also lacks data as to the potential clinical utility of the Glasgow equation to evaluate progressive retinal diseases. Further correlation between GL ratios 104 and disease stage or genotype/phenotype will be needed to explain the broad range of ON/OFF ratios obtained in patients suffering from retinitis pigmentosa. We already know that mutations on at least 75 different genes can cause RP (Rivolta & al., 2002). Also, the pattern of progression of the disease varies grandly between individuals, all reasons that could explain the differences observed in our study. If the project was to be continued, genetic testing as well as repeated PH recordings and analysis over several years and even decades, would probably be the best way to approach the problem. However, during the course of this study, we also realized that the model has its limitations as it was impossible to fit luminance-response function for nearly extinguished ERG (10-15 V or less). We thus have to develop new methods of analysis for ERGs of low amplitude. In that matter, our team is already exploring new avenues to assess this challenging problem, one of them being the Continuous Wavelet Transform analysis (Jauffret & al., 2008). 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In this first study, Marianne Rufiange and I performed all the ERG recordings together. Marianne Rufiange constructed most of the figures for this study, some of which I modified for the manuscript. I produced the remaining figures myself, analyzed the results and wrote the manuscript. Dr Lachapelle helped in revising the manuscript. Manuscript 2 (Chapter 3) Garon ML, Rufiange M, Hamilton R, McCulloch DL, Lachapelle P. Asymmetrical growth of the photopic hill during the light adaptation effect In this second study, I performed all the ERG recordings and data analysis by myself and wrote the manuscript. Dr Marianne Rufiange, then a PhD student, taught me the rudiments of ERG recordings. Drs Ruth Hamilton and Daphne L. McCulloch provided the mathematical model used to fit the Photopic Hill function and helped in revising the manuscript along with Dr Pierre Lachapelle. Manuscript 3 (Chapter 4) Garon ML, Lachapelle P. Estimating the ON and OFF retinal pathways function with the Glasgow equation: Normative Data and Clinical Applications. In this third study, either Marianne Rufiange or myself performed the ERG recordings for the normal subjects. The pathological ERGs were obtained from the database of the Montreal Children’s Hospital Electrophysiology Clinic. Marianne Rufiange, Julie Racine and Allison Dorfman, as technicians working in the clinic, performed the ERG recordings on the patients. I analyzed all the data by myself and wrote the manuscript. Dr Pierre Lachapelle helped in revising the manuscript.