Download The Photopic ERG Luminance-response Function

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
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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
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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.
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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.
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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.
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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.
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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).
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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]
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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).
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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
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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. The photopic
hill combined with the LAE could therefore represent new investigative means to assess,
in a dynamic way, the functional integrity of the ON and OFF retinal pathway with the
use of the short flash ERG paradigm, which is that most frequently used clinically.
66
Acknowledgements: This study was supported by McGill University-Montreal Children’s
Hospital Research Institute, the Canadian Institute for Health Research, the Foundation
Fighting Blindness (USA) and FRSQ-Réseau-Vision.
67
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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.
In summary, use of the GL ratio distinctly segregated normal subjects from patients
affected with stationary retinopathies, presumably by quantifying the retinal ON and OFF
92
pathway contribution to the building of the short flash ERG. Normal subjects, CSNB-1
and CPCPA patients presented with typical GL ratios, while RP patients showed a broad
range of ratios. Clearly more research is needed to determine the significance of the
Glasgow model analysis in progressive retinal disorders.
93
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Captions to illustrations
FIGURE 1
Glasgow equation fitting of the Photopic Hill. 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).
In conclusion, the photopic luminance-response function proved to be useful in studying
both normal and pathological human retinal physiology. Although some questions
remained unanswered concerning the Glasgow equation, it would be relevant to include
luminance-response recording in routine electrophysiologic evaluation, especially since it
is very easy to incorporate to testing protocols.
105
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APPENDIX
CONTRIBUTION OF AUTHORS ON CO-AUTHORED PAPERS
Manuscript 1 (Chapter 2)
Garon ML, Rufiange M, Lachapelle P. Spectral electroretinography : What does the
luminance-response function reveal?
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.