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FURTHER CHARACTERIZATION OF THE RETINOPATHY,
GLOBE ENLARGED (rge) CHICKEN

By

Gillian Curtis Shaw

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Comparative Medicine and Integrative Biology

2007

ABSTRACT

FURTHER CHARACTERIZATION OF THE RETINOPATHY, GLOBE ENLARGED
(rge) CHICKEN

By

Gillian Curtis Shaw

Previous work has shown that retinopathy, globe enlarged (rge) affected chicks
eventually become functionally blind and subsequently develop globe enlargement.
Electroretinographic (ERG) abnormalities include increased thresholds, a “supernormal
b-wave” in response tO brighter flashes, a lack of oscillatory potentials and ERG
responses that are present long after functional blindness. Screening of several positional
candidate genes was performed; however, another research group identified the causative
mutation in GNB3, the [3 subunit Of a heterotrimeric G-protein involved in
phototransduction in cone photoreceptors. Pharmacological dissection of the rge ERG
using aspartate, APB and PDA demonstrated decreased cone sensitivity but also
suggested abnormalities of the inner retinal ON and OFF pathways. GNB3 has not
definitively been shown to be involved in inner retinal pathways, however these ERG
results suggest it may play a role in such pathways. Preliminary immunohistochemistry
identified GNB3 immunoreactivity in cone cell bodies, outer plexiform layer synaptic
terminals and a population of cells in the inner nuclear and ganglion cell layers in normal
chicken retina. These data suggest GNB3 may also be important in inner retinal

signaling. Further studies are necessary to explore this hypothesis.

ACKNOWLEDGEMENTS

I would like to acknowledge all of the people who not only facilitated my getting
involved with the Comparative Ophthalmology Laboratory but also who helped me as l
tackled the work for this project. Dr. John Baker deserves a big thank you because he
facilitated the establishment of the NIH T-32 program at Michigan State University for
veterinary students to take a year from vet school and work toward a Master’s degree.
Without this program, I never would have pursued a Master’s degree. Dr. Vilma
Yusbasiyan-Gurkan, the director of the Comparative Medicine and Integrative Biology
program, was very supportive as I made my way through the course of this Master’s
project. And I never would have gotten involved with the lab had it not been for my
friend, Lexi Mentzer, who first introduced me to the lab and its various goings-on.

The folks in the Comparative Ophthalmology Laboratory were instrumental in my
successful completion of this project, and without their help I never would have made it.
Michelle Curcio patiently taught me how to do most of the molecular work included in
this project. She didn’t even seem to mind when I asked her how to dilute PCR primers
for the 97th time! Janice Forcier was an ever-present companion in the dark and expert
chicken wrangler, anesthetist and artificial inseminator. Who would have thought doing
ERGs at 2am would have been so MUCH FUN! Nalinee Tuntivanich was also a patient
teacher who willingly shared all of her vast ERG knowledge and expertise with me. She
always had a kind word and helpful advice for the various stumbling blocks I
encountered.

I would also like to thank Fabiano Montiani-Ferreira whose rge chicken torch I

continued to carry after he had finished his PhD work. He taught me quite a bit about the

iii

rge chicken and helped me along as I was getting started with the rge chickens. His PhD
thesis was my bible as I worked on this project and I slept with it under my pillow each
night. His statistical knowledge and willingness to help was greatly appreciated in the
interpretation of my results and the statistical analysis. He has become a good friend and
I will always consider him to be a mentor as I move onto new things in life.

My committee members, Drs. Hans Cheng, Matti Kiupel, Pat Venta and Art
Weber, also deserve acknowledgement as they helped to guide me through the process of
the research and the completion of this Master’s project. Special thanks go to Dr. Hans
Cheng whose advice and laboratory staff helped immensely with the molecular part Of
this project.

Lisa Allen and all of the Vivarium staff deserve a big thank you for taking care of
the precious rge chickens and putting up with all Of my crazy ideas to improve their
husbandry.

' My mom and dad, Jane and Darrel Shaw, must be mentioned as I would not be in
the position I am today without their love, encouragement and support.

Finally, I would like to thank Simon Petersen-Jones my principal investigator and
mentor, who not only took me on as a student but also patiently explained the principles
of ERGs and phototransduction at least 800 times throughout the course of this project. I
thank him for working around the difficulties I encountered as I completed this dual
degree program. He was ever patient as I struggled to understand the pathology behind
the rge chicken while also working towards my DVM. I find it unbelievable how far I’ve
come since I joined the lab in 2004 and I couldn’t have done it without Simon’s

encouragement and support. MUCH THANKS TO ALL!

iv

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vii

LIST OF FIGURES ............................................................................... viii
CHAPTER 1 — INTRODUCTION

1.1. Structure of the chicken globe ....................................................... 1

1.2. Structure and function of the retina ................................................. 3

1.3. Chicken retinal structure .............................................................. 6

1.4. Signaling within the retina ............................................................ 6

1.4.1. Guanine-nucleotide binding proteins .................................... 6

1.4.2. Visual transduction ......................................................... 7

1.4.3. ON and OFF pathways ................................................... 11

1.5. Retinal dystrophy models ........................................................... 13

1 .6. Electroretinography .................................................................. 15

1.6.1. The electroretinogram .................................................... 15

1.6.2. The origins of the ERG waves .......................................... 18

1.6.3. Light adaptation status and the electroretinogram .................... 25

1.6.4. Circadian ERGs ........................................................... 25

1.6.5. The ERG in disease states ................................................ 27

1.7. The rge chicken .................................................................. -. . ..27

1.7.1. Original rge characterization ............................................ 27

1.7.2. Globe enlargement ........................................................ 28

1.7.3. Lacquer crack lesions ..................................................... 31

1.7.4. Retinal changes ............................................................ 34

1.7.5. Vision testing .............................................................. 40

1.7.6. Electroretinographic characteristics ..................................... 41

1.7.7. Investigation of several candidate genes ............................... 48

1.8. Hypotheses ............................................................................ 49

1.9. The scope of this project ............................................................ 49

CHAPTER 2 — INVESTIGATION OF CANDIDATE GENES

2. 1. Introduction ........................................................................... 52
2.1.1. Background information for the chosen genes ...................... 55

2.2. Materials and Methods ............................................................... 58

2.2.1. Design of primers ......................................................... 58

2.2.2. DNA isolation ....................................... . ...................... 62

2.2.3. PCR amplification .................................... - ..................... 6 3

2.2.4. Purification of PCR products for sequencing ......................... 64

2.2.4.a. Sodium acetate and isopropanol PCR product
Purification ...................................................... 64
2.2.4.b. Purification of PCR products out from gel ................. 64

2.2.5; Amplification of genes from retinal cDNA ........................... 66

2.2.5.a. RNA Extraction ................................................ 69
2.2.5.b. cDNA Synthesis ................................................ 70
2.2.6. Analysis of sequencing results .......................................... 72
2.2.7. Investigation of SNPs identified in candidate genes ................. 72
2.2.8. Investigation of GNB3 ................................................... 72
2.2.8.3. Development of PCR RE test to genotype birds for
GNB3 mutation ................................................. 74
2.2.8.b. GNB3 immunoreactivity in the normal chicken
retina ............................................................. 77
2.2.8.b.i. Chicks ................................................ 77
2.2.8.b.ii. Retina Collection .................................. 77
2.2.8.b.iii. Fixation, Embedding and Staining ............. 78
2.3. Results .......................................................................... . ...... 79
2.4. Discussion ............................................................................. 90

CHAPTER 3 — FURTHER ELECTRORETINOGRAPHIC STUDIES OF THE rge

CHICKEN
3. 1 . Introduction ........................................................................... 94
3.2. Materials and Methods ............................................................... 96
3.2.1. Chicks ...................................................................... 96
3.2.2. ERG Recording ............................................................ 97
3.2.2.a. Long flash ERG ................................................ 98
3.2.3. Pharmacological dissection of the ERG ............................... 99
3.2.4. Circadian electroretinograms ........................................... 100
3.2.5. Data analysis ............................................................. 101
3.3. Results ................................................................................ 102
3.3.1. Long flash ERGS ......................................................... 102
3.3.2. APB Injection ............................................................ 104
3.3.3. PDA Injection ............................................................ 108
3.3.4. Aspartate Injection ....................................................... 112
3.3.5. Circadian ERGs ......................................................... 115
3.4. Discussion ........................................................................... 122

CHAPTER 4 — CONCLUSIONS AND FUTURE STUDIES

4.1. Conclusion ........................................................................... 128
4.2. Future studies ........................................................................ 135
REFERENCES .................................................................................... 138

vi

LIST OF TABLES

Table 2.1. Genes examined in this investigation .............................................. 54
Table 2.2. Genomic primers used to sequence the candidate genes ..................... 59-61
Table 2.3. cDNA primers used to sequence the candidate genes ........................ 67-68
Table 2.4. Sequence analysis results of the twelve genes examined ........................ 80
Table 2.5. Polymorphisms found during investigation .................................... 81-83
Table 3.1. Summary of short flash ERG protocol ............................................. 98

vii

LIST OF FIGURES

Figure 1.1. Histological and gross structural characteristics of the chicken eye ............ 2
Figure 1.2. A simple diagram of the organization of the retina ................................ 4
Figure 1.3. Plastic-embedded retinal section of a normal chicken at 270 days of age. . ....5

Figure 1.4. A simplified diagram of the rod phototransduction cascade and

visual cycle ......................................................................... 10
Figure 1.5. Synapses between rods, cones and their associated bipolar cells ............... 12
Figure 1.6. The ERG waves ....................................................................... 17
Figure 1.7. The long flash ERG .................................................................. 22
Figure 1.8. Short flash ERG with oscillatory potentials .................. , .................... 23
Figure 1.9. Commonly measured parameters of the ERG waveform ........................ 24

Figure 1.10. Gross ophthalmic photographs from representative rge and
control birds ................................................................................. 30

Figure 1.1 1. Posterior eyecup from a 49-day-old rge bird showing lacquer cracks ....... 32

Figure 1.12. Plastic and resin-embedded retinal sections from rge birds

demonstrating morphologic details of the lacquer crack lesions ............ 33
Figure 1.13. Semithin sections of outer retina .................................................. 35
Figure 1.14. Mislocalization of glycogen deposits ............................................. 37
Figure 1.15. EM images of photoreceptor synaptic terminals ................................ 39
Figure 1.16. Representative ERG responses from a control and an rge chick ............. 44

Figure 1.17. Light and dark adapted mean a-wave amplitudes and implicit times for 7

day old chicks ..................................................................... 45
Figure 1.18. Light and dark adapted mean b-wave amplitudes and implicit times for 7

day old chicks ..................................................................... 46
Figure 2.1. Area of interest on chicken chromosome one .................................... 53

viii

Figure 2.2. Schematic drawing of GNB3 cDNA with primers and mutation .............. 73
Figure 2.3. Restriction enzyme test to establish GNB3 status ............................... 76
Figure 2.4. GNB3 amino acid sequence alignment ............................................ 86
Figure 2.5. Results of the GNB3 RE test ........................................................ 87
Figure 2.6. GNB3 immunoreactivity in normal chicken retina .............................. 89
Figure 2.7. A computational model of chicken (Gallus gallus) GNB3 protein ............ 92
Figure‘3.1. Long flash ERG ...................................................................... 103
Figure 3.2. Mean long flash ERG wave amplitude comparisons ........................... 103
Figure 3.3. APB effect on short flash ERG ................................................... 106
Figure 3.4. APB effect on long flash ERG .................................................... 107
Figure 3.5. PDA effect on short flash ERG ................................................... 110
Figure 3.6. PDA effect on long flash ERG .................................................... 1 11
Figure 3.7. Aspartate effect on short flash ERG .............................................. 1 13
Figure 3.8. Aspartate effect on long flash ERG ............................................... 114
Figure 3.9. Mean dark-adapted circadian a-wave amplitudes .............................. 1 16
Figure 3.10. Mean dark-adapted circadian b-wave amplitudes ............................. 1 16
Figure 3.11. Mean light-adapted circadian a-wave amplitudes ................... ' .......... 118
Figure 3.12. Mean light-adapted circadian b-wave amplitudes ............................. 118
Figure 3.13. Mean dark-adapted circadian a-wave implicit times ...... ' .................... 120
Figure 3.14. Mean dark-adapted circadian b-wave implicit times .......................... 120
Figure 3.15. Mean light-adapted circadian a-wave implicit times .......................... 121
Figure 3.16. Mean light-adapted circadian b-wave implicit times ......................... 121

Figures in this thesis are presented in color.

ix

CHAPTER 1

INTRODUCTION

1.1. Structure of the chicken globe

The chicken eye, like many avian eyes, is proportionately larger than that of
mammals and takes up a larger volume of the head. The outermost wall of the globe is
made up of the avascular transparent cornea and the fibrous sclera. The sclera contains
ossicles (bones) rostrally and a cartilage cup caudally, both of which help to maintain the
shape of the eye. The uveal tract, the middle layer consists of the iris, ciliary body and
the choroid. The avian pupil, unlike the mammalian pupil, is controlled by striated
muscle as opposed to smooth muscle, and is responsible for regulating the amount of
light that is allowed to reach the retina. The choroid contains blood vessels responsible
for supplying nutrients to and removing wastes from the retina and the retinal pigmented
epithelium (RPE), which is associated with the outer segments of the photoreceptors. An
important job of the RPE is to remove spent portions of outer segments of the
photoreceptors as they are released. The RPE’s relationship to the photoreceptor outer
segments changes in a circadian rhythm. During the daylight hours, the RPE cells have
processes, which are particularly developed in the avian and fish retinas, that envelope
the outer segments; at night these processes retract. The innermost layer of the globe is
the neurosensory retina. It is responsible for the conversion of light energy into a
Chemical and then electrical signal (phototransduction), which, when sent to the brain,
allows visual perception. A unique feature of the avian eye globe is the pecten, which is

a highly vascular and pigmented structure projecting into the vitreous from the back of

the globe (Figure 1.1), whose complete functions have merely been hypothesized. The
most widely accepted theory of its function is that it helps to provide nutrients to the

retina (Meyer, 1977).

 

 
  

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Figure 1.1. Histological and gross structural characteristics of the chicken
eye. A) Paraffin-embedded cross retinal section of a chicken eye at 7 days of
age, the names of the main structures are indicated in the figure. Note the
comb-like appearance of the pecten, located anteriorly, overlying the optic
nerve head. Stain = hematoxylin/eosin. Bar - 2.5 mm. B) Gross photograph of
the posterior eyecup of a 60-day-old chicken eye after removal of the anterior
segment, lens and vitreous. The retina is the transparent layer covering the
eyecup. Note that the pecten projects into the inferior portion of the posterior
chamber. The dorsal part of the pecten is always tilted to the temporal aspect of
the eye. Thus, B shows a retinal cup from a right eye. Bar - 5 mm. Figure used
with permission from Montiani-Ferreira PhD Thesis 2004.

1.2. Structure and function of the retina

The retina lines the back of the eye globe. It is positioned between the vitreous
humor and the choroid. The layers of the retina from the outer to inner retina are as
follows: retinal pigmented epithelium (RPE), photoreceptors, outer limiting membrane
(OLM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer
(INL), inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer (NFL)
and inner limiting membrane (ILM) (Figures 1.2 and 1.3). The photoreceptor layer is
composed of the inner and outer segments (IS and OS) of the rods and cones. The outer
segments contain the photoactive pigments and phototransduction proteins that are
capable of converting light energy into an electrical signal.

Rods are capable of perceiving very low light levels but their responses become
saturated with bright lights. There are several different types of cones, which have lower
sensitivities than rods but do not become saturated as easily as rods and are responsible
for perceiving a broader range of wavelengths of light than rods, thus allowing for color
vision. The outer and inner limiting membranes are, in fact, not membranes but are made
up of the endfeet of Miiller glial cells and appear as dense lines at the light microscopic
level. The outer nuclear layer contains the cell bodies of the rods and cones. The
innermost part of the photoreceptors, the rod spherules and cone pedicles, make the
synaptic connections with the second order neurons (bipolar cells and horizontal cells) in
the outer plexiform layer.

The bipolar cells stretch from the outer nuclear layer to the inner plexiform layer,
whereas the horizontal cells relay information laterally in the outer plexiform layer

(typically from rods to cones or from cones to cones). The bipolar cells communicate

with the ganglion and amacrine cells in the inner plexiform layer. The inner nuclear layer
contains the cell bodies of the amacrine, ganglion and Miiller cells. Finally, the axons of
the ganglion cells make up the nerve fiber layer, which ultimately makes up the optic
nerve. The Miiller cells are the primary glial cell of the retina and span the retina from

the outer nuclear layer to the nerve fiber layer.

 

Figure 1.2. A simple diagram of the organization of the retina. Key: RPE —
retinal pigment epithelium; MiiC — Muller cell; HzC — horizontal cell; BC —
bipolar cell; AC — amacrine cell; GC — ganglion cell; NFL — nerve fiber layer;
ILM — inner limiting membrane. (Source www.webvision.med.utah.edu)

 

 

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Figure 1.3. Plastic-embedded section of a normal chicken retina at 270 days
of age. The oil droplets, present in some of the cone photoreceptor inner
segments can be seen adjacent to the RPE (arrows). The hyperboloid and the
paraboloid (arrowheads) with their characteristic metachromatic appearance in
toluidine blue can also be seen. Stain - toluidine blue; Bar — 20 um. Key: BM
— Bruch’s Membrane; RPE+OS — Retinal Pigment Epithelium + Outer
Segment; IS — Inner Segment; OLM — Outer Limiting Membrane; ONL —
Outer Nuclear Layer; OPL — Outer Plexiform Layer; INL — Inner Nuclear
Layer; IPL — Inner Plexiform Layer; GCL+NFL — Ganglion Cell Layer +
Nerve Fiber Layer; ILM — Inner Limiting Membrane. Figure used with
permission from Montiani-Ferreira PhD Thesis 2004.

 

1.3. Chicken retinal structure

Chickens, like most other avian species, fish, reptiles and amphibians, have a
cone-dominant retina, whereas nearly all mammals, including humans, have rod-
dominant retinas. 'They possess two types of cones, single and double cones, which exist
as either a single cell (single) or tightly associated accessory and principal cell (double).
It has been shown that the accessory and principal cells of the double cones are
electrically coupled (Smith et al., 1985). Their cones are unique in that they possess oil
droplets in the inner segment (ellipsoid) that act as filters through which light must pass
before reaching the visual pigment (Bowmaker et al., 1997), a feature only found in birds
and some reptiles. There are several different types of cones categorized both by the type
of oil droplets and the visual pigment they contain. The oil droplets and visual pigments
are characterized by the wavelength of light to which they are most sensitive. There are
four different cone pigments with maximal absorbance wavelengths of 415, 460, 505 and
562nm (Fager and Fager, 1981; Yen and Fager, 1984; Yoshizawa T & Fukada Y, 1993).
The various types of cones are accompanied by oil droplets of different colors, which

help to narrow the spectral sensitivities of the photopigments (Bowmaker et al., 1997).

1.4. Signaling within the retina
1.4.1. Guanine—nucleotide binding proteins

Guanine nucleotide binding proteins (G-proteins) are involved in many cell
signaling cascades in many tissues of the body. They play various roles in regulating the
activity of enzymes, ion channels and vesicular transport through their interaction with G

protein-coupled receptors (GCPRs) (Neer, 1995; Krapivinsky et al., 1995; Helms, 1995).

 

Despite their great array of “jobs” they all share the same heterotrimeric structure. The 0t
subunit is responsible for binding GTP and converting it to GDP and together the B and y
subunits act as a single unit. When activated, the a subunit exchanges GDP for GTP,
dissociates from the By dimer and goes on to stimulate other pathways. In some systems,
such as phototransduction, the a-GTP complex is active, while in other instances the By
subunit is active. An example ofthe By dimer directly initiating a signaling pathway is a
G-protein-activated inwardly rectifying K+ channels (GIRK2) that is activated by GBly2
when co-expressed in Xenopus oocytes (Kofuj i et al., 1995). When the GTP is
hydrolyzed to GDP, the By dimer binds to the Qt subunit once again to reform the

holoenzyme. There are different forms of the three subunits that are found in different

cell types in various combinations (Hamm and Gilchrist, 1996).
Of particular significance to this project is the B3 subunit. B subunits are made up
of an alpha helix and 7 repeating WD domains that form “propellers.” The 0t and y

subunits bind to the B subunit at opposite sides of the molecule.

1.4.2. Visual transduction

As previously mentioned, phototransduction is the process by which light is
converted into a chemical signal and is one of the best characterized cell-signaling
cascades involving G-proteins. This visual transduction cascade has been well
characterized in rod photoreceptors, but has not yet been fully elucidated in cones,
although it is thought to involve a similar process. Figure 1.4 is a simplified schematic of

phototransduction in rods.

In rods, a photon of light converts the l l-cis-retinal molecule to all-trans-retinal,
which is then combined with opsin to become the visual pigment rhodopsin, thus
“bleaching” rhodopsin. One of the intermediates is metarhodopsin II, which is
responsible for activating transducin (Emeis et al., 1982). Metarhodopsin II (Rho*)
interacts with the GDP-bound form of transducin (GaBy trimer) and initiates the
exchange of GDP for GTP thus dissociating the now-activated a-subunit from the By
dimer (Molday, 1998). One Rho* can interact with many transducin complexes thus
amplifying the response. Ga-GTP then interacts with cGMP phosphodiesterase (PDE), a
heterotrimer consisting of aB catalytic subunits and two inhibitory y subunits. Ga
interacts with the y subunits (of PDE) causing the release of the OLB subunits. The
catalytic site of PDE-aB is then exposed and lowers the concentration of cGMP in the
cytoplasm, which in turn leads to closure of the cGMP-gated ion channels in the cell
membrane. This stops the current of Na+ and Ca2+ ions which steadily flows into the cell
in the dark. The closure of the cGMP-gated channels results in hyperpolarization of the
cell and a decrease in the release of glutamate from the photoreceptor terminal. To
reiterate, when stimulated with light, the photoreceptors hyperpolarize, which is unusual
amongst excitable cells.

Inactivation of the phototransduction cascade is accomplished by several
pathways. Phosphorylation of activated rhodopsin by rhodopsin kinase (RK) reduces the
enzymatic activity of activated rhodopsin and generates an affinity of rhodopsin to bind
to arrestin (Arr) (Alloway and Dolph, 1999). Arrestin binding to rhodopsin prevents
future transducin activation. Hydrolysis of Ta-GTP into Tor-GDP results in deactivation

of PDE (Vuong and Chabre, 1991) by releasing PDEy molecules, which can then rebind

to the PDEaB complex. In the mean time, Ton-GDP is deactivated by rebinding to the
TBy complex. A decrease of intracellular Ca2+ due to closure of cGMP-gated channels
mediates recoverin (RC), a Ca2+-binding protein, to relieve inhibition of guanylate
cyclase (GC) (Venkataraman et al., 2003). Activated guanylate cyclase synthesizes
cGMP, and is activated by guanylate cyclase activating protein (GCAP) (Palczewski et
al., 1994). Because activated GC re-synthesizes cGMP, an elevation of cGMP
concentration results in a re-opening of some of the cGMP-gated ion channels, which
consecutively increases the inward cation flow responsible for the dark current, leading to

rod depolarization and an increase in glutamate release.

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1.4.3. ON and OFF pathways

The photoreceptors synapse in various ways with several different types of cells
including ON-center (rods and cones) and OFF-center (cones only) bipolar cells. When
the photoreceptors are stimulated with light, they hyperpolarize reducing glutamate
release at the synaptic terminal. This affects the ON and OFF bipolar cells in opposite
ways; ON-center bipolar cells respond by depolarizing and OFF-center bipolar cells
respond by hyperpolarizing (Dowling and Werblin, 1969; Werblin, 1991). Thus ON
bipolar cells are often referred to as depolarizing bipolar cells (DBC) and OFF bipolar
cells are referred to as hyperpolarizing bipolar cells (HBC) (Figure 1.5).

The type of synapse between the photoreceptors and the bipolar cells is associated
with the type of bipolar cell. Synapses between photoreceptors and bipolar cells that are
characterized as invaginating ribbon—type synapses are associated with ON bipolar cells
and create a sign-inverting synapse (i.e. when photoreceptors hyperpolarize, ON bipolar
cells depolarize). Both rods and cones synapse with bipolar cells with this type of
ribbon-synapse, meaning there are rod ON bipolar cells and cone ON bipolar cells.
These synaptic connections are made through metabotropic glutamate receptors,
specifically mGluR6 receptors, which signal through a G-protein coupled cascade (Nawy
and Jahr, 1990; Nawy, 1999).

Cones (but not rods) also synapse with OFF bipolar cells with a basal junction
connection and create a sign-preserving synapse because they depolarize when the
photoreceptors depolarize in response to light. These synaptic connections are made

through ionotropic glutamate receptors (iGluR) (Slaughter and Miller, 1983).

11

cone
-?f\5.o.~“o. .,
/ l ‘\.{ 0°
. l ’ ~.\"*/-.\' ‘

 

 

I
I .
é .

perpolarizing a

t ' AF)?

J—L_

depolarizing hyperpolarizing depolarizing

 

 

Figure 1.5. Synapses between rods, cones and their associated bipolar cells.
Rod photoreceptors only synapse with an invaginating synapse with ON
bipolar cells creating a sign inverting response — rod ON bipolar cells
depolarize in response to rod hyperpolarization. Cones synapse with both
basal (OFF bipolar cells) and invaginating synapses (ON bipolar cells). Cone
ON bipolar cells are also sign inverting in that they depolarize in response to
cone hyperpolarization, whereas cone OFF bipolar cells have sign preserving
responses in that they hyperpolarize in response to cone hyperpolarization. HC
—Horizontal Cell (Source: www.webvision.med.utah.edu)

ON and OFF bipolar cells form single excitatory synapses with specific ganglion cells in
the inner plexiform layer in a highly stratified configuration (Raviola and Raviola, 1982).
ON cells synapse in layers closer to the ganglion cell layer, while OFF cells synapse in
layers closer to the inner nuclear layer (Gouras, 1971; Famiglietti, Jr. and Kolb, 1976;
Nelson et al., 1978); therefore, ON bipolar cells synapse with ON ganglion cells and OFF
bipolar cells synapse with OFF ganglion cells. Similar to the ON/OFF bipolar cells, ON
ganglion cells depolarize in response to stimulation and OFF ganglion cells

hyperpolarize.

1.5. Retinal dystrophy models

There are hundreds of animal models of genetic retinal dystrophies, in species
from Drosophila to dogs to primates. Some of them are naturally occurring
(spontaneous), while some of them have had various mutations induced intentionally
(knock out). There are currently many mutant mice and several mutant dogs that are
being used to study retinal diseases. One example is the RPE65 mutant dog from the
Briard breed, a model of Leber’s Congenital Amaurosis Type II, in which there is a
failure of formation of l l-cis retinal in the RPE meaning there is a lack of visual pigment
formation. The lack of visual pigment formation, has a severe effect on vision. There is
an accumulation of all trans retinyl esters that leads to a slowly progressive degeneration
of the retina (Narfstrom et al., 1989; Veske et al., 1999; Narfstrom et al., 2003). Another
example is the retinal degeneration (rdl) mouse, a model of retinitis pigmentosa, in
which a nonsense mutation of the beta-subunit of cGMP phosphodiesterase leads to an

accumulation of cGMP and eventually photoreceptor degeneration (Pittler and Baehr,

l3

1991). Both of these models have been used in gene therapy trials (Acland et al., 2005;
Pang etal., 2006).

There are also several naturally occuring chicken models of retinal diseases
including blindness, enlarged-globe (beg), delayed amelanotic (DAM), retinal dysplasia
and degeneration (rdd) and retinal degeneration (rd). The rdd chicken was discovered in
1979 in Scotland and further described in the early 1980’s (Randall and McLachlan,
1979; Wilson MA, 1982; Randall et al., 1983). Affected chicks have reduced vision at
hatch and eventually become blind by 15 weeks of age. Histopathological changes of
this model include severe retinal dystrophy involving thinning of the RPE and of all
layers of the retina, and near complete loss of the outer nuclear layer and photoreceptors.
This genetic abnormality has been shown to be a sex-linked recessive trait, the disease
interval of which is homologous to human chromosomes 9 and 5 (Burt et al., 2003).

The blindness, enlarged globe (beg) chick was characterized in the early 1980’s as
having vision abnormalities at hatch, abnormal coordination and eventually developing
globe enlargement and blindness (Pollock et al., 1982). No further research has been
published regarding the beg chicken. Another chicken model of a retinopathy is the
delayed amelanotic (DAM) chicken, which suffers a gradual depigmentation due to
defective melanocytes and involves the feathers and the choroid and thus the retinal
pigmented epithelium (Boissy et al., 1983). The disrupted retinal pigmented epithelium
is unable to maintain contact with the outer segments of the photoreceptors and
eventually leads to retinal detachment (Fite et al., 1985). Additionally, the abnormal RPE

cells are no longer able to phagocytize photoreceptor outer segments, which leads to

14

impaired function of the photoreceptors and eventually the entire retina (Lahiri and
Bailey, 1993).

The retinal degeneration (rd) chick is blind at hatch but has a morphologically
normal retina indicating normal retinal development. The photoreceptors begin
deteriorating at 2-3 weeks of age until eventually very few intact photoreceptors remain
at 6 months and the photoreceptor cell bodies are replaced by Muller cell processes
(Ulshafer et al., 1984; Ulshafer and Allen, 1985). The causative mutation is a null
mutation in the guanylate cyclase 1 gene (GCl), which encodes the enzyme involved in
cGMP synthesis. Without GC 1, the abnormally low amount of cGMP may lead to
permanent closure of the cGMP-gated cation channels located in the photoreceptor cell
membrane and elimination of the dark current and chronic hyperpolarization of the
photoreceptors (Semple-Rowland et al., 1998). The rd chicken is considered a model of

Leber’s Congenital Amaurosis Type I.

1.6. Electroretinography
1.6.1 . The electroretinogram
When stimulated with light, the cells of the retina react in various ways and
produce electrical currents. This electrical activity is detectable at the cornea’s surface
and the recording of the electrical response is called an electroretinogram (ERG). This
allows the retina to be evaluated using a minimally invasive method. The
electroretinogram represents the summation of the responses from the entire retina and

most of its cell types, some of which have opposing responses. The ERG generally

15

consists of an initial corneal negative a-wave followed by a corneal positive b-wave, and
under certain circumstances a c-wave and a d-wave.

Some of the earliest work with electroretinography was done by Einthoven and
Jolly in 1908. They found when the retina was stimulated with light, three waves
appeared at various times after stimulation. They named the initial negative wave the a-
wave, the second positive wave the b-wave and the last, slower positive wave the c-wave
(Figure 1.6) (Einthoven and Jolly, 1908). These wave designations have been used since
then. In 1933, Ragnar Granit recorded electroretinograms from an anesthetized cat and
found as the anesthesia deepened, the electroretinogram changed. He named the waves
P-I, P-11 and P-III for the order in which they disappeared as ether anesthesia deepened
(Figure 1.6). P-I is a slow cornea positive wave and disappeared first. P-II has an initial
fast and sharp positive wave, which then drops to an intermediate potential until the light
stimulus is terminated. P-III is a negative wave that appears before the other two and was
the most resistant to anesthesia. P-III has been shown to have two components, a fast
component and a slow component (Granit, 1933). Since then, Granit’s three waves have
been associated with Einthoven and Jolly’s waves as follows: the a-wave is the fast
component of P-III, the b-wave consists of P-11 and the slow component of P-III, and the

c-wave is P-I.

l6

 

 

 

Lights on Lights off

lb #

a 100pV l

0 50 100 150 200 250
mSec

 

 

 

Figure 1.6. The ERG waves. A) Diagrammatic representation of the Granit
waveforms (I, II and III) which make up the electroretinogram (ERG). Note
the superimposed representation of a-, b- and c-waves correlating with the
waveforms III, II and 1, respectively. B) An actual ERG from a 90 day old
chicken. The arrow indicates the timepoint at which the eye was stimulated by
a brief flash of light. Note the chicken ERG has the same basic components of
a mammalian ERG. The a- and b-waves are marked. Time scales are not equal
for A and B. Figure used with permission from Montiani-Ferreira PhD Thesis

2004.

17

1.6.2. The origins of the ERG waves

Much work has been done since the birth of electroretinography to elucidate the
origins of the waves of the electroretinogram. Two methods have been used historically;
recording of responses with electrodes placed within various layers of the retina and
dissociated cells, and pharmacological dissection using agonists and antagonists to alter
the response of certain cell types. More recently, knockout models of various essential
retinal components have furthered the elucidation of ERG waveform origins.

The a-wave, the first to appear after light stimulation, corresponds with the
leading edge of Granit’s P-III (the fast component) and has since been found to largely
originate from the photoreceptors. When stimulated with light the phototransduction
cascade leads to the closure of the cation channels and the hyperpolarization of the
photoreceptors, which results in the corneal negative a-wave. The a-wave is visible until
the corneal positive b-wave appears. Intraretinal recording revealed that the a-wave is the
“light current” from the photoreceptors and is due to the loss of the dark current as
cGMP-gated channels close; thus, the a-wave represents the photoreceptors’ response to
light stimulation (Penn and Hagins, 1969; Sillman et al., 1969). Furthermore, when 2-
amino-4-phosphonobutyric acid (APB — a glutamate agonist, also known as L-AP4) is
applied, the response of some second order neurons is prevented and the a-wave is the
only wave remaining (Slaughter and Miller, 1981; Slaughter and Miller, 1983; Stockton
and Slaughter, 1989). Evidence that the a-wave, particularly the cone response, also
contains post-receptor contributions was elucidated when N-methyl-D-aspartic acid

(NMDA — a glutamate receptor antagonist that decreases the response of third order

18

retinal neurons) was found to decrease the amplitude of the a-wave (Robson and
Frishman, 1996).

The b-wave has been the subject of much debate among electrophysiologists. The
results of many studies have shown that the b-wave corresponds to P-11 and the slower
component of P-III and that it originates from both the bipolar cells and possibly from the
Miiller cells. The hyperpolarization of the photoreceptors in response to light decreases
the amount of neurotransmitter (glutamate) that is released onto the second order bipolar
cells, which in turn either depolarize (ON bipolar cells) or hyperpolarize (OFF bipolar
cells), which in turn alters the extracellular potassium concentration thus affecting Miiller
cells. One study using APB, which selectively blocks the response of ON bipolar cells,
demonstrated that the b-wave was eliminated after application of APB (Gurevich and
Slaughter, 1993). Another study has shown that the b-wave is the summation of the
response from both the ON (depolarizing) and OFF (hyperpolarizing) bipolar cells, which
create opposite electrical potentials in response to light (Sieving et al., 1994). They
coined the phrase the “push-pull model” of the electroretinogram in reference to the
competing responses of the bipolar cells in response to light.

Barium, an ion that completely blocks the potassium permeability of Muller cells,
has also been used to study the b-wave (Newman, 1989; Reichelt and Pannicke, 1993;
Linn et al., 1998). The Miiller cells’ involvement in the b-wave was disproved when
barium ions were injected intravitreally; the b-wave was augmented instead of being
eliminated, which is what would be expected if Muller cells contributed positively to the
b-wave (Lei and Perlman, 1999). Other research indicates that third order neurons also

contribute to the b-wave because drugs that enhance or decrease the response of third

19

order neurons also respectively enhance or decrease the b-wave (Awatramani et al.,
2001).

The c-wave’s origin does not appear to be quite as convoluted as that of the b-
wave. It has been shown to originate from the retinal pigmented epithelium (RPE).
Several methods have been used to come to this conclusion. Intracellular recordings
from the RPE in response to light stimulation had an identical shape and time course as
the c-wave of the ERG (Steinberg et al., 1970). Additionally, when the retina is
separated from the RPE, light stimulation produces a- and b-waves, but not the c-wave
(Pepperberg et al., 1978). The c-wave is dependent on the potassium concentration
changes induced by the photoreceptors upon light stimulation (Oakley and Green, 1976;
Oakley, 1977). So, although the c-wave originates from the RPE, the photoreceptors
must be functional in order to produce the wave.

The d-wave becomes apparent only when the length of the light stimulus is 100ms
or greater (i.e. a long flash) because it appears at the off set of the light stimulus (Figure
1.7). When the stimulus is shorter, the b-wave and the d-wave are overlapped. Current
source density analysis showed that the d-wave originates from the hyperpolarizing
(OFF) bipolar cell (Xu and Karwoski, 1994). Pharmacologic dissection using cis 2,3-
piperidine-dicarboxylic acid (PDA — a glutamate antagonist) suppresses the light
responses of OFF bipolar cells and eliminates the d-wave of the ERG, suggesting the d-
wave originates from the transmission between photoreceptors and OFF-center bipolar
cells (Stockton and Slaughter, 1989). Other research suggests that third order neurons
(amacrine and ganglion cells) also contribute to the d-wave. Baclofen, a drug that

enhances the light responses of third order neurons, enhanced the d-wave, whereas drugs

20

that decrease the response of third order neurons decreased the d-wave (Awatramani et

aL,2001)

21

Q.
50 pVOlt

50 msec

 

b
a
_
Figure 1.7. The long flash ERG. Representative ERG from a normal chicken

in response to a prolonged flash of light (150 msec). The a-, b- and d-waves

are labeled. Heavy black line indicates length of flash. Notice the d-wave
appears at lights-off.

22

Another relevant component of the ERG is the oscillatory potentials (OPs). These
are small amplitude “wavelets” that appear on the rising edge of the b-wave when a
bright light is used as a stimulus (Figure 1.8). In order to evaluate them more closely, a
band-pass filter can be used to isolate the DPS. The exact cellular origin of CPS has yet
to be determined, but some information has been gathered. Intraretinal recordings reveal
that the amplitude is the greatest when the electrode is in the inner retina, specifically the
inner plexiform layer (Brown, 1968; Ogden, 1973). It is thought the OPs are generated
by extracellular electrical currents created by negative feedback pathways between
amacrine, ganglion and bipolar cells (Wachtmeister and Dowling, 1978). OP amplitudes
are decreased in conditions causing retinal ischemia and because of this, their amplitudes
have been used to evaluate diabetic retinopathy, a condition characterized by localized
retinal ischemia (Frost-Larsen et al., 1980; Simonsen, 1980; Bresnick and Palta, 1987;

Asi and Perlman, 1992; Kizawa et al., 2006).

 

 

3:
O
>
:1.
c
o
F

50 msec

Figure 1.8. Short flash ERG with oscillatory potentials. Representative short
flash ERG from a normal chicken. The OP’s are marked with large black
arrow heads. The small arrow indicates the brief light stimulus.

23

When studying electroretinograms, several parameters are used to evaluate the
waveforms (Figure 1.9). The amplitude, which is measured in microvolts (uV), of the a-
wave is measured from the baseline to the trough (peak) of the a-wave. The b-wave
amplitude is measured from the trough of the a-wave to the peak of the b-wave. The
implicit time is the amount of time from stimulus onset to the peak of the wave and is

measured in milliseconds (msec). The latency of a wave is the amount of time from

stimulus onset to the beginning of the wave and is also measured in milliseconds.

E— a!

 

 

 

Figure 1.9. Commonly measured parameters of the ERG waveform. A-wave
amplitudes are measured from baseline to the trough. B-wave amplitudes are
measured from the trough of the a-wave to the peak. Implicit times (denoted
Lal and Lb) are measured from light stimulus to the peak of the wave. (Source:
www.webvision.med.utah.edu)

24

1.6.3. Light adaptation status and the electroretinogram

Because ERG waveforms reflect the responses of the photoreceptors, the
receptors that are active under the stimulus conditions are those that ultimately shape the
ERG waveform; therefore when the stimulus intensity is low and/or the retina is “dark-
adapted,” the rod response dominates whereas when the stimulus intensity is bright but
the retina is dark-adapted, the response is a mixed rod/cone response. Lighting
conditions that elicit a mixed rod/cone response are called mesopic. An ERG performed
when a subject is light adapted is termed a photopic ERG and when dark adapted, a
scotopic ERG. Photopic ERGs are performed after a subject has been light adapted to a
“rod-saturating” background light, which means the rods are incapable of responding to a
light stimulus. In addition to stimulus intensity and the light/dark adaptation status, the
ERG is also affected by the proportion of rods and cones in a retina; some species have a
cone dominant retina (chicken, ground squirrel), whereas some have a rod dominant

retina (humans, dogs, rats and mice).

1.6.4. Circadian ERGs

Retinal morphology and function has been shown to exhibit circadian variation in
many species. Photoreceptors in chicks and rats shed outer segments in a circadian
rhythm -— rods shed their outer segments soon after lights on and cones shed theirs soon
after lights off (LaVail, 1976; Young, 1978). Retinal and behavioral sensitivity of
zebrafish are also circadian in nature in that sensitivity thresholds change throughout the
day (Li and Dowling, 1998). In chickens, research using electroretinograms has shown

that rod function is greater at night and is reduced during the day (Schaeffel et al., 1991).

25

This differential activity of photoreceptors is known as the retinal Purkinje Shift,
which is characterized by rods and cones being active at opposite times of day (rods
active at night, cones active during the day) or in different light adaptation (rods after
dark adaptation and cones after light adaptation). Further research has shown that b-wave
amplitude is rhythmic — the amplitude is larger in the aftemoon and that exogenous
melatonin administered during the day reduced the b-wave amplitude (Lu et al., 1995).
The human’s visual threshold is significantly increased 1.5 hours after lights-on, which
corresponds with the time that rods are maximally shedding their outer segments (Birch
etal., 1984).

Other research has shown that chick’s and pigeon’s a- and b-wave amplitudes
were larger, implicit times were shorter and sensitivity was lower during the day time.
Short implicit times and lower sensitivity are considered to be specific to the behavior of
cones (McGoogan and Cassone, 1999; Wu et al., 2000). This was attributed to the avian
retina being cone-dominant and thus a larger number of photoreceptors were able to
respond during the day (when the cones were active) than at night (when only rods were
active) (McGoogan et al., 2000). Melatonin is one substance that has been implicated in
the circadian rhythm of the ERG and visual system in that when exogenous melatonin is
administered, the rhythmicity of a- and b-wave parameters are abolished (Peters and
Cassone, 2005). Reseach has shown that melatonin is manufactured in and released from
both the pineal gland and in the retina (Takahashi et al., 1980; Zawilska and Wawrocka,
1993; Bernard et al., 1997). The work of another research laboratory contradicts that of
McGoogan and Cassone’s in that they found that quail’s b-waves are larger at night than

during the day (Manglapus et al., 1998). This laboratory implicates dopamine as being a

26

key regulator in the circadian rhythms of the retina because when retinal dopamine (D2)

receptors were blocked during the day the b-wave amplitude is increased (Manglapus et

al., 1999).

1.6.5. The ERG in disease states

Because the components of the ERG waveform are created by the various cell
types under varying conditions, alterations in the ERG waveform found with specific
diseases may offer a clue as to which cells are affected. ERG waves can be normal,
reduced in amplitude, supemormal (increased amplitude), absent, or have an abnormal
shape or implicit time. Generally speaking, diseases that affect photoreceptors will have
reduced a- and b-wave amplitudes. Specifically, diseases affecting rods will present with
smaller a- and b-wave amplitudes under scotopic conditions, whereas those that affect
cones will cause smaller amplitudes under photopic conditions. Conditions that only
affect second order neurons or the transmission of signals from the photoreceptors to the
second order neurons will present with normal a-waves and abnormal b-waves. If third
order neurons are affected preferentially, the DPS and possibly the b- and d-waves will be

affected.

1.7. The rge chicken
1.7.1. Original rge characterization

The focus of this thesis is the retinopathy, globe-enlarged (rge) chicken and much
work has already been done to characterize its phenotype. The rge chicken was

identified in commercial flocks in the United Kingdom in the early 1980’s. Affected

27

birds were detectable by 3 weeks of age but became obvious at 8 weeks of age when
poultry house equipment was moved as the affected birds appeared lost when their
normal counterparts scattered. Affected birds were unable to peck at food particles,
although they were able to respond to large moving objects and to bright lights. The
birds also exhibited the bizarre behavior of peeking at the air. They had a reduced
pupillary reflex, which corresponded to the severity of their functional visual impairment.
Fundus examination revealed prominent choroidal vasculature and linear white lesions in
the retina, Older birds often developed cataracts. Histopathology revealed an overall
thinning of the retina, a reduction in the number of photoreceptor outer segments and
nuclei, a thinner inner nuclear layer and a reduced number of ganglion cells (Curtis et al.,
1987; Curtis et al., 1988).

The condition was considered autosomal recessive as heterozygous individuals
were indistinguishable from homozygous normal individuals. Test matings between an
affected and a carrier suggested that there might be a degree of reduced embryonic

survival as ratios were not 1:1 as expected (Ingleheam et al., 2003).

1.7.2. Globe enlargement

In addition to the visual impairment and histological abnormalities, the affected
birds invariably develop enlarged globes without an increase in intraocular pressure as
they get older (non-glaucomatous) (Figure 1.10). This resulted in increased exposure of
the sclera at the medial canthi of affected birds. The enlarged globes also had a loss of
the normal corneal curvature and appeared flatter (Figure 1.10). Both the axial length

and the weight of affected eyes were greater than unaffected birds of the same age. The

28

anterior chamber of affected birds became very shallow and the anterior surface of the
lens was almost in contact with the posterior corneal surface. The vitreal chamber of
affected birds gradually became significantly larger and the pecten appeared atrophied as
they aged (Montiani-Ferreira et al., 2003; Inglehearn et al., 2003). These changes are
considered to be secondary in response to the visual deficits as alterations in globe size
have been experimentally linked to induced visual stimulus deprivations (Hodos and

Kuenzel, 1984).

29

     

control" w .133

Figure 1.10. Gross ophthalmic photographs from representative rge and control
birds. A) Appearance of freshly enucleated eye globes of a control and an affected
chick, at 180 days of age. Note the difference in radial diameter of the globes. B)
Freshly enucleated eye globes of a control and an rge chick, at 270 days of age.
Note the difference in axial length of the globes. Profile view of the cornea and
anterior chamber of a control (C) and an rge chick (D), at 180 days of age. Note the
flatter cornea and very shallow anterior chamber of the rge eye. E) Detail of the eye
position inside the orbit of a control and an rge bird at 45 days of age. Note the mild
lateral strabismus causing greater exposure of the sclera at the medial canthus in the
rge chick. Figure used with permission from Montiani-Ferreira PhD Thesis 2004.

30

1.7.3. Lacquer crack lesions

Many affected birds develop white linear lesions in the fundus located close to the
pecten (Figure 1.11). Histopathologic examination of these lesions revealed focal and
severe thinning of the retina in which the photoreceptor inner and outer segments, outer
nuclear, outer plexiform and retinal pigment epithelial layers were totally absent, the
inner nuclear layer was present but disorganized and the inner retinal layers (nerve fiber,
ganglion cell and inner plexiform layers) were mostly normal (Curtis et al., 1988). These
lesions have since been characterized as lacquer crack lesions, which are also associated
with complications of severe human myopia (Klein and Curtin, 1975; Klein and Green,
1988; Czepita D., 2002). They have also been experimentally induced in form
deprivation myopia in chicks and in chicks continuously exposed to light; two situations
in which globe enlargement is a resulting feature (Li et al., 1995; Hirata and Negi, 1998).

A more detailed light microscopic evaluation of these lesions revealed mild focal
fibrosis at the level of the inner choroid, focal absence of the retinal pigment epithelium,
and an accumulation of eosinophilic hyaline material in the subretinal space.
Additionally, the overlying photoreceptor inner and outer segments were disorganized
and there was thinning and displacement of the inner and outer nuclear layers (Figure
1.12). Semithin sections additionally demonstrated a rupture in Bruch’s membrane (the
basement membrane of the RPE located between the RPE and the choroid) with scar
formation and deposition' of collagen fibers (Figure 1.12). These changes were
hypothesized to be a result of the abnormal globe enlargement causing the retina to

stretch and then break (Montiani-Ferreira et al., 2004).

31

 

 

 

 

 

Figure 1.11. Posterior eyecup from a 49-day-old rge bird showing lacquer cracks.
The lacquer crack lesions appear as multiple white to gray linear lesions (white
arrows), extending from the pecten to the periphery. Additionally, an area of
subretinal hemorrhage (white arrowheads) is present. Figure used with permission
from Montiani-Ferreira PhD Thesis 2004.

32

 

 

 

Figure 1.12. Plastic and resin-embedded retinal sections from rge birds
demonstrating morphologic details of the lacquer crack lesions. A) Eye of an rge
bird at 49 days of age. Cross section through a region of retina adjacent to the lesion
shown in B. The retinal architecture is relatively normal with a normal retinal
pigment epithelium layer and normal photoreceptor-retinal pigment epithelium
interface. However, there is a mild dilation of the photoreceptor inner and outer
segments, which is typical of early stage changes observed in the rge phenotype. B)
Cross section through one of the lesions shown in Figure 1.10 from the central
retina. Note the mild focal fibrosis at the level of the inner choroid (black
arrowheads) and the absence of retinal pigment epithelium (white arrowheads).
There is an accumulation of eosinophilic material in the subretinal space. The
overlying photoreceptor inner and outer segments are disorganized and there is
thinning and displacement of the outer and inner nuclear layers (black arrow).
Stained with H&E. Size bar = 150 um. C & D) Eye of an rge bird at 336 days of
age. C) This shows the region adjacent to the lesion that is shown in D. Bruch’s
membrane is intact (arrow) and is lined by the RPE layer on one side and the
choriocapillaris on the choroidal side. D) Details of a linear lesion showing an
absence of the normal retinal pigment epithelium layer, rupture of Bruch’s
membrane (arrow), scar formation with deposition of collagen fibers and the
abnormal presence of retinal pigment epithelium cell melanosomes on the choroidal
side of Bruch’s membrane. Stained with toluidine blue. Size bar = 20 pm. Figure '
used with permission from Montiani-Ferreira PhD Thesis 2004.

33

1.7.4. Retinal changes

A more detailed examination using light microscopy, electron microscopy and
immunohistochemistry provided a more complete description of the progression of the
pathological changes affecting the rge retina. This study showed that at hatch, the rge
retinas were the same thickness as age-matched control retinas, but they then gradually
became thinner as time progressed with all layers except 'the RPE gradually becoming

significantly thinner than controls (Montiani-Ferreira et al., 2005) (Figure 1.13).

34

 

Figure 1.13. Semithin sections of outer retina. Sections from control (A & C) and
rge birds (B & 11)) at one day (A & B) and 270 days (C & D) of age. A. Control
retina has a well organized two-layered arrangement of photoreceptor synaptic
termini in the ONL (arrows). B. The mutant retina has dilated photoreceptor cell
bodies and disorganization of the OPL architecture (white arrow). Bars for A & B =
10 um. C. Control retina at 270 days has glycogen deposits only in the ISs
(external to the outer limiting membrane). These are associated with the rod
hyperboloid (white arrowheads) and with the cone accessory cell paraboloid (black
arrowheads). D. The mutant retina at 270 days of age is thinned with shortened
photoreceptors. Larger glycogen deposits are present and were quite often displaced
internal to the outer limiting membrane of the accessory cells of the double cones
(arrow). Bars for C & D = 20 um. Adapted from Montiani-Ferreira et a1 (2005).

.35

The earliest changes found in rge retinas were found in the outer plexiform layer
and included a disorganization of the photoreceptor synaptic terminal organization and
abnormal location of the endoplasmic reticulum (ER) of the photoreceptors (Figure 1.14).
The bilayered arrangement of the rod spherules and cone pedicles in control birds was not
present in rge retinas from an early age. This disorganization became more severe as the
birds aged. There also appeared to be fewer synaptic ribbons in the synaptic terminals of
photoreceptors from affected retinas although the number of synaptic ribbons in the inner
plexiform appeared to be similar to that of control retinas. The endoplasmic reticulum of
the photoreceptors was frequently found in the cell bodies rather than in its normal
location in the inner segments. Accumulations of glycogen were found in the perinuclear
region of the photoreceptors associated with the abnormally located ER (Figure 1.14).
No evidence of apoptosis was found using TUNEL staining, caspase 3 staining or

morphological criteria (Montiani-Ferreira et al., 2005).

36

. l ‘ i V i ‘-
1§<T 1‘ ‘ g:l-’;; ’1}?
1‘ 0"}
r<£lfl

 

Figure 1.14. Mislocalization of glycogen deposits. Semi-thin (A) and ultra-thin
(B, C & D) sections of rge (A, B & D) and control (C) retina at 7 days of age. A.
Semi-thin section showing the detail of a large glycogen deposit (dashed white
square) in the perinuclear cytoplasm. Note the disrupted OPL. Bar = 20 um. B.
Ultrastructural detail of a deposit in the perinuclear cytoplasm of an accessory cell
of double cone of a GNB3 mutant bird where it is possible to observe the abnormal
accumulation of glycogen (dashed white square). Bar = 5 11m. C. Control retina
showing evenly-arranged SER in the IS (paraboloid). Small glycogen granules can
be seen among the cisterns. Bar = 1 pm. D. Higher magnification of section in B to
show abnormal glycogen accumulation associated with ER. Bar = 1 pm. Adapted
from Montiani-Ferreira et a1 (2005).

37

Ultrastructural analysis showed further abnormalities in the photoreceptor
terminals. At seven days of age, the photoreceptor pedicles and spherules were increased
in size and frequently contained numerous small vesicles and multivesicular bodies.
Dense glial cell processes were found between the photoreceptors at 7 days of age and
their presence became more prominent as the birds aged (Figure 1.15). The
morphological abnormalities in the synaptic terminals of photoreceptors suggest
abnormal physiological (specifically synaptic) function and may contribute to the vision

loss in the rge birds (Montiani-Ferreira et al., 2005).

38

 

Figure 1.15. EM images of photoreceptor synaptic terminals. A. Synaptic terminal
of a cone pedicle from a normal control chick at 7 days of age. B. A typical example
of a synaptic terminal of a cone pedicle of a rge chick at 7 days of age. The
cytoplasm is less densely stained. Note the disruption in the architecture of the
synaptic terminals (small black arrows). Also note the presence of electron dense
glial bodies separating the photoreceptors (white arrowheads) and multivesicular
bodies (black arrowheads). C. Higher power detail of a rge chick retinal section at 7
days of age demonstrating one of the sets of numerous flattened (tubuliform)
vesicles (white arrow) in a cone pedicle. Size bars = 1 pm. Adapted from Montiani-
Ferreira et al (2005).

39

lmmunohistochemical staining using a panel of antibodies showed numerous
differences between rge and control retinas. Tyrosine hydroxylase (TH) staining
revealed the morphology and distribution of amacrine cells were similar between affected
and control retinas but in older rge birds, there was an increased density of TH positive
dendrites. Staining with antibodies against opsin and rhodopsin (found in rods) revealed
an increased amount of rhodopsin immunoreactivity was present in the inner segments of
rge retinas compared to controls. In sections from older rge birds, the rods appeared
swollen with loss of the normal architectural organization and rhodopsin
immunoreactivity was mislocalized to the outer nuclear layer and outer plexiform layer.
Mislocalization of opsin is a common finding in retinal dystrophy models (Gao et al.,
2002; Nishimura et al., 2004; Rohrer et al., 2005; Beltran et al., 2006). A panel of
antibodies to various glial cell and phagocyte components revealed that rge retinas
contained many activated glial and microglial cells and that phagocytosis was occurring.
Staining for synaptic vesicle protein 2 (found in synaptic vesicles) confirmed the
disorganization of the OPL found ultrastructurally in that rge retinas lacked the normal

organized stratification of the OPL (Montiani-Ferreira et al., 2005).

1.7.5. Vision testing

In addition to functional testing like the ability to evade cathre and peek at food
particles (somewhat subjective), the functional vision of the rge birds was also evaluated
using an optokinetic device to more objectively characterize the loss of vision as they
aged. This device consisted of a drum lined with black and white bars of varying widths

that revolved around the chicken being tested. A positive result consisted of the chicken

40

following the bars as they moved from side to side. The rge chicks showed decreased
acuity, which was worse in dim lighting. lntriguingly, some chicks had positive
responses to the optokinetic device only when wide stripes were used (indicating reduced
visual acuity) long after they were no longer able to peek at food particles (Montiani-

Ferreira et al., 2003).

1.7.6. Electroretinographical characteristics

The rge chicken has been evaluated electroretinographically using short flash
electroretinography. Initial electroretinographic work done in the UK reported the ERG
waveform of the affected chicks had reduced amplitudes (Curtis et al., 1988), but upon
further examination at MSU, this was found to be incorrect for bright stimuli in the
younger birds. Preliminary studies have shown that young affected birds have an
elevated response threshold (require brighter stimulus intensities to elicit a response),
similar photopic and scotopic waveforms, a decrease in oscillatory potentials and most
intriguing, a supemormal b-wave amplitude in response to brighter stimuli (Figure 1.16).
In a short flash electroretinography intensity series protocol, the rge a-waves showed
decreased amplitudes and increased latencies to all flash intensities. The b-waves of the
rge chicks demonstrated higher thresholds, but in response to brighter flashes had greater
amplitudes when compared to control chicks, thus it was termed a “supemormal” b-wave.
Figures 1.17 and 1.18 contain compiled a- and b- wave amplitudes and latencies in 7 day
old rge and control birds. There was also evidence that the rod photoreceptors were
affected early on in the disease, which correlated with the results of the optokinetic

testing in which the rge birds had decreased visual acuity in dim light. The ERG

41

responses of affected birds slowly deteriorate over time but this slow decline does not
correspond to functional vision loss; ERG responses are still recordable months after the
birds no longer have functional vision. This is unusual among the other chicken retinal
dystrophy models and models in other species in which ERG responses are extinguished

concurrently with vision loss (Montiani-Ferreira et al., 2007).

42

Figure 1.16. Representative ERG responses from a
control and an rge chick. Responses from a control (A
and B) and an rge mutant (C and D) chick at 7 days post
hatch under dark-adapted (A and C) and light-adapted
(B and D) conditions. Note that due to the cone-
dominated retina that the dark-adapted and light-adapted
ERG tracings of the normal chick are similar at the
higher intensities. Also there is a diminution of the b-
wave with increasing stimulus intensity due to a
photopic hill effect. The ERG responses of the rge
chick are quite different from the control. As with
normal chicks dark- and light-adapted responses are
similar (although the light-adapted amplitudes are
slightly lower). However, the response thresholds are
elevated compared to the control. The shape of the ERG
is quite abnormal: the a-wave tends to have a greater
implicit time at the higher flash intensities; the b-wave is
smoother than in normal chicks, due to a lack of
oscillatory potentials; and the b-wave amplitude is
greater than that of the control (“supemormal”) at the
higher flash intensities. Flash intensities for each panel
(from top to bottom) were: -2.4, -2.0, -1.42, -l.l9, -0.79,
-039, 0.00, 0.39, 0.85, 1.4, 2.3 & 2.9 log cdS/mz. x-
axis = time (mSec). Figure used with permission from
Montiani-Ferriera PhD Thesis 2004.

43

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46

The supemormal b-wave is an unusual, but not unique, feature of the rge chick
phenotype. This ERG abnormality has been reported in humans who have the enhanced
S-cone syndrome (ESCS). The ERGs of these patients are described as having delayed
b-waves that were supemormal in amplitude to brighter flashes but lower in amplitude
and markedly delayed in response to weaker flashes (Gouras et al., 1983; Jacobson et al.,
1990). The causative mutation appears in the NR2E3 gene, which is a nuclear receptor
that is responsible for determining photoreceptor cell fate, and causes an abnormally
large number of S-cones to be formed in the retina and a reduction in rod photoreceptors
(Haider et al., 2000; Haider et al., 2001). These S-cones are sensitive to short.
wavelengths of light. Therefore, these patients have enhanced responses to short
wavelengths of light (i.e. blue) (Hood et al., 1995; Haider et al., 2000). This pathogenesis
was excluded for the rge chicken by doing ERGs with different wavelengths of light.
None of the rge responses of colored light flashes showed increased amplitudes
compared to controls. Additionally, histopathology did not show obvious differences in
rod to cone ratios (Montiani-Ferreira, 2004).

Another technique used previously with the rge chicks is pharmacological
dissection of the electroretinogram. This technique involves injecting a pharmacological
agent into the vitreous and performing pre- and post-injection electroretinograms. If the
agent is known to affect only certain receptors, then its effect on the ERG can provide
information as to what receptor/cell types are contributing to the various ERG
components. This is potentially useful when one is trying to elucidate which cell types

are being affected by retinal disease. Several agents were used previously to dissect the

47

rge ERG. APB (0.1 mM) was previously used and eliminated the b-wave of the control
birds leaving only the a-wave, but did not affect the ERG of the rge chicks. PDA (5 mM)
was also previously used to dissect the ERG. It truncated the a-wave (smaller amplitude
and smaller slope of second half of a-wave) and increased the implicit time of the b-wave

of the control birds, but did not affect the rge ERG (Montiani-Ferreira, 2004).

1.7.7. Investigation of several candidate genes

In order to rule out the guanylate cyclase gene mutation that is responsible for the
rd chicken phenotype, an RT-PCR was performed using primers specific to the guanylate
cyclase gene. Products were obtained from this RT-PCR indicating that the rge mutation
is different from the rd mutation (Montiani-Ferreira, 2004). Previous work has been
done to find the causative gene responsible for the rge phenotype. A multipoint linkage
analysis was used to map the gene to chicken chromosome 1. It was further localized to
between ADL0314 and MC W01 1 2, an interval of 243cM, and represents 99% confidence

limits. This region contains between 100 and 200 genes. Within that interval the gene

was most closely linked to LE10071. This position on chicken chromosome 1 shows - ~

homology with several regions in the human genome: 12p13, 22ql3, 7q35 and 21q22
(Ingleheam et al., 2003). Ingleheam’s group also examined and excluded several genes
known to contain mutations causing human retinal disease: ABCA4, mutated in Stargardt
macular dystrophy in humans (Allikmets et al., 1997); IMPDHl, the gene underlying the
RPlO retinal dystrophy in humans (Bowne et al., 2002); and TIMP3, a gene mutated in

humans with Sorsby’s Fundus Dystrophy (Weber et al., 1994).

48

The interval was further refined by Hans Cheng’s laboratory at the USDA Avian
Disease and Oncology Laboratory in East Lansing, MI using different microsatellites to
perform a linkage analysis. An additional microsatellite marker (MC W03 1 8 at ~80.5MB)
was found to cosegregate with the rge gene (Hans Cheng, personal communication
2003). Additionally, it was concluded that human 12p11-13 exhibits the highest amount
of conservation for that map region. One additional gene was sequenced and analyzed
for mutations. Vampl, also known as synaptobrevin, codes for a protein that is part of
the SNARE protein complex (Sherry et al., 2001; Yang et al., 2002; Sherry et al., 2003).
The gene was sequenced and no differences between the rge and control birds were

found, making it unlikely to be responsible for the rge phenotype (Montiani-Ferreira,

2004).

1.8. Hypotheses

Based on the previous findings of the retinopathy, globe-enlarged chicken, the
premise of this research was based on two basic hypotheses. One is that the gene
responsible for the phenotype codes for a retinally expressed protein that is located on
chicken chromosome one. The second is that both the rod and cone pathways of the

retina are affected and specifically that the inner retina has abnormal function.

1.9. The scope ofthis project

The ERG work that had been done previously demonstrated that the rge chicks
have several unique features (supemormal b-wave and persistence of ERG waveforms

long after functional blindness), which warranted a more detailed analysis. Several

49

techniques were used to further evaluate the rge chick including additional
pharmacological dissection using specific agonists and antagonists of various cells’
responses in the retina, long flash ERG to separate the ON from the OFF responses and
circadian ERGs to determine if rge chicks’ retinas have normal circadian rhythms. The
results of these more detailed tests will provide information about how specific cell types
in the retinal are functioning, which will hopefully aid in determining which cells in the
rge retina are most affected, and thus could help to narrow the search for the causative

gene.

Additionally, a positional candidate gene approach was used to select and
sequence potential genes that could be responsible for the rge phenotype. The region
between 78 and 82 MB on chicken chromosome 1 was evaluated and candidate genes
were chosen. The first draft of the chicken genome was published in 2004 and greatly
facilitated the molecular work (International Chicken Genome Sequencing
Consortium2004). Nevertheless, toward the end of the research period for this project,
the Molecular Vision Group, a competing laboratory in the UK led by Chris Ingleheam,
discovered the causative gene. The rge phenotype was found to be caused by an in-frame
three base-pair deletion in a highly conserved region of GNB3, which is part of a guanine
nucleotide binding protein found in the retina (Tummala et al., 2006). An aspartate
amino acid residue is deleted in the affected birds. It is predicted that this amino acid
deletion alters the tertiary structure of the GNB3 protein by abolishing [3 sheets in
propellers 1 and 5 of the GNB protein. It was also hypothesized that this mutation would

create an unstable protein susceptible to premature proteolysis. This was supported by

50

finding a 70% reduction in GNB3 protein immunoreactivity in retinas from affected birds

compared to controls (Tummala et al., 2006).

GNB3 encodes the [3 subunit of cone-transducin, which is part of the
heterotrimeric guanine nucleotide binding protein involved in phototransduction in cones.
Transducin is coupled to phosphodiesterase (Peng et al., 1992), which means it is
essential for phototransductiOn. Thus GNBB plays an important role in regulating the

response of the cone photoreceptors to light stimulation.

In light of Tummala et al’s findings, immunohistochemistry was performed to

localize GNB3 expression in the retina of normal chickens.

51

CHAPTER 2

INVESTIGATIONS OF CANDIDATE GENES

2.1. Introduction

Previous work had mapped the rge locus to chromosome 1 (Ingleheam et al.,
2003) in a region syntenic to human chromosomes 12p13 and 7q35. Microsatellite
marker (MCW0318 at ~80.5MB on chicken chromosome one) was shown to co-
segregate with the rge locus using the rge flock maintained at MSU (H. Cheng, personal
communication, 2003). Using this mapping information, genes were selected using a
positional candidate gene approach. Twelve genes flanking MCW0318 were chosen,
directly sequenced and analyzed for polymorphisms (Figure 2.1 and Table 2.1).

Positional candidate genes were selected for their known expression in the eye or
central nervous system, known involvement with retinal or neuronal function or
dysfunction and cell signaling function. Table 2.1 shows a complete list of the genes,
their locations on chicken chromosome one and in the human genome and a summary of
their known functions. The chicken genome (initially the February 2004 assembly)
located on the University of California Santa Cruz’s Genome Browser website was used

to identify genes that mapped to the region flanking MCW0318.

52

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54

2.1.1. Background information for the chosen genes

TULP3 (Tubby-like protein 3) is a member of the tubby multigene family, which
also contains TUB (tubby), TULPl and TULPZ. It is a small gene family that plays an
important role in maintenance and function of neuronal cells during development and
post-differentiation (Ikeda et al., 2002). Additionally, tubby-like proteins might function
as heterotrimeric-G-protein-responsive intracellular signaling factors (Carroll et al.,
2004). TULPl mutations cause some forms of retinitis pigmentosa in humans and the
gene is abundantly expressed in the retina (Heikenwalder et al., 2001). An expressed
sequence tag (EST) for TULP3 (EST DR429568) was isolated from whole eye mRNA
extracts, supporting its possible role in the retina. At the time it was chosen, its exact
chromosomal location in the chicken was unknown, however it was known that it
mapped to chromosome one of chicken and was on the syntenic region of chromosome
12 of human. At the time this dissertation was written, its location had been assigned
(see Table 2.1 for details).

PHC‘l (polyhomeotic-like 1) is a member of the polycomb group (PcG) of gene
products, which form multimeric protein complexes and contribute to anterior-posterior
specification via the transcriptional regulation of Hox cluster genes (Isono et al., 2005).
This gene was selected in an attempt to establish a left-hand boundary for the rge
interval.

COPS7A (COP9 complex subunit 7a) is a subunit of the COP9 signalosome,
which is a multiprotein complex of the ubiquitin-proteasome pathway necessary in

eukaryotic development (Schwechheimer, 2004). It is widely expressed in many organs

55

in the chicken including the cerebrum, cerebellum and hypothalamus/pineal gland (EST
BU274813, CD215816, BU345751). It has many functions and is associated with both a
target for kinase activity and also associates with and coordinates activity of kinases
(Harari-Steinberg and Chamovitz, 2004).

GPR162 (G protein-coupled receptor 162 isoform 2) is a recently characterized
protein in the Rhodopsin family of G protein-coupled receptors. It is found in many
human tissues including in the brain and eye (EST DA356952.1, BM665884.1) and
orthologues have been found in other species including chicken (Gloriam et al., 2005).

Enolase is an enzyme of the glycolytic pathway catalyzing the dehydratation
reaction of 2-phosphog1ycerate (Piast et al., 2005). EN02 (gamma neuronal enolase 2) is
found in the interval of interest on chicken chromosome one and was thus chosen.
Additionally, auto-antibodies to alpha-enolase have been associated with an acquired
retinopathy (Magrys et al., 2007). Messenger RNA for EN02 was found in extracts in
whole chicken eye (EST DR427176).

ATNl (atrophin 1) is a gene that is associated with dentatorubral pallidoluysian
atrophy, which is a rare neurodegenerative disorder characterized by cerebellar ataxia,
myoclonic epilepsy, choreoathetosis, and dementia. The disease is reportedly caused by
the expansion of a trinucleotide repeat within this gene, and the number of repeats has
been correlated with the severity of the disease (Ikeuchi et al., 1995). The protein is
expressed in human brain and eye (EST CD671073. 1, AA985328.1).

A deficiency of PTPN6 (protein tyrosine phosphatase non-receptor type 6) is a
phosphatase that has been associated with the "viable motheaten" phenotype of mice.

This phenotype is associated with immune dysfunction, hyperproliferation of myeloid

.56

cells, regenerative anemia and retinal degeneration. This protein is expressed in murine
and human hematopoietic cells (Lyons et al., 2006).

The calsyntenin protein family has been localized in the postsynaptic membrane
of excitatory central nervous system (CNS) synapses. CLSTN 3 (calsyntenin 3) has been
found expressed at the highest levels in GABAergic neurons of the nervous system
(Hintsch et al., 2002). Its expression has been documented in chicken eyes (EST
DR424960).

Ephrins and ephrin related receptors are receptors in the protein-tyrosine kinase
family, which have been implicated in mediating developmental events, particularly in
the nervous system and specifically the retina (Mann et al., 2004). Both 13le (ephrin
receptor EphAl) and CEK9 (chicken embryo kinase 9, or ephrin receptor B6) are
receptors whose genes are found in the interval of interest on chicken chromosome one.
Additionally, expression of CEK9 has been found in both the cerebrum and cerebellum of
chicken (EST BU272681, BU353591).

Zyxin is a zinc-binding phosphoprotein that concentrates at focal adhesions and
along the actin cytoskeleton (Hoffman et al., 2006). It has been associated with actin
assembly at the tip of nerve growth cones during filopodial protrusion (Jay, 2000). It has
been found in the chicken central nervous system (EST BU273767.1).

GNB3 is the beta subunit of the guanine-nucleotide binding protein (beta-
transducin) of cone photoreceptors. It is an important signaling protein of cone
phototransduction (Peng et al., 1992). During screening of the selected positional
candidate genes another research group discovered a mutation in the gene coding for

GNB3 that is responsible for the rge chick phenotype (Tummala et al., 2006). At that

57

point, further screening of candidate genes was stopped. We then developed a PCR
restriction enzyme test to allow rapid genotyping of birds within our rge flock. We also

obtained an antibody to GNB3 and used it to localize GNB3 expression within the normal

chicken retina.

2.2. Materials and methods

The published chicken genome was used to select candidate genes using a
positional candidate approach from the regions flanking the microsatellite marker
MCWO318 (~80.5MB) and twelve genes were chosen. Refer to Table 2.1 for the genes,
their unabbreviated names, locations and functions. The strategy used was to screen each
gene by sequencing the coding region and intron/exon boundaries and analyze the

sequences for polymorphism.

2.2.1 Design of primers

The published sequence of each gene was obtained from the University of
California Santa Cruz’s genome browser (www.genome.ucsc.edu/index.html). Intronic
primers were designed using Integrated DNA Technology’s PrimerQuest program (based
on the Primer 3 program, located at http://www.idtdna.com/Scitools/
Applications/Primerquestl) to amplify each exon and intron/exon boundary of each gene.
Primers were purchased from Integrated DNA Technology (Coralville, IA) and were
generally 24 bp in length and had melting temperatures between 55 and 60°C. See Table

2.2 for a list of the genomic primers and their locations on chicken chromosome one.

58

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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61

2.2.2. DNA isolation

The DNA from one affected and one carrier bird was isolated from whole blood

using the Puregene DNA purification system (Gentra, Minneapolis, MN) and used in the

reactions. Whole blood was collected from the jugular vein of the birds and used to

isolate DNA. The exact contents and concentrations of the Puregene kit’s solutions were

proprietary and not possible to provide.

40 uL of whole blood added to 6 mL of Cell Lysis solution and vortexed
2 mL of Protein Precipitation solution added and vortexed

Centrifuged for 10 minutes at 2000 x g, supernatant poured into 10 mL of ice cold

100% ethanol and mixed until DNA pellet formed

Centrifuged for 5 minutes at 2000 x g, supernatant discarded and 10 mL 70%

ethanol added to the tube and mixed to wash the DNA pellet

Centrifuged for one minute at 2000 x g, the supernatant discarded and pellet

allowed to dry for 10 minutes
500 uL of DNA hydration solution added to pellet and DNA allowed to rehydrate
over night at room temperature

DNA concentration established using a NanoDrop (ND-1000

Spectrophotoemeter, NanoDrop Technologies, Wilmington, DE)
If needed, the sample was diluted to approximately 100 ng/uL and then it was

stored at -20°C until needed.

62

2.2.3. PCR amplification

The polymerase chain reactions (PCRs) were carried out using the Taq DNA

Polymerase kit (Invitrogen, Carlsbad, CA). Each reaction included the following

components:

5 uL of 10X bovine serum albumin

0 5 uL 10X PCR buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl)
0 5 uL 2 mM dNTP’s

o 1.5 uL of 50mM MgCl

0 1.5 uL (15 pmoles) 10X forward primer

0 1.5 uL (15 pmoles) 10X reverse primer

0 1 uL of template DNA (~100 ng)

o 0.25 uL of T aq polymerase

0 29.25 uL of molecular grade water for a total reaction volume of 50uL

PCR conditions were optimized for each primer set, and in general were close to
the following: 94°C for 5 minutes, (94°C for 30 seconds, 57°C for 30 seconds, 72°C for
30 seconds) 35 times, 72°C for 30 seconds and then held at 4°C. 5 uL of PCR product
was run on a 1% agarose gel with ethidium bromide for visualization in TAE (tris-
acetate-EDTA) buffer. Gels were documented with an Eagle Eye System (Stratagene,

LaJolla, CA).

63

2.2.4. Purification of PCR products for sequencing
2.2.4.a. Sodium acetate and isopropanol PCR product purification
If the PCR product was robust and contained one single band, it was purified with
isopropanol and sodium acetate.
0 4.5 uL of 3 M sodium acetate added to the remaining PCR product (usually 45 uL
remained) and mixed by pipetting
0 31.5 uL of 100% isopropanol added to the tube, mixed and incubated for 5
minutes at room temperature
0 Centrifuged at 20,800 x g for 10 minutes, supernatant discarded and the pellet
allowed to dry for 10 minutes
0 40 uL molecular water added to the tube and the pellet allowed to resuspend for

24 hours at room temperature, then stored at -20°C until needed

2.2.4.b. Purification of PCR products cut from gel

When the PCR resulted in more than one product, the band of the predicted size
was cut from the gel and the PCR product was isolated using the QIAquick Gel
Extraction Kit (Qiagen, Valencia, CA) as summarized below. The exact contents and

concentrations of the Qiagen kit’s solutions were proprietary and not possible to provide.

0 Band cut from gel under UV illumination, transferred to a 1.5 mL centrifuge tube

and weighed

0 3 volumes of Buffer QG to one volume of gel were added to the tube and

vortexed

64

0 Tube incubated at 50°C for 10 minutes until the gel slice had dissolved

o The resulting solution was applied to the provided column in a clean
microcentrifuge tube and then centrifuged at 20,800 x g for one minute

0 Flow-through was discarded, an additional 500 uL of Buffer QG applied to the
tube, recentrifuged for one minute and the flow-through was discarded

0 750 uL of Buffer PE (wash buffer) applied to the column (provided with the
Qiagen kit) and allowed to incubate for three minutes at room temperature.

0 Column and tube were spun again for one minute, the flow-through discarded and
then centrifuged again for one minute

0 Column placed into a clean 1.5 mL microcentrifuge tube, 30~50 uL of molecular
grade water was applied to the column and allowed to stand at room temperature
for one minute

0 Column and tube centrifuged for one minute to elute the PCR product, which was

stored at -20°C until needed

The purified products (whether sodium acetate purified or cut from the gel) were
then run on a 1% agarose gel with ethidium bromide in TAE buffer again and the
concentrations were estimated by comparison to a standardized DNA ladder. The
products were then sent to Michigan State University’s Genomics Technology Support
Facility for direct sequencing using the appropriate primer (from those used to create the
PCR product) to separately sequence forward and reverse sequences. The sequences
were aligned and analyzed using Sequencher 4.0 (Gene Codes Corporation, Ann Arbor,

' MI). Differences between the affected and carrier sequences were recorded.

65

2.2.5. Amplification of genes from retinal cDNA

Reverse transcriptase polymerase chain reaction (RT-PCR) was used with chicken
retinal mRNA to confirm expression in the retina and to check for the possibility of
alternate splicing. cDNA sequences for each gene were generated using mRNA extracted
from wild type retinas (and carrier and affected retinas for GNB3) using a similar
protocol for the genomic sequences (as far as primer design — except exonic primers were
selected) and RT-PCR. Table 2.3 contains the primer sequences used for the cDNA

amplification and sequencing.

66

Table 2.3.

Legend follows table (see below)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Gene Forward Primer Reverse Primer Product

Size

(b9)

1nqc1 TCAGCCAAGCTCTTGGGATTCTGT TGAACCTGATCTGTGTCTGCATGG 642
Chr 1: 79291731-79291754 Chr 1: 79297276—79297296

ffliCll ACGCAGTCTGTTCTCCTTGGGAAT TGGTGATGAATGGCAATCTGCTGC 330
Chr 1: 79296115-79296138 Chr 1: 79299311—79299334

IfiiCl AGACTGTGGGCATGAACCTTACCA TGACTCTGCCTTGCGCTTTATTGC 357
Chr 1: 79298837-79298860 Chr 1: 79302122-79302145

[q1C1 ATGGCATCAAcccrorciiccier iciccrAATGCCACGAGCCTTTGA 808
Chr 1: 79306654-79306677 Chr 1: 79307659—79307682

(XDPS7A. TCTTCCACCTGCTCACCATCTTCG bLLL1llbbLtlibbAAbiL11L11b 603
Chr 1: 80303413—80303436 Chr 1: 80305254-80305278

(X)PS7A. GTTAGCCGAGCCAACCAACACAAA TCCAAGCTTCTGTGATGCCCTACA 620
Chr 1: 80304784-80304807 Chr 1: 80305938—80305961

(3PRJ62 TATCATTCATCCTCTCCACCCTGC TACCATTCTGTGCTTGCCTCGTCT 919
Chr 1: 80384229—80384252 Chr 1: 80388082—80388105

(3PRJ62 ACAACAATTCACTGTGGTGGCTGG ACCAAAGCCCAGACCTATCTTGCT 497
Chr 1: 80383842-80383865 Chr 1: 80384315-80384338

GPHB3 AGATCCTGATTCAAuoriccccAC lLllleleLLlelLlLAATGT 584
Chr 1: 80406258-80406272 Chr 1: 80408300-80408323

Gpugg TGTGCACTCTGGGACATTGAGACA lblGLllbbLLUATGALUleilb 597
Chr 1: 80408288-80408310 Chr 1: 80410424—80410447

EPMDZ ACCAGCACTTTCTCAGTCTCTCGT AAGCTGGCACAGGAAGGATGAGAT 493
Chr 1: 80444868-80444891 Chr 1: 80450554—80450574

Ebfljz ACAAGGCTGGCTACACAGACAAGA TGATGTGGGAAAGAGCTGAAGGGT 657
Chr 1: 80451899-80451922 Chr 1: 80453209—80453232

EhKJZ AACTGCCTCCTGCTCAAAGTCAAC ACTGCACATGCTTCGGCATACAAC 640
Chr 1: 80452342—80452365 Chr 1: 80453509—80453532

EhKDZ ACCCTTCAGCTCTTTCCCACATCA ibllbLlllbLibAbLblleCTG 805
Chr 1: 80453209-80453232 Chr 1: 80454003—80454026

A11q1 AGGTGACTGTAGTAATCCTCCTGGGT AGGTTCAACAAGCACCTGGATCGT 935
Chr 1: 80482104—80482615 Chr 1: 80479498-80479521

PTTdQ6 AAGGCACCTCTCCAGAAACAGAGT TGAAGTGCTCCACAAGATCTGCCA 847
Chr 1: 80498148-80498171 Chr 1: 80502806—80502829

pjjnq6 CCGTCTCAAAGTCACCCACATCAA CGCATTTGGAATGCTCTCCTGCTT 859
Chr 1: 80502065—80502088 Chr 1: 80504949-80504972

pjjnq6 ATGATAGTGAGGCTGTGCGTGAGA CACACCATCACACACTGGCATA 1145
Chr 1: 80504841-80504865 Chr 1: 80507484—80507505

CLSTTJ3 CCGCTGTTTGCACTGGACAAAGAT AGGCAGCAAGTCAATCTCTCCTGA 816
Chr 1: 80570804-80570827 Chr 1: 80575125-80575148

CLSTTJ3 AGGGCTGGAACAAGAGGATTGAGT TGTCATGAATGAGGGCAGGGTCAT 675
Chr 1: 80574496-80574517 Chr 1: 80575897-80575920

Cszqq3 ACTTCACCCTGTCTGTGTGGATGA AAGCGCAGGGAATTCATGTAAGCG 688
Chr 1: 80575294—80575317 Chr 1: 80578308—80578331

Cszqq3 TTGGAGAGCLUUUHUULLHLLUHA 11LL1L1LATAAAGAGCAGCGCCA 722
Chr 1: 80577956—80577979 Chr 1: 80581125-80581148

CLSTTQ3 TATTTCTCACCAGGTGGAGGCCAA TTACCTGCTCTGCCCTCACAGTTT 777
Chr 1: 80579008-80579031 Chr 1: 80583285-80583308

zyj( AGACTCTGCCAGTGCAGTTGGTTA ACAGTGGTCGGACAGCGTAGTTAT 905

 

Chr 1: 80671593-80671616

 

Chr 1: 80670711-80670734

 

 

 

67

 

Table 2.3. Continued

 

 

 

 

 

 

 

 

 

 

 

 

 

ZYX TATGCTCCACGCTGCTCAGTATGT ACTCTGCTTCCACACTCTTACCCA 747
Chr 1: 80672539-80672562 Chr 1: 80671202-80671225

IZYQ( ACCTCAGCCTCCCAATTTCACCTA ACACGCACTGTCTCATCTTTCCCA 657
Chr 1: 80673952—80673975 Chr 1: 80672492-80672515

zyg( ACCCACAGAAGAAATTTGCACCCG TGCAGTGGGTCTACGCATGTCTTT 854
Chr 1: 80678473-80678497 Chr 1: 80673500-80673524

(33(9 AGTATCGCAAGTTCACCTCCTCCA AGGTGCTTGTGGTCTGTCCAAACT 870
Chr 1: 81094865-81094888 Chr 1: 81088237-81088260

(13(9 AATTTGGAGAGGTGTGCTTTGGGC GAGCAGATGGCTTGCGGATCATTT 777
Chr 1: 81099360—81099383 Chr 1: 81094469-81094492

CIH<9 TGTCATCTGCAAGGAATGCCCAAC AGGAACTCTCGACGCTGTTCATCA 924
Chr 1: 81109684—81109707 Chr 1: 81099272—81099295

(Ingg AGTGTCCAGCTGTGGTGAAAGGAT AGGCCAAGACAGTGTGATGCTACT 806
Chr 1: 81127028-81127051 Chr 1: 81104640-81104663

(13(9 GCTCTGGCTGGTTTGCTTCTTTCA CAGTTGTGCTTGCCTCTTCAGCAT 692
Chr 1: 81143172-81143195 Chr 1: 81126929-81126952

Table 2.3. cDNA primers used to sequence candidate genes. The table includes the

gene, the forward and reverse primer, the length of the expected product and the location

on chicken chromosome one (based on version 2.1 of the draft chicken genome assembly
from May 2006).

68

 

2.2.5.a. RNA Extraction

Retinas were collected from adult chickens, flash frozen in liquid nitrogen and

then stored at -80°C until needed. The RNeasy Mini-Kit (Qiagen, Valencia, CA) was

used to isolate the RNA from the tissues. All reusable materials (mortar and pestle,

spatulas, etc) were washed with RNAse Away (Invitrogen Corp., Carlsbad, CA) and

baked at 200°C for 24 hours to remove RNAses. The exact contents and concentrations

of the Qiagen kit’s solutions were proprietary and not possible to provide.

Frozen retina ground under liquid nitrogen in a mortar and pestle and then the

powder was transferred to a microcentrifuge tube
600 11L Buffer RLT added and the sample was mixed/disrupted using a 22
gauge needle on a 3 mL syringe

Sample centrifuged at 16,100 x g for 3 minutes and the supernatant transferred

via pipette to a clean tube

1 volume of 70% ethanol was added and mixed by pipetting

Mixture placed on an RNeasy spin column in a clean tube, centrifuged for 15
seconds and the flow-through discarded

700 11L of Buffer RWl added to the column, recentrifuged for 15 seconds to
wash the column and then the flow-through discarded

500 uL Buffer RPE was added, recentrifuged for 15 seconds to wash the
column again and the flow-through was discarded

An additional 500 uL Buffer RPE was added, column centrifuged for 2

minutes and the flow-through discarded

69

0 Column transferred to a clean tube and then recentrifuged for one minute to

remove any remaining Buffer RPE
0 Column again transferred to clean microcentrifuge tube, 30 11L of RNase-free

water was applied to the column and then centrifuged 1 minute to elute the

RNA

0 RNA stored at -80°C until needed

The RNA was treated to remove DNA using DNase enzyme (Roche Diagnostics
Corporation, Indianapolis, IN). Briefly, 1 11L of DNase enzyme and l uL of 10X
Reaction Buffer (Roche — contents proprietary) with magnesium chloride was added per
8 uL of RNA solution. This solution was incubated at 37°C for one hour and then stored

at —80°C until needed.

2.2.5.b. cDNA Synthesis

The DNA-free RNA was then used to create cDNA using the Fermentas First
Strand cDNA Kit (Fermentas Inc., Hanover, MD). The exact contents and concentrations
of the Fermentas kit’s solutions were proprietary and not possible to provide.

6 2 uL mRNA (of unknown concentration) combined with 0.5 ug of oligo(dT).g

primer and nuclease free water to reach 11.5 11L
0 Mixture incubated at 70°C for 5 minutes and then chilled on ice
0 4 11L 5X reaction buffer for reverse transcriptase, 2uL dNTP Mix (lOmM

each), 0.5 11L RiboLockTM Ribonuclease Inhibitor (20u), and then DEPC-

70

treated water to reach '18 11L added to the tube and incubated at 37°C for 5
minutes

0 40 units of reverse transcriptase (M-MuLV Reverse Transcriptase) was added
to the reaction, it was incubated at 37°C for 60 minutes, then 70°C for 10
minutes and then chilled on ice

0 This cDNA was then stored at -20°C until needed

Primers were created as described previously and used to generate products,
which were then sequenced and analyzed with Sequencher 4.0. Table 2.3 shows the
cDNA primers. The cDNA sequences were used to verify expression in the retina and to

check exon boundaries.

71

2.2.6. Analysis of sequencing results

Using Sequencher 4.0, reading frames were determined and polymorphisms were
noted, the majority of which were single nucleotide polymorphisms (SNPs). Nucleotide
variations that resulted in amino acid changes were analyzed by comparing the deduced
amino acid sequence with that of human or other available species and/or run through
SIFT (Sorting Intolerant from Tolerant) (http://blocks.fhcrc.org/sifi/SIFT.html) and/or
Polyphen (http://www.bork.embl-heidelberg.de/PolyPhen/) programs. Aligning amino
acid sequences across species was accomplished with the CLUSTALW program at the

Multiple Sequence Alignment website (http://align.genome.jp/).

2.2.7. Investigation of SNPs identified in candidate genes

Birds of known status (30 homozygous affected and 39 obligate carriers) were
genotyped for one SNP from each of the genes EN02, COPS7A and GPR1’62 using
pyrosequencing at the USDA Avian Disease and Oncology Laboratory in Dr. Hans
Cheng’s lab. This was performed to assess whether the genes were in linkage
disequilibrium with the rge status, indicating whether it was possible for the gene in

question to be a candidate gene.

2.2.8. Investigation of GNB3
GNB3 was also sequenced from retinal cDNA from wild type and affected birds
as previously described to confirm Tummala et al’s findings. Figure 2.2 is a schematic

diagram of the GNB3 cDNA sequence with cDNA primers and mutation marked.

72

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73

2.2.8.a. Development of PCR RE test to genotype birds for GNB3 mutation

A custom made computer program designed to develop mismatch restriction
enzyme digest tests (Petersen-Jones 2003 unpublished) was used to create a restriction
enzyme test for rapid genotyping of birds for the GNB3 mutation. As there were no
restriction enzymes that could naturally be used in differentiating the normal allele from
the affected allele, a mismatch primer was made that preserved a restriction enzyme
cleavage site at the deletion site in the resulting PCR product 6from the normal birds, but
removed the cleavage site from the affected ones (Figure 2.3). The restriction enzyme
prIII188 has a restriction site sequence of TCNNGA, and when a PCR was run with a
mismatch primer, the product from the normal allele had an pr111188 cleavage site
associated with the site of interest, while the mutant allele did not because of the deletion.
The PCR product was 192 bp for the wild type allele and 189 bp for the rge allele. After
restriction enzyme digestion of the PCR product, affected birds had a single product of
189 bp, normal birds had two products — one that was 161 bp and another that was 31 bp
in length. The carriers had three products (189, 161 and 31 bp).

The PCR with the mismatched primer was run with an annealing temperature of
55°C. A sample of the PCR product was run in a 1% gel to ensure that the PCR
amplified the product and that the product was the correct size (192 bp) and then the rest
of the PCR product was digested with the prIIIl88 enzyme. For a 20 uL digestion
reaction the following components were used:

0 12 11L of PCR product
0 0.25 uL of pr111188 enzyme (New England Biolabs, Ipswich, MA)

0 2 uL of 10X NEBuffer 4 (provided with the enzyme, contents proprietary)

74

0 2 11L 10X bovine serum albumin

0 3.75 uL molecular grade water.

These components were mixed and allowed to digest at 37°C for one to two hours. The

product of this incubation was run in a 4% agarose gel. The higher percentage gel was

required to differentiate the size difference in the bands.

75

GENOMIC DNA FROM THE AREA OF INTEREST OF GNBB
Exon 5CGCGTGAAGGCAACGTCAAAGTGAGCAGGGAACTCTCAGCTCATACAth
Gagtgttctgcctaccagtggcctgggtgtctctgcttccaaatctcact
cttctaaccctgctctgtctctcatactcactggttcctgctccccagGT
Exon 6TACCTCTCCTGCTGCCGGTT(TCTTGA)TGACAACAGTATTGTGACTAGC
TCTGGAGATACCACATthaagtattatcctctatcttattgacatgagc

GAT = deleted codon from affected birds
(TCTTGA) = prlBBIII cleavage site

DESIGN OF ASSAY
Exon 5CGCGTGAAGGCAACGTCAAAGTGAGCAGGGAACTCTCAGCTCATACAth
gagtgttctgcctaccagtggcctgggtgtctctgcttccaaatctcact
cttctaaccctgctctgtctctcatactcactggttcctgctccccagGT
Exon 6 TACCTCTCCTGCTGCCGGTT(TCTTGA)TGaCAACAGTATTGTGACTAGC
TCTGGAGATACCACATthaagtattatcctctatcttattgacatgagc

 

 

ACGTCAAAGTGAGCAGGGAACTCT = forward primer
GcCAACAGTATTGTGACTAGCTCTGGA = mismatch reverse primer

 

 

PCR PRODUCT - WILD TYPE ALLELE
ACGTCAAAGTGAGCAGGGAACTCTCAGCTCATACAthgagtgttctgcc
Taccagtggcctgggtgtctctgcttccaaatctcactcttctaaccctg
ctctgtctctcatactcactggttcctgctccccagGTTACCTCTCCTGC
TGCCGGTT(TCTTGA)TGcCAACAGTATTGTGACTAGCTCTGGA

prlBBIII cleavage site present, so two bands created after
digestion (161 bp and 31 bp)

 

 

PCR PRODUCT AFFECTED ALLELE:
ACGTCAAAGTGAGCAGGGAACTCTCAGCTCATACAGgtgagtgttctgcc
taccagtggcctgggtgtctctgcttccaaatctcactcttctaaccctg
'ctctgtctctcatactcactggttcctgctccccagGTTACCTCTCCTGC
TGCCGGTTTCTTGCCAACAGTATTGTGACTAGCTCTGGA

No prlBBIII cleavage site present, so only one band of
189 bp present after digestion

 

 

Figure 2.3. Restriction enzyme test to establish GNB3 status. This figure shows the
restriction enzyme test designed to distinguish normal (+/+) from carrier (rge/+) from
affected (rge/rge) birds. A mismatch reverse primer removes the pr188III cleavage site
from the affected bird sequence but doesn’t affect the site in the normal bird sequence.
Thus the PCR products from normal birds have two bands (161 and 31 bp), those of
carrier birds have three bands (189, 161, 31 bp) and those of affected birds have only one
band (189 bp). UPPERCASE = exonic sequence, lowercase = intronic sequence.

76

2.2.8.b. GNB3 immunoreactivity in the normal chicken retina
2.2.8.b.i. Chicks

Chicks used in the immunohistochemistry studies were from the rge colony of
chickens, which is housed under 12 hour lightzdark cycles in the Vivarium facility at
Michigan State University’s College of Veterinary Medicine. Fertile eggs were produced
in two ways; natural insemination between male and female carriers and artificial
insemination between affected roosters and carrier females. The eggs were collected
once a day, stored at 50°F in a humidified cooler and incubated in batches. The eggs
were hatched in incubators (Hova-Bator, G.Q.F. Manufacturing Co., Savannah, GA).
The chicks were typed (control or affected) via electroretinogram or their MCWO318
status was determined (Hans Cheng’s Laboratory). All procedures were conducted in
accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research and approved by the Michigan State University All-University Committee on

Animal Use and Care.

2.2.8.b.ii. Retina Collection

Immediately following euthanasia using an overdose of pentobarbital (Fatal Plus,
Vortech Pharmaceutical, Dearbom, MI), bilateral enucleation was performed. The eyes
were hemisected at the equator, the vitreous body removed and the posterior segment of

the eye immersed into fixative.

77

2.2.8.b.iii. Fixation, Embedding and Staining

For IHC analyses, retinal samples were fixed in 4% paraforrnaldehyde, 3%
sucrose in 0.1 M phosphate buffer for 48 hours at 4°C and then dehydrated in ethanol
from 65 to 100% concentration, rinsed twice in xylene, and embedded in paraffin blocks
prior to sectioning. Five-um thick sections were cut and mounted on double-gelatinized
glass slides and dried at 65°C for 20 minutes. After deparaffination in xylene, sections
were rehydrated gradually in ethanol (100% then 95%) and, finally in distilled water.

The sections were incubated in a preheat antigen retrieval buffer (Citrate buffer,
DakoCytomation, Carpinteria, CA) for 20 minutes at 97°C. After the section had been
cooled to 50°C, it was incubated in 50mM TRIS-buffered saline (TBS; pH 7.6) for 5
minutes, followed by 10 minutes incubation with a protein-blocking agent
(DakoCytomation) prior to application of the primary antibody. Goat anti-rabbit
secondary antibody from the Labeled Streptavidin-Biotin 2 System and Horseradish
Peroxidase (LSABZ System-HRP, DakoCytomation) was used to reveal primary
antibody-positive immunoreactivity. Immunoreaction was visualized with 3,3’-
diaminobenzidine substrate (Liquid DAB substrate chromogen system,
DakoCytomation), and the sections were counterstained with hematoxylin (Gill III
formulaTM, Surgipath Medical Industries Inc., Richmond, IL) for 10 minutes. The
sections were then washed in distilled water, rinsed in tap water for 5 minutes, blued with
0.04% lithium carbonate, washed again in distilled water, dehydrated in 100% and 95%
ethanol then in xylene before being coverslipped.

The primary antibody used was a polyclonal anti-GNB3 antibody raised in rabbits

against the peptide (ADITLAELVSGLEW) and affinity purified (Invitrogen

78

Corporation, Carlsbad, CA) (received as a gift from Dr. Anand Swaroop, University of
Michigan) (Yu et al., 2004). Immunostaining was carried out at MSU’s Diagnostic

Center for Population and Animal Health by the technicians in the Histopathology

Laboratory.

2.3. Results

A compilation of the polymorphisms found in the twelve sequenced genes can be
found in Table 2.4. The sequencing of each gene in its entirety was not completed
because this area of the work stopped once the causal gene and mutation were identified.
At. the time the molecular work was being completed, the actual number of exons in
chicken TULP3 was unknown; however, 4 exons were sequenced. The cDNA sequence
of EphAl was unobtainable after repeated trials using retinal cDNA from an adult
chicken and therefore it was assumed that it was not expressed in the adult retina.

A total of 128 polymorphisms were found. The majority of them (126) were
single nucleotide polymorphisms (SNPs), with 94 of them being intronic and 32 of them
being exonic. Additionally, two insertion/deletions were found. Most of the exonic
SNPs were “synonymous” meaning they did not cause an amino acid change because
they were in the third codon position (wobble position). However, 5 of the exonic SNPs
were “non-synonymous” meaning they caused an amino acid change. One non-SNP

polymorphism was an exonic 6 bp deletion in both carrier and affected chickens in CEK9

compared to the published mRNA sequence (U23783.1). A 3 bp deletion in the coding

region of the GNB3 gene was found in the affected retinal cDNA (as described by

Tummala et al 2006) but not in the wild type transcript.

79

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80

 

 

Table 2.5. Legend follows table (see below)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Gene SNP Affected Carrier Exon/lntron Position
PHCl c/t c/c c/t Intronic 79294985
PHCl c/t c/c c/t Intronic 79295134
PHCl a/ g a/ g g/ g Intronic 79301971
PHCl c/t c/t t/t Intronic 79302263
PHCl G/C G/G G/C Exonic 79303210
COPS7a G/C C/C G/C Exonic 80304771
COPS7a g/a a/a g/a Intronic 80304924
COPS7a G/A A/A G/A Exonic 80305939
GPR162 c/t c/c c/t Intronic 80386502
GPR162 t/c t/t t/c Intronic 80386919
GPR162 G/A G/G G/A Exonic 80386959
GPR162 g/a a/a g/a Intronic 803871 11
GPR162 c/t c/c c/t Intronic 803 87154
GPR162 c/t c/c c/t Intronic 80387167
GPR162 a/g a/a a/g Intronic 80387210
GPR162 C/T C/C C/T Exonic 803 88044
GPR162 g/c c/c g/c Intronic 803 88162
GNB3 GAT mutation Exonic 80407769-

in rge bird 80407771
GNB3 G/A A/A G/A Exonic 80407596
GNB3 C/T T/T C/T Exonic 80409299
GNB3 T/C C/C T/C Exonic 80410288
En02 t/c t/t t/c Intronic 80448998
En02 g/a g/ g g/a Intronic 80449226
En02 t/c t/t t/c Intronic 80450390
En02 g/a g/ g g/a Intronic 80451299
En02 T/C T/T T/C Exonic 80451573
En02 a/ g a/ a a/ g Intronic 80451673
En02 c/t c/c c/t Intronic 8045 l 708
En02 t/ g t/t t/ g Intronic 80451790
En02 a/ g a/a a/ g Intronic 8045 l 846
En02 a/g a/a a/g Intronic 80452093
En02 t/a t/t t/a Intronic 80452533
En02 t/c t/t t/c Intronic 80452663
En02 t/c t/t t/c Intronic 80452967
En02 c/t c/c c/t Intronic 80453035
En02 t/c t/t t/c Intronic 80453228
ATNl C/T C/C C/T Exonic 80477586
ATNl c/t c/c c/t Intronic 80477679
ATNl g/a a/a g/a Intronic 80477833
ATN 1 OT C/C C/T Exonic 80479960
ATNl C/T C/C C/T Exonic 80479963
ATN 1 GM G/G G/A Exonic 80480044
ATN] c/g g/ g c/g Intronic 80481707
ATNl t/a a/ a t/a Intronic 80481763
PTPN6 A/G A/A A/G Exonic 80498822
PTPN6 a/g a/a a/g Intronic 80499374

 

 

 

 

 

 

81

 

 

Table 2.5. Continued

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PTPN6 a/g g/ g a/ g Intronic 80499755
PTPN6 g/a g/ g g/a Intronic 80502917
PTPN6 t/c c/c t/c lntronic 80503178
PTPN6 g/t t/t g/t lntronic 80503406
PTPN6 t/a t/t t/a Intronic 80503412
PTPN6 a/ g g/g a/g Intronic 80503498
PTPN6 c/a c/c c/a Intronic 80506546
PTPN6 a/t a/a a/t Intronic 80506547
CLSTN3 a/ g a/a* g/a Intronic 80570927
CLSTN3 G/C G/G* C/G Exonic 80573569
CLSTN3 A/T A/A* T/A Exonic 80573671
CLSTN3 a/g a/a* g/a Intronic 80576883
CLSTN3 t/a, t/t* t/a Intronic 80576896
CLSTN3 c/a c/c* c/a lntronic 80576937
CLSTN3 a/g a/a* g/a lntronic 80576938
CLSTN3 g/t g/g* t/g Intronic 80576954
CLSTN3 c/t c/c* t/c lntronic 80577189
CLSTN3 T/C C/C T/C Exonic 80578867
CLSTN3 a/ g g/g a/g Intronic 80579147
CLSTN3 c/ g g/g c/g Intronic 80579255
CLSTN3 a/g g/g a/g Intronic 80582879
CLSTN3 G/A A/A‘l' G/A Exonic 805831 13
CLSTN3 t/c c/c t/c Intronic 80583181
CLSTN3 c/t t/t c/t Intronic 80583200
CLSTN3 c/t t/t c/t lntronic 80583215
CLSTN3 c/t t/t c/t Intronic 80583233
CLSTN3 A/G G/G'l' A/G Exonic 8058331 1
EphAl g/a a/a g/ g Intronic 80635136
EphAl a/c c/c a/c lntronic 80639103
EphA 1 OT C/C C/T Exonic 80653703
EphAl G/A G/G G/A Exonic 80653727
EphAl g/t g/ g g/ g Intronic 80653 799
EphAl t/c t/t t/c Intronic 80658597
EphAl g/a a/a g/a Intronic 80658612
EphAl c/t t/t c/t lntronic 80659402
EphAl c/t c/c c/t lntronic 80659672
EphAl c/g c/c c/ g Intronic 80659679
EphAl t/c t/t t/c lntronic 80659710
EphAl G/A A/A'l' G/A Exonic 80659816
EphAl c/t t/t c/t Intronic 80660491
EphAl g/a g/ g g/a lntronic 80660520
EphAl t/c c/c t/c Intronic 80660948
EphAl C/T C/C C/T Exonic 80661294
EphAl a/t t/t a/t lntronic 80661840
EphAl c/t t/t c/t Intronic 80661844
EphAl t/c t/t t/c Intronic 80661887
EphA 1 t/c t/t t/c lntronic 80661938
EphAl c/ g c/c c/ g Intronic 80661941

 

 

 

 

 

 

82

 

 

Table 2.5. Continued

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

E hAl g/c g/ g g/c lntronic 80662030
EphAl C/T C/C C/T Exonic 80662702
EphA 1 g/a a/a g/a lntronic 80662915
EphA 1 g/c g/ g g/c Intronic 80662981
EphAl a/t t/t a/t lntronic 80663419
EphAl G/ A G/G G/A Exonic 80665007
EphAl A/G G/G A/G Exonic 80665253
ZYX a/ g a/a a/ g Intronic 80676221
ZYX c/ g c/c c/ g lntronic 80676108
ZYX t/c t/t t/c lntronic 80675843
ZYX C/T T/T C/T Exonic 806741 19
ZYX g/c g/ g g/c lntronic 80673643
ZYX c/t c/c c/t lntronic 80672793
ZYX a/c c/c a/c lntronic 80672759
ZYX a/ g a/a a/g lntronic 80672658
ZYX t/c c/c t/c lntronic 80672592
CEK9 t/c t/t t/c lntronic 81 143482
CEK9 C/T C/C C/T Exonic 81 127231
CEK9 G/A A/A G/A Exonic 81127131
CEK9 CTGCCA Exonic 81 1 14604-
indel 81 1 14609
CEK9 T/C C/Ci‘ T/C Exonic 81 114581
CEK9 T/C T/T T/C Exonic 81 1 14430
CEK9 c/t t/t c/t lntronic 81 109959
CEK9 t/c c/c t/c lntronic 81 109905
CEK9 g/a g/g g/a lntronic 81 103132
CEK9 c/t t/t c/t Intronic 81102932
CEK9 t/c t/t t/c lntronic 81 102282
CEK9 t/g g/g t/g lntronic 81100282
CEK9 C/G C/CT C/G Exonic 81093332
CEK9 c/t t/t c/t Intronic 81093232
CEK9 c/t t/t c/t Intronic 81093159
CEK9 g/a a/a g/a Intronic 81093031
CEK9 g/a g/g g/a Intronic 810931 12
CEK9 G/A A/A G/A Exonic 81088082

 

 

 

 

Table 2.5. Polymorphisms found during investigation. *=affected same as published. '1'=SNP
changes amino acid in affected bird (see text for description of change).

83

 

The affected bird was heterozygous for two of the intronic SNPs in PHCl,
suggesting a crossover event between the PHC 1 locus and the disease locus.

Pyrosequencing revealed that each of the SNPs analyzed from the three genes
(EN02, COPS7A and GPR162) was in linkage disequilibrium with disease status
meaning that all of the carriers were heterozygous and all of the affected birds that were
tested were homozygous at the locus.

The non-synonymous SNP in EphAl caused a cysteine to tyrosine amino acid
change in the affected bird. The SIFT program described the substitution as “tolerable”
and the Polyphen program reported the change as “benign.” In addition to the SIFT and
Polyphen results, the amino acid substitution is not in a well-conserved region (across
species) suggests the change may not affect protein function.

The first non-synonymous SNP in the CEK9 gene changes an isoleucine residue
to a threonine residue in the affected birds. SIFT predicted that this substitution would be
“tolerated” and Polyphen predicted the change to be “benign.” This coupled with the fact
that it is not a well-conserved region (across species) suggests the amino acid substitution

may not affect protein function. The second amino acid changing SNP in CEK9 was a

“wild type” SNP meaning the affected sequence was identical to the published genomic

and mRNA sequences (NM_001004387, U23783.1) and the carrier sequence is the one

that differed. The wild type and affected sequence coded for a glutamine and the carrier
sequence indicated that the homozygous normal bird sequence would code for glutamic
acid. The 6 bp indel found in CEK9 was very close to the first SNP (Ile to Thr
substitution) and was thus in a region of the gene that is not well conserved. The

published mRNA sequence for CEK9 (U23783.1) contained 6 bp (CTGCCA — leucine

84

and proline residue) whereas both of the carrier and affected DNA samples were
“missing” the 6 bp in the genomic sequence, as was the wild type retinal cDNA sample
when compared to the published mRNA sequence (U23783.1). The published genomic
sequence (NM_001004387) was also “missing” these 6 bp. The area of the protein
sequence that is affected by these insertion/deletions is not well conserved across species,
therefore this polymorphism is difficult to evaluate. This evidence suggests that either
there is an error in the published mRNA sequence or that it is insignificant.

GNB3’s sequence from retinal cDNA confirmed Tummala et al’s findings; a 3 bp
deletion of the 153rd codon in the affected birds was found. Three synonymous exonic
SNPs were also found. The 3 bp deletion was in frame and removed an aspartic acid
residue. When this deletion was entered in the CLUSTALW program
(http://align.genome.jp/) and compared with mouse, rat and human GNB3, it became
apparent that this amino acid deletion occurred in a highly conserved region across
species (Figure 2.4), suggesting that its deletion was potentially significant.

The restriction enzyme test was developed successfully, allowing typing of chicks
of unknown status. Figure 2.5 shows a gel with the results of the restriction enzyme test
of a normal, a carrier and an affected bird. Note that the 31 bp band is not visible on the

gel due to its small size.

85

Mouse_NM_013530_GNB3
Rat_NM_021858_GNB3
Human_NM_00207S_GNB3
RGE_Chicken
Normal_Chicken_GNBB

Mouse_NM_Ol3S30_GNBB
Rat_NM_021858_GNB3
Human_NM_OOZO75_GNB3
RGE_Chicken
Normal_Chicken_GNB3

Mouse_NM_Ol3530_GNB3
Rat_NM_021858_ GNB3
Human_NM_OO2075_GNBB
RGE_Chicken
Norma1_Chicken_GNB3

Mouse_NM_013530_GNB3
Rat_NM_021858_ GNB3
Human_NM_002075_GNB3
RGE_Chicken

Normal_Chicken_GNB3

Mouse_NM_013530_GNB3
Rat_NM_O21858_ GNB3
Human_NM_002075_GNB3
RGE_Chicken
Normal_Chicken_GNBB

Mouse_NM_013530_GNB3
Rat_NM_021858_ GNB3
Human_NM_002075_GNBB
RGE_Chicken

Normal_Chicken_GNB3

Mouse_NM_Ol3S30_GNB3
Rat_NM_021858_ GNB3
Human_NM_00207S_GNB3
RGE_Chicken

Normal_Chicken_GNB3

MGEMEQLRQEAEQLKKQIADARKACADITLAELVSGLEVVGRVQMRTRRT
MGEMEQLKQEAEQLKKQIADARKACADITLAELVSGLEVVGRVQMRTRRT
MGEMEQLRQEAEQLKKQIADARKACADVTLAELVSGLEVVGRVQMRTRRT
MGEMEQMKQEAEQLKKQIADARKACADTTLAQIVSGVEVVGRIQMRTRRT
MGEMEQMKQEAEQLKKQIADARKACADTTLAQIVSGVEVVGRIQMRTRRT

*‘k'k'k'k'k: :******************* *‘k‘k: :***:*****:*******

LRGHLAKIYAMHWATDSKLLVSASQDGKLIVWDTYTTNKVHAIPLRSSWV
LRGHLAKIYAMHWATDSKLLVSASQDGKLIVWDTYTTNKVHAIPLRSSWV
LRGHLAKIYAMHWATDSKLLVSASQDGKLIVWDSYTTNKVHAIPLRSSWV
LRGHLAKIYAMHWSTDSKLLVSASQDGKLIVWDTYTTNKVHAIPLRSSWV
LRGHLAKIYAMHWSTDSKLLVSASQDGKLIVWDTYTTNKVHAIPLRSSWV

*‘k'k'k'k‘k'k'k'k'k'k'k'k:*******************:****************

MTCAYAPSGNFVACGGLDNMCSIYNLKSREGNVKVSRELSAHTGYLSCCR
MTCAYAPSGNFVACGGLDNMCSIYSLKSREGNVKVSRELSAHTGYLSCCR
MTCAYAPSGNFVACGGLDNMCSIYNLKSREGNVKVSRELSAHTGYLSCCR
MTCAYAPSGNFVACGGLDNMCSIYNLKTREGNVKVSRELSAHTGYLSCCR
MTCAYAPSGNFVACGGLDNMCSIYNLKTREGNVKVSRELSAHTGYLSCCR

************************ .**:**********************

O

FLDDNNIVTSSGDTTCALWDIETGQQKTVFVGHTGDCMSLAVSPDYKLFI
FLDDNNIVTSSGDTTCALWDIETGQQKTVFVGHTGDCMSLAVSPDYKLFI
FLDDNNIVTSSGDTTCALWDIETGQQKTVFVGHTGDCMSLAVSPDFNLFI
FL-DNSIVTSSGDTTCALWDIETGQQKTVFLGHTGDCMSLAVSPDFKLFI
FLDDNSIVTSSGDTTCALWDIETGQQKTVFLGHTGDCMSLAVSPDFKLFI

** ** .************************:**************: :***

SGACDASAKLWDVREGTCRQTFTGHESDINAICFFPNGEAICTGSDDASC
SGACDASAKLWDVREGTCRQTFTGHESDINAICFFPNGEAICTGSDDASC
SGACDASAKLWDVREGTCRQTFTGHESDINAICFFPNGEAICTGSDDASC
SGACDATAKLWDVREGTCRQTFSGHESDINAICFFPNGEAICTGSDDATC
SGACDATAKLWDVREGTCRQTFSGHESDINAICFFPNGEAICTGSDDATC

******:***************:*************************:*

RLFDLRADQELTAYSQESIICGITSVAFSLSGRLLFAGYDDFNCNVWDSL
RLFDLRADQELTAYSHESIICGITSVAFSLSGRLLFAGYDDFNCNVWDSL
RLFDLRADQELICFSHESIICGITSVAFSLSGRLLFAGYDDFNCNVWDSM
RLFDLRADQELIVYSHESIICGITSVAFSRSGRLLLAGYDDFNCNIWDSL
RLFDLRADQELIVYSHESIICGITSVAFSRSGRLLLAGYDDFNCNIWDSL

*********** :*:************* *****:*********:***:

KCERVGILSGHDNRVSCLGVTADGMAVATGSWDSFLKIWN
KCERVGVLSGHDNRVSCLGVTADGMAVATGSWDSFLKIWN
KSERVGILSGHDNRVSCLGVTADGMAVATGSWDSFLKIWN
KAERVGILSGHDNRVSCLGVTADGMAVATGSWDSFLKIWN
KAERVGILSGHDNRVSCLGVTADGMAVATGSWDSFLKIWN

* **++.+66++6¢¢é$+¢¢++++$6¢+++*6dv+*~b+*‘+

Figure 2.4. GNB3 amino acid sequence alignment. This figure shows the amino acid
sequence alignment of GNB3 aligned using the CLUSTAL program. The amino acid
sequences from mouse, rat and human were obtained from the UCSC Genome Browser
and the rge chicken sequence was obtained from a translation of sequenced retinal cDNA.
This shows how well conserved the protein is between the 4 species. The deleted amino

acid is an aspartic acid residue (D — in gray with ‘b above it).

86

M rge/rge rge/+ +/+

 

Figure 2.5. Results of the GNB3 RE test. This figure shows a 4%
agarose gel with the final results of the pr188111 restriction enzyme
test for affected (rge/rge), carrier (rge/+) and normal (+/+) birds. The
normal bird has one band 161 bp in length, the carrier has two bands
161 and 192 bp in length and the affected bird has one band 189 bp in
length. The 31 bp band is not visualized on the gel. M = molecular
ladder (100 bp DNA Ladder N3231S, New England BioLabs, Ipswich,
MA)

87

Immunohistochemistry for GNB3 in the normal chicken retina (Figure 2.6) shows
GNB3 immunoreactivity in several retinal locations. The natural pigmentation in the
retina (dark brown) must be differentiated from the lighter brown chromogen. In addition
to cone outer segments, there is also some immunoreactivity in the region of cone cell
bodies and in synaptic terminals in the outer plexiform layer. A population of cells
within the inner layer of the inner nuclear layer, (the region where amacrine cells are

located) and ganglion cell layer were also immunoreactive.

88

 

RPE

OS

6

ONL

OPL ,

INL ‘

 

 

 

Figure 2.6. GNB3 immunoreactivity in normal chicken retina. This photomicrograph
displays GNB3 antibody IHC using a rabbit anti-GNB3 polyclonal antibody (Gifi
fiom Dr. Anand Swaroop, U of Michigan). The upper image is the outer retina. The
brown chromogen used is a slightly lighter brown than the RPE pigmentation but the
similarity in color does make the immunoreactivity of cone outer segments (*) a little
more difficult to appreciate. In addition to immunoreactivity of cone outer segments
some immunoreactive (IR) structures between the photoreceptor cell bodies can be
seen (arrows). IR synaptic termini are present in the OPL (arrowheads). The lower
image is the inner retina. A subpopulation of cells in the inner aspect of the INL are
IR to this GNB3 antibody (arrows). The cell bodies of the ganglion cell layer are also
IR to this GNB3 antibody (arrowheads). Key: RPE = retinal pigment epithelium; GS
= photoreceptor outer segments; IS = photoreceptor inner segments; ONL = outer
nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; GCL =
ganglion cell layer; NFL = nerve fiber layer. This figure is presented in color.

89

2.4. Discussion

The majority of polymorphisms found in the positional candidate genes were
intronic SNPs, which is expected as introns are not as highly conserved as exons. The
majority of exonic SNPs were in the codon’s third position (wobble position) and so did
not alter the amino acid that was ultimately expressed. Each of the SNPs analyzed with
pyrosequencing was in linkage disequilibrium with the disease status, meaning affected
birds were homozygous and carriers were heterozygous. This means those three genes
(EN02, COPS7A and GPR162) were considered to be within the disease interval.

The discovery of the SNPs in PHCl that were heterozygous in the affected and
not the carrier means the gene is_not in linkage disequilibrium with the rge locus.
Because of this, PHCI is in a region that can be considered a left hand boundary for the
disease interval. As stated previously the location of TULP3 was unknown at the time
the molecular work was being done. Once the updated version of the chicken genome
was published, TULP3’s location was assigned to an area upstream from PHC].
Consequently, due to PHCl’s lack of linkage disequilibrium with the disease status,
TULP3 was determined to be located outside of the interval for the rge gene locus,
although no SNPs were found to confirm this.

The 3 bp deletion in GNB3, which removes an aspartic acid, is present only in the
affected chicken sequence. When compared to mouse, rat and human GNB3 amino acid
sequences, it is in a conserved region (Figure 2.2). All three of the other species GNB3
amino acid sequences contain the aspartic acid residue that the rge chicken is missing,
which strongly suggests that this is the rge mutation. This region is also conserved across

four of the five GNB proteins identified in humans (GNBI: NM_002074; GNB2:

90

NM_002075; and GNB4: NM_021629). GNBS’s amino acid sequence is not well

conserved across species.

GNB3 is the beta subunit of a G-binding protein and is made up of an amino-
terminal (it-helical segment followed by 7 repeating units called WD repeats that form a
propeller structure (Sondek et al., 1996). Each WD-repeat consists of 40-60 amino acid
residues bordered by 3 GH (glycine-histidine) and a WD (tryptophan-aspartic acid).
Each propeller is composed of a four-stranded anti-parallel B-sheet (Neer, 1995). The
four strands are labeled a, b, c and d. The aspartic acid deletion in the rge mutant is
positioned in a beta hairpin between the a and b B strands of the third propeller; therefore,
the deletion of this amino acid residue could possibly affect the folding of this protein
(Figure 2.7). The amino acid deletion was modeled with the “What if” computer
program using bovine GNBl as a close homologue of GNB3, and the results suggested
that this deletion abolishes [3 sheets in propellers l and 5 of the GNB protein (Tummala et
al., 2006). The CASPS committee (Critical Assessment of Methods for Protein Structure
Prediction) predicted that the mutant GNB3 protein would be unstable and liable to
premature proteolysis. This prediction was supported by the finding that there was a 70%
reduction in GNBB protein immunoreactivity in affected retinas when compared to age-

and sex-matched normal retinas (Tummala et al., 2006).

91

 

Figure 2.7. A computational model of chicken (Gallus gallus) GNB3
protein. The protein has a 7 propeller arrangement characteristic of G-
protein beta subunits. The side chain of the 153Asp deleted in the rge
chicken is shown (black arrow). It is in the beta hairpin between the a and
b beta strands of the third propeller. Figure created with the help of
William J. Wedemeyer.

92

As previously stated, when it became known that Tummala et a1 had found the
gene and causative mutation, the molecular work was abandoned except for confirming
their findings and developing the restriction enzyme test to quickly and accurately test
chicks as they hatched.

Additionally, immunohistochemistry was done in paraffin embedded control
chicken retina to establish GNB3 ’5 locations in the retina. The results obtained are
similar to those obtained by other research groups in that GNB3 immunoreactivity is
found in cones and in the inner retina (inner nuclear layer) (Peng et al., 1992; Huang et
al., 2003). Ganglion cells were also found to have GNB3 immunoreactivity, which has
not been reported elsewhere. The moderate amount of background staining that appeared
is probably due to this particular antibody’s lack of specificity for chicken GNB3. The
sequence the antibody was raised against is ADITLAELVSGLEVV (from mouse GNB3)
and the corresponding sequence in chicken GNB3 is ADTTLAQIVSGVEVV, which is

only a 73% match.

93

CHAPTER 3

FURTHER ELECTRORETINOGRAPHIC STUDIES OF THE rge CHICKEN

3.1. Introduction

The retinopathy, globe-enlarged (rge) chick has an unusual autosomal recessive
retinal dystrophy whose unique electroretinographic features have previously been
partially characterized. Previous work has shown that rge chicks have a progressive
deterioration in vision from hatch such that they are functionally blind by approximately
one month of age. Electroretinographic responses of the rge chicks are abnormal from
hatch with altered b-wave shape and reduced oscillatory potentials (OPS). In the first few
weeks of life the rge ERGs have supemorrnal b-wave amplitudes in response to brighter
flashes, which is of particular interest. The ERG responses slowly deteriorate with age
and surprisingly are maintained for a considerably longer time than functional vision,
which is unusual amongst retinopathies (Montiani-Ferreira etal., 2007).

The use of ERG as a diagnostic tool has been further enhanced by greater
knowledge of the retinal processes underlying the different components of the ERG,
mainly when different techniques, such as the use of drugs (pharmacological dissection)
and intracellular recordings, were introduced. Since then, it has commonly been assumed
that cells in the distal part of the sensory retina (photoreceptors, bipolar cells and Muller
cells), are the main contributors to the ERG, while proximal processes contribute less to
the response. The pharmacological dissection approach also has been used to investigate

the ERG of animal models of inherited retinal disease.

94

Several different pharmacological compounds were used in this study in an

attempt to determine the origin of the supemorrnal b-wave:

o APB (2-amino-4-phosphonobutyric acid, also known? as L-AP4), a
glutamatergic receptor agonist that acts on metabotropic glutamate receptors,
has been shown to isolate the OF F-hyperpolarizing responses thus removing
the majority of the b-wave of the ERG (Slaughter and Miller, 1981; Stockton
and Slaughter, 1989).

o PDA (cis-2,3-piperidinedicarboxylic acid), a glutamatergic receptor antagonist
isolates the response of the photoreceptors and ON-depolarizing bipolar cells
by blocking transmission from photoreceptors to OFF bipolar cells and
horizontal cells and transmission from bipolar cells to third order neurons
(Slaughter and Miller, 1983; Stockton and Slaughter, 1989).

o Aspartate blocks all post-receptoral responses; thus, it reveals the P111
response or the fast component of the a-wave, which originates from the

photoreceptors (Cervetto and MacNichol, Jr., 1972; Murakami et al., 1975).

In contrast to the standard short flash ERG in which the ON and OFF responses
are super—imposed on one another, the long flash ERG allows the separation of the ON
responses from the OFF responses due to the length of the flash. It was utilized in this
study with the addition of the various previously mentioned pharmacological agents in an

attempt to analyze the ON and OFF components of the rge chick.

95

Avian species, chickens included, have been shown to have circadian rhythms in
their ERG waveforms, although the literature is contradictory about what differences are
seen between day and night and why (Schaeffel et al., 1991; Manglapus et al., 1998; Wu
et al., 2000). In order to establish whether the rge chicks maintained a circadian rhythm
in the face of visual deficits, the effect of circadian rhythm on ERG was also investigated

in both control and affected chicks.

3.2. Materials and methods
3.2.1. Chicks

Chicks used in the ERG studies were from an experimental colony of rge
chickens, which is housed under 12 hour lightzdark cycles in the Vivarium facility at
Michigan State University’s College of Veterinary Medicine. Fertile eggs were produced
in two ways; natural insemination between male and female carriers and artificial
insemination between affected roosters and carrier females. The eggs were collected
once a day, stored at 50°F and incubated in batches. The eggs were hatched in incubators
(Hova-Bator, G.Q.F. Manufacturing Co., Savannah, GA). The chicks were typed
(control or affected) via electroretinogram or later in the project by restriction enzyme
test specific for the phenotype causing mutation. Previous studies have shown that
heterozygous birds had no ERG abnormalities when compared to homozygous normal
chicks (Montiani-Ferreira et al., 2007); therefore both heterozygous carriers and
homozygous normal birds were used as controls. All procedures were conducted in

accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision

96

Research and approved by the Michigan State University All-University Committee on

Animal Use and Care.

3.2.2. ERG recording

All electroretinograms were performed between the hours of 8am and 5pm
(except the night time circadian ERGs) to avoid circadian effects on the ERG. For all of
the electroretinographic studies, the chicks were dark adapted for one hour and then
anesthetized with isoflurane delivered in 100% oxygen. Body temperature was
maintained with a water blanket. The left eye was typically used to record
electroretinographic responses. Their pupils were dilated with topical 1% vecuronium
bromide (ESI Lederle, Philadelphia, PA). A Burian-Allen bipolar corneal contact lens
(Hansen Labs, Coralville, IA) lubricated with 2.5% hydroxypropyl methylcellulose
(Goniosol; Alcon Laboratories, Inc., Fort Worth, TX) was used to record the responses.
A ground electrode was inserted subcutaneously in the hind limb.

Full-field (Ganzfeld) flash intensity-series and long flash ERGs were recorded
with a UTAS-E 3000 Electrophysiology unit (LKC Technologies Inc, Gaithersburg, MD)
with the bandpass set between 1 and 500 Hz. The stimulus was delivered by a Ganzfeld
unit consisting of a spherical chamber painted with reflective white paint in which the
anesthetized chicks were placed with the test eye (and lens) exposed to the interior of the
bowl. Short flash series were done with the LKC Ganzfeld, which can produce a wide
range of light intensities from discharge of xenon flash tubes. Dark-adapted and light-

adapted intensity series were done following the protocol outlined in Table 3.1. After the

97

dark-adapted series had been recorded, the chicks were light adapted (while anesthetized)

to a white background light of 30 cd/m2 and then the light-adapted series was performed.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Intensity (cdS/mz) # Flashes Averaged # Seconds between flashes
-3.19 10 1
-2.80 10 1
-2.60 10 1
-2.0 10 1
-1.6 10 l
-l.19 10 l
-0.80 10 1
-0.40 10 1
0.00 3 (10) 5 (1)
0.39 2 (5) 30 (5)
0.86 2(5) 45 (10)
1.36 1 (3) 120 (30)

1.90 1 (3) 18%45)
2.39 l (2) 240 (60)
2.82 1 (2) 360 (60)

 

Table 3.1. Summary of short flash ERG protocol. Dark-adapted series utilized all of the
listed flash intensities whereas light-adapted series only utilized -2.0 cds/m2 and brighter
flashes. Numbers in parentheses were the values used for light-adapted series when they
differed from the dark-adapted series.
3.2.2.a. Long flash ERG

Long flash ERGs were performed with a customized Ganzfeld stimulator, which
can create an adjustable duration of light stimulus with a rod saturating background light
(43 cd/mz) (Sieving et al., 1994) and recorded with the previously mentioned UTAS-E
3000 electrophysiology unit (LKC Technologies Inc; Gaithersburg, MD). The stimulator
can produce longer flashes of light (50-300 msec), and has a maximum intensity of 180

cd/m2. The long flash recordings were typically done following the light-adapted series.

98

 

Thus, the anesthetized chicks were transferred to the long flash Ganzfeld and were
allowed to light adapt to the background light for 10 minutes before recording. Flashes
were either 150 or 200 msec in length and the responses to 10 flashes were averaged with
5 seconds between flashes. In order to evaluate the long flash response, 5 control and 5
affected chicks were used to record long flash responses (these were the day-time long
flash responses from the circadian ERGs). Pharmacological dissection was also
employed in concert with the long flash technique (see section 3.2.3 for details of
technique). All ERG waveforms were averaged, stored and displayed by LKC software

for further analysis.

3.2.3. Pharmacological dissection of the ERG

The pharmacological agents were injected intravitreally to attain the following
final vitreal concentrations: APB, 3 mM; PDA, 7 mM; sodium aspartate, 50 mM. These
concentrations were selected based on the results of studies in other species. All of the
drugs were obtained from Sigma (St. Louis, MO) and the solutions were made by
dissolving the compounds in balanced salt solution (Alcon Laboratories, Fort Worth,
TX). The drugs were injected into the vitreous body of anesthetized chicks with a 30
gauge needle attached to a Hamilton syringe in a volume of 0.02 mL. The injected eye
was not reused for any electroretinography or histopathology.

For the pharmacological dissection, the pre-injection ERG was performed as
previously stated. APB and aspartate injection ERGs included dark-adapted and light-
adapted series and a long flash response, while the PDA ERGs only included the light-

adapted series and the long flash response because PDA only effects cone-driven

99

pathways. The dark-adapted and light-adapted intensity series were done according to
Table 3.1. Following the long flash ERG recording, the pharmacological agent was
injected and then the chick was allowed to recover from anesthesia in the dark until the
post—injection dark-adapted ERG was begun. The post-injection ERG was repeated one
to two hours after the injections of APB, PDA and aspartate to allow diffusion of the
agent to the retina, time for it to find its target and exact its effect.

APB injection and ERGs were done with 4 controls, which ranged in age from 14
to 26 days, and 4 affected birds, which ranged in age from 13 to 23 days. PDA injection
and ERGs were done with 7 control birds, which ranged in age from 13 to 177 days, and
8 affected birds, which ranged in age from 24 to 50 days. Aspartate injection and ERGs
were done with 4 control birds, which ranged in age from 14 to 56 days, and 4 affected

birds, which ranged in age from 11 to 26 days.

3.2.4. Circadian electroretinograms

Both daytime and nighttime ERGs were performed to evaluate circadian ERG
differences and consisted of dark-adapted and light-adapted series and a long flash
response (as outlined above). The daytime ERG was started around 1pm (halfway
between lights on in the morning and lights off in the evening), and the nighttime ERG
was started around lam (halfway between lights off in the evening and lights on in the
morning). Circadian ERGs were done with 5 control birds and 5 affected birds all of

which were between 15 and 22 days of age.

100

3.2.5 Data analysis

Raw data was imported into and analyzed with Microsoft Excel 2003 (Microsoft
Corporation, Redmond, WA). Pharmacological dissection ERG waveforms were plotted
using Excel and visually analyzed. Peak a- and b-wave amplitudes of circadian and long
flash ERGs and d-waves of long flash ERGs and implicit times of circadian ERGs were
measured and averaged. A-wave amplitudes were measured from the baseline to the
lowest trough of the a-wave and b-wave amplitudes were measured from the trough of

the a-wave to the highest peak of the b-wave. A 5 pVolt criterion threshold was used,

meaning any wave with an amplitude of less than 5 uVolt was excluded. Implicit times
are measured from the time of light onset to the peak of the wave being analyzed.
Affected and control long flash mean a-, b-, and d-wave amplitudes were
compared using a t-test. Several comparisons were made for the circadian ERGs.
Circadian implicit times and amplitudes were averaged for each light intensity. As a first
step in the statistical analysis a descriptive test that included a skewness of the
distribution analysis (PROC MEANS, SAS 2001—version 8.2. SAS Institute Inc., Cary,
NC, USA) was run on the implicit time and amplitude data. Kolmogorov-Smirnov test of
normality also was performed. The distribution of the data revealed to be right-skewed
(around +2) and therefore considered as non-Gaussian. The data was then log-
transformed and the mean values were compared between day and night using a paired t-
test. Emphasis was placed on day/night differences within the groups (affected and
control) because previously published results demonstrated significant differences in the

ERG responses between control and affected birds (Montiani-Ferreira et al., 2007).

101

Results were considered statistically significant when P values were less than 0.05. The

resulting data were back-transformed to be shown on graphs and tables.

3.3. Results
3.3.1. Long flash ERGs

The long flash ERG of both the control and the affected birds contain the same
components: a negative a-wave at lights on followed by a positive b-wave, a return to
baseline and then a positive d-wave at lights off. The d-wave of the control birds’ long
flash ERGs quickly rises to a peak, then, following an initial rapid drop from that peak, it
slowly returns to baseline. The wave amplitudes of the rge long flash a-, b- and d-waves
were greatly reduced compared to control birds (P<0.0001 for each wave). Figure 3.1
shows representative long flash ERG waveforms from an affected and a control bird.

Figure 3.2 shows the mean a-, b- and d-wave amplitudes of both control and affected

birds.

102

=
>°

:l
O
In

50 msec

Control

 

 

_ Affe cte d

Figure 3.]. Long flash ERG. Representative long flash ERG waveforms from a
control (top wave) and an affected bird (bottom wave) of comparable ages. Note
the markedly attenuated amplitudes of the a-, b- and d-waves of the rge chicks.
The heavy black bar indicates the length of the flash stimulus (200 mSec).

100
90 1
so .
7o

60‘

Amplitude (pVolt)

N
O

     

A—wave B—wave D-wave

Figure 3.2. Mean long flash ERG wave amplitude comparisons. Stippled
columns are amplitudes of affected birds, while black columns are amplitudes of
control birds. There was a significant difference between affected and control
amplitudes for each wave. (*) indicates a significant difference between affected
and controls (P<0.0001).

103

3.3.2. APB Injection

The control chicks’ post-APB dark-adapted and light-adapted ERG waveforms
were similar in that the b-wave was eliminated, which allowed the a-wave to reach a
more negative potential. The peak of the a-wave was followed by a slow return to
baseline over the recording time (Figure 3.3 A and B). The return to baseline happened
more quickly with the lower intensities than it did at the higher intensities.

APB also eliminated the b-wave of the long flash ERG of the control birds
(Figure 3.4 A). It increased the amplitude of the a-wave, which began slowly returning to
baseline until lights off. The ON bipolar cell components which are removed by APB are
displayed by subtracting the post-injection waveform from the pre-injection waveform
(Figure 3.4). In the case of APB injection in the control birds, the subtraction displays
the ON response and includes a small negative deflection then a b-wave that does not
return to baseline until lights off and a small residual d-wave.

The rge chicks’ post-APB dark-adapted and light-adapted ERG waveforms were
also similar to each other, but differed from those of the control chicks. The APB
increased the amplitude of the a-wave and made it wider and had a slight but variable
effect on the b-wave (Figure 3.3 C and D). In some birds, the b-wave had a slightly
larger amplitude post-injection whereas some birds’ b-waves were not altered post-
injection.

APB also increased the rge a-wave amplitude in the long flash ERG; however, it
returned to baseline more rapidly than in the control (Figure 3.4 B). At lights off a

positive d-wave was present. The subtraction of the rge pre- and post-APB long flash

104

waveform to reveal the ON bipolar cell response showed a positive going “b-wave” that

returned to baseline more rapidly than in the control.

105

Normal: Dark-adapted B Normal: Light-adapted

 

 

 

 

C. Mutant. Dark-adapted D. Mutant. Light-adapted

 

 

 

 

 

100 ”Volt

 

50 mSec

Figure 3.3. APB effect on short flash ERG. Representative ERGs showing the
effect of intravitreal APB on the ERG of control (A and B) and mutant (rge) (C
and D) chicks under dark-adapted (A and C) and light-adapted (B and D)
conditions. The dotted lines represent the responses prior to intravitreal
injection and the solid lines the responses after APB was administered. Note that
in the control chick the b-wave is eliminated by APB revealing a portion of the
P111 response, whereas in the mutant chick APB has very little effect on the b-
wave amplitude but does slightly enhance the a-wave. Flash intensity from top:
0.39, 1.36 and 2.39 log cdS/mz. Final concentration of APB was 3 mM.

106

Control

Subtraction

%

>|___
3

0

L0

50 msec

 

     
     
   

Affected

Subtraction

L__

50 msec

10 uVOlt

Figure 3.4. APB effect on long flash ERG. Representative ERGs showing the
effect of intravitreal APB on the long flash ERG of control (A) and affected (B)
chicks under light adapted conditions. Heavy black lines represent length of
light flash. The thin tracings represent the responses prior to intravitreal
injection and the heavier tracings the responses after APB was administered.
The tracings labeled “Subtraction” are the pre-injection ERG minus the post-
injection ERG and in this case show the response from the ON bipolar cells.
Note that the scale for the affected tracings is different from the control tracings.
Note that in the control chick the b-wave is eliminated by APB revealing the P111
response, whereas in the mutant chick APB reduces the amplitude of the b-wave
but doesn’t entirely eliminate it. The effect on the b-wave allows the affected
chick’s a-wave to reach a larger amplitude than before the injection. Final
concentration of APB was 3 mM.

107

3.3.3. PDA injection

PDA blocks a negative component of the control chick ERG resulting in a greatly
decreased a-wave amplitude and an increased b-wave amplitude (Figure 3.5 A).

The results of the long flash ERG afier PDA injection in control birds is similar to
the “short flash in that the a-wave is almost completely eliminated and the b-wave
amplitude larger. Additionally, the d-wave is almost completely eliminated (Figure 3.6).
The components removed by PDA are displayed in the “subtraction” waveform (obtained
by subtracting the post-injection waveform from the pre-injection waveform). In the case
of PDA injection in the control birds, the subtraction displays the OFF response and
includes a negative potential at lights on, which would contribute to the generation of the
a-wave and decrease the b-wave amplitude, and a positive wave at lights off, which forms
the major portion of the d-wave. In other words, the OFF bipolar cells, the horizontal
cells and third order neurons have opposite potentials at lights on (and oppose the b-
wave) and a positive response at lights off to form most of the d-wave.

PDA consistently made the slope of the a-wave shallower, increased the a-wave
implicit time and decreased the amplitude of the b-wave of the rge chicks’ ERGs (Figure
3.5 B).

PDA decreased the rge a-wave amplitude, increased the implicit time and delayed
the beginning of the downward slope of the a-wave in the long flash (Figure 3.6 B).
Although both waves were still present post-PDA injection, the amplitude of both the b-
and d-waves was greatly decreased. The subtraction waveform displays the contributions

of the OFF bipolar cells, horizontal cells and the third order neurons and in the case of the

108

rge birds includes components of a-, b- and d-waves and is quite different from that of

the control birds.

109

Normal: Light-adapted Mutant: Light-adapted
A. .3.

  

 

 

 

 

100 uVolt

 

Figure 3.5. PDA effect on short flash ERG. Representative ERGs showing the effect of
intravitreal PDA on the ERG of control (A) and rge (B) chicks under light-adapted
conditions. The dotted lines represent the responses prior to intravitreal injection and the
solid lines the responses after PDA was administered. A negative component of the control
chick ERG is removed resulting in a reduction in a-wave amplitude. The photopic hill
effect is also removed. The result in the mutant bird is quite different in that PDA appears
to remove a positive component of the b-wave thus reducing the b-wave amplitude. It also
results in a delay in the a-wave. Flash intensity from top: 0.39, 1.36 and 2.39 log cdS/mz.
Final concentration of PDA was 7mM.

110

 
   
 

Control

 

 

Subtraction

L__

50 msec

50 uVOIt

 

Affected

'H \ ' .MWA-t

Subtraction

10 uVolt

50 msec

Figure 3.6. PDA effect on long flash ERG. Representative ERGs showing the effect of
intravitreal PDA on the long flash ERG of control (A) and affected (B) chicks under light adapted
conditions. Heavy black lines represent length of light flash. The thin tracings represent the
responses prior to intravitreal injection and the heavier tracings the responses after PDA was
administered. The tracings labeled “Subtraction” are the pre-injection ERG minus the post-
injection ERG and in this case show the response from the OFF bipolar cells and any downstream
responses from second and third order neurons. Note that the scale for the affected tracings is
different from the control tracings. Note that in the control chick a negative component at lights-
on is eliminated, which almost completely eliminated the a-wave and greatly increased the b-
wave. Additionally, the d-wave is greatly decreased at lights-off. In the affected chick, PDA
greatly reduces the amplitude of all three waves (a-, b- and d-waves) and delays the beginning of
the down slope of the a-wave. Final concentration of PDA was 7mM.

lll

3.3.4. Aspartate injection

Aspartate completely eliminated the b-wave of both the control chicks’ dark-
adapted and light-adapted ERGs, thus revealing the true amplitude of the P111 response
(Figure 3.7 A and B).

Aspartate had similar effects on the control chicks’ long flash ERG in that it
eliminated the b-wave and in doing so greatly increased the a-wave amplitude and
implicit time (Figure 3.8 A). At lights off, a d-wave appeared, but it had a longer implicit
time and was wider than the pre-injection d-wave, much like the d-wave post—APB
injection. As expected, the aspartate subtraction included an immediate negative
potential at lights on (which must contribute to the a-wave), a positive potential (which
makes up the b-wave), and a positive response at lights off.

Aspartate had the same effect on the rge ERG waveforms as it did on the control
chicks’ ERG waveforms in that it eliminated the b-wave in both the dark-adapted and
light-adapted ERGs, thus increasing the a-wave amplitude and implicit time (Figure 3.7 C
and D). However, unlike the control birds, the remaining amplitude in the light—adapted
series was smaller in amplitude than that of the dark—adapted series.

The post-aspartate long flash of the rge chicks was similar in shape to that of the
control chicks but lower in amplitude. The b-wave was eliminated thus increasing the a-
wave amplitude and implicit time and the d-wave amplitude was decreased and appeared
wider (Figure 3.8 B). The subtraction waveform was also similar to the control birds, but

lower in amplitude.

112

Normal: Dark-adapted Normal: Light-adapted

 

 

Mutant: Dark-adapted Mutant: Light-adapted

 

 

 

 

 

O.
.......
.............

 

 

100 uVolt L

 

50 mSec

Figure 3.7. Aspartate effect on short flash ERG. Representative ERGs showing
the effect of intravitreal aspartate on the ERG of control (A and B) and GNB3
mutant (C and D) chicks under dark-adapted (A and C) and light-adapted (B and
D) conditions. The dotted lines represent the responses prior to intravitreal
injection and the solid lines the responses after aspartate was administered.
Aspartate removes the post-receptoral responses in both control and GNB3 mutant
birds leaving the P111 (photoreceptor) response. Flash intensity from top: 0.39,
1.36 and 2.39 log cdS/mz. Final vitreal concentration of aspartate was 50mM.

113

G) Control

100 uVOlt

  
 
   

50 msec

Subtraction

 

Affected

'VM

Subtraction

l_

50 msec

1o uVOlt ‘

Figure 3.8. Aspartate effect on long flash ERG. Representative ERGs showing the effect
of intravitreal aspartate on the long flash ERG of control (A) and affected (B) chicks
under light adapted conditions. Heavy black lines represent length of light flash. The thin
tracings represent the responses prior to intravitreal injection and the heavier tracings the
responses after PDA was administered. The tracings labeled “Subtraction” are the pre-
injection ERG minus the post-injection ERG and in this case show the photoreceptor
response. Note that the scale for the affected tracings is different from the control
tracings. In both chicks, the b-waves were eliminated leaving the P111 response, but a
component of the d-wave was spared. Final concentration of aspartate was 50mM.

114

3.3.5. Circadian ERGs

Figure 3.9 displays the mean dark-adapted a—wave amplitudes of both control and
affected birds during the nighttime and daytime. There were no statistically significant
differences between nighttime and daytime amplitudes for either group.

Figure 3.10 contains the averaged dark-adapted b-wave amplitudes of both
control and affected birds during the nighttime and daytime. The affected birds’ dark-
adapted b-wave amplitudes were larger than those of the control birds at the higher
intensities (the “supemorrnal” b-wave) (Figure 3.10), which has been reported previously
(Montiani-Ferreira et al., 2007). The control birds’ dark-adapted b-wave amplitudes
reached a plateau at relatively low light intensities, whereas the affected birds’ dark-
adapted b-wave amplitudes peaked at a much higher intensity. When the overall day and
night dark-adapted b-wave amplitudes were compared they were found to be significantly

different (P = 0.017).

115

160

 

Q 140 l
. § 120 1
1 3
1 .3 100 *‘1‘
3
E 80 -
g. l
< 60 ‘
5 40
0
E 20 . i
-9- ._ L l
-4 -2 O 2 4 ‘

Intensity (log cdS/m‘Z)

Figure 3.9. Mean dark-adapted circadian a-wave amplitudes. Gray tracings indicate
daytime amplitudes and black tracings indicate nighttime amplitudes. Heavy tracings
with circles are control chicks and those with triangles are affected chicks. Error bars
display standard error of the mean. None of the differences between day and night

were significant.

 

 

250
§ 200~
13-
81509
1%
.0- w
E 100‘
1“ ‘
5
g 50‘
0. ‘ __
—4 -2 0 2 4

Intensity (log cdS/m"2)

Figure 3.10. Mean dark-adapted circadian b-wave amplitudes. Gray tracings indicate
daytime amplitudes and black tracings indicate nighttime amplitudes. Heavy tracings
with circles are control chicks and those with triangles are affected chicks. Error bars
display standard error 0f the mean. (* indicates a significant difference between by the
overall day and night amplitudes for the control birds only (P = 0.017)).

116

Figure 3.11 displays the mean light-adapted a-wave amplitudes of both control
and affected birds during the nighttime and daytime. There were no statistically
significant differences between nighttime and daytime amplitudes for control or affected
birds.

Figure 3.12 displays the mean light-adapted b-wave amplitudes of both control
and affected birds at nighttime and daytime. The control birds’ b-wave amplitudes
exhibit a “photopic hill” in that the highest amplitudes are reached at intermediate
intensities after which point the amplitudes decrease. The affected birds’ light-adapted b-
wave amplitudes are larger than those of control birds at the higher intensities (the
“supemorrnal b-wave”) as reported previously (Montiani-Ferreira et al., 2007). Control
birds’ light-adapted b-wave amplitudes were slightly greater at night than during the
daytime, but the differences were not statistically significant. Affected birds’ light-
adapted b-wave amplitudes were similar at night and during the day with no statistically

significant differences.

117

Mean Amplitude (“V010
(D
O

140
120
100 '

 

Intensity (log cdS/m‘2)

Figure 3.11. Mean light-adapted circadian a-wave amplitudes. Gray
tracings indicate daytime amplitudes and black tracings indicate
nighttime amplitudes. Heavy tracings with circles are control chicks and
those with triangles are affected chicks. Error bars display standard error
of the mean.

' v _ ,, , , ._ ,

 

250
1 g 200 1 I ‘
I 3 ' I‘ll/‘1‘ r
l 3 150 .
a .
1 €- ‘\ *
. \
a E: 100 , \l
C ' “tall
J g 50 - / »
0-—~ -_____1,_.__ ~ 1 ~ —
.4 -2 0 2 4
Intensity (log cdS/m"2)

Figure 3.12. Mean light-adapted circadian b-wave amplitudes. Gray
tracings indicate daytime amplitudes and black tracings indicate
nighttime amplitudes. Heavy tracings with circles are control chicks and
those with triangles are affected chicks. Error bars display standard error
of the mean.

118

Figure 3.13 displays the mean dark-adapted a-wave implicit times of both control
and affected birds at night and during the day. The affected birds’ a-wave implicit times
were longer than those of the control birds at all intensities, which was previously
reported (Montiani-Ferreira et al., 2007). Notice that for both groups, the higher the
intensity, the shorter the implicit time. There were only two intensities at which there
was a significant difference between day and night a-wave implicit time for the affected
birds (P <0.05). There were no other significant differences between day and night for
the groups.

Figure 3.14 shows dark-adapted b-wave implicit times of both control and
affected birds at night and during the day. Figure 3.15 shows light-adapted a-wave
implicit times of both control and affected birds at night and during the day. Figure 3.16
shows light-adapted b-wave implicit times of both control and affected birds at night and
during the day. There were several intensities for which the affected birds had significant
differences in b-wave implicit times between day and night, but for the most part, there

were no significant differences between day and night.

119

40

 

’g 35; —
m E
g 30-
: 201i
1.2 l
.315.
E
1: 10;
m 1
é’ 5*
0. _ _ _T__
4 -2 o 2 4

Intensity (log cdS/mAZ)

Figure 3.13. Mean dark-adapted circadian a-wave implicit times. Gray tracings
indicate daytime amplitudes and black tracings indicate nighttime amplitudes.
Heavy tracings with circles are control chicks and those with triangles are affected
chicks. Error bars display standard error of the mean. ("' indicates a significant
difference between day and night for the affected birds only.)

 

 

70
. E? 60 3 .
s g x I
’ v 50~ 1
‘8' l 1
1
p 401
s: l
' L3 30 ~«
! a -
‘ § 20-
: C 1
l 3 10
i 2 ,
; 4 -2 o 2 4

Intensity (log cdS/mAZ)

Figure 3.14. Mean dark-adapted circadian b-wave implicit times. Gray tracings
indicate daytime amplitudes and black tracings indicate nighttime amplitudes. Heavy
tracings with circles are control chicks and those with triangles are affected chicks.
Error bars display standard error of the mean. (* indicates a significant difference
between day and night for the affected birds only.)

120

30
25 ~
20 -

15~

 

10‘,

Mean Implicit Time (msec)

3-2.151 2: 4;
Intensity (log cdS/m‘2)

Figure 3.15. Mean light-adapted circadian a-wave implicit times. Gray tracings
indicate daytime amplitudes and black tracings indicate nighttime amplitudes.
Heavy tracings with circles are control chicks and those with triangles are
affected chicks. Error bars display standard error of the mean. (* indicates a
significant difference between day and night for the affected birds.)

70
60 g *
y .

50

40-

 

301

20‘

10

‘Mean Implicit Time (msec)

o 1
-3 -2 -1 o 1 2 3 4

Intensity (log cdSImAZ)

Figure 3.16. Mean light-adapted circadian b-wave implicit times. Gray
tracings indicate daytime amplitudes and black tracings indicate nighttime
amplitudes. Heavy tracings with circles are control chicks and those with
triangles are affected chicks. Error bars display standard error of the mean. (*
indicates a significant difference between day and night for the affected birds.)

121

3.4. Discussion

The long flash ERG of both the control and the affected birds contain the
expected components (a-, b- and d-waves); however, the affected birds had greatly (and
significantly) reduced amplitudes compared to the controls. The brightest intensity that
the long flash Ganzfeld stimulator is capable of producing is 180 cd/m2, which is not
bright enough to elicit the “supemormal b-wave” seen in the brighter short flash
intensities. Although the amplitudes were lower in the affected birds, the long flash does
indeed contain both an ON and an OFF response meaning that those responses were
intact in the rge birds.

As was found previously, APB eliminated the b-wave in both the dark-adapted
and light-adapted ERGs of control chicks, thus the a-wave was prolonged and enhanced
(Montiani-Ferreira, 2004). APB, a glutamatergic receptor agonist that acts on
metabotropic glutamate receptors, has been shown to isolate the OFF-hyperpolarizing
responses by maintaining the ON bipolar cells in a hyperpolarized state, thus removing
the majority of the b-wave of the ERG (Slaughter and Miller, 1981; Stockton and
Slaughter, 1989; Xu et al., 2003; Sharma et al., 2005). The results of the long flash are
similar in that the b-wave was eliminated.

The subtraction waveform revealed that the ON bipolar cells contribute to each of
the basic ERG components (a-, b- and d-waves). It is interesting that the ON bipolar cells
appear to make up part of the d-wave considering the d-wave is usually considered to be
the OFF response. The APB-sensitive portion of the d-wave appears to be quick to rise
and quick to decline, which would appear to shape the d-wave by shortening the implicit

time and beginning a return to baseline. This finding regarding the d-wave suggests that

122

ON bipolar cells play some sort of role in the d-wave. Ueno et a1 (2006) found that both
ON bipolar cells and cone photoreceptors contribute to the d-wave of the
electroretinogram, which may help to explain the finding that the APB-sensitive ON
bipolar cells appears to contribute to the chicken d-wave.

APB did not eliminate the rge b-wave, which is similar to a previous attempt
(Montiani-Ferreira, 2004). This lack of response of the rge b-wave suggests that it either
does not originate from ON bipolar cells or that the rge ON bipolar cells are incapable of
responding to APB the way normal ones are. The long flash results show that there is
some reduction in b-wave. The quick return to baseline after the a-wave in the post-
injection APB long flash could potentially be considered solely the response of the
photoreceptors; however, when the post-APB long flash is compared to the post-aspartate
long flash (Figures 3.4 B and 3.8 B), the isolated photoreceptor response after aspartate
returns to baseline much slower than that of APB. This suggests that the return to
baseline after the a-wave in the post-APB long flash is composed of inner retinal
contributions (such as ON or OFF bipolar cells).

The post-APB d-wave is slightly larger than the pre-injection d-wave. Therefore,
it appears that the ON bipolar cells in the rge chicks contribute partially to the b-wave
and compete with the positive-going d-wave (OFF response). Unlike in the control birds
in which the ON bipolar cells appear to contribute positively to the d-wave, the rge birds’
ON bipolar cells appear to contribute only negatively to the OFF response.

The post-APB injection results may help to explain the rge chicks’ lack of

oscillatory potentials (OPs). OPs are thought to originate from negative feedback from

123

amacrine cells to second order neurons. If the input from ON bipolar cells to amacrine
cells is disrupted in rge birds it might prevent the development of CPS.

PDA, a glutamatergic receptor antagonist, isolates the response of the
photoreceptors and ON-depolarizing bipolar cells by blocking transmission from
photoreceptors to OFF bipolar cells and horizontal cells and transmission from bipolar
cells to third order neurons (Slaughter and Miller, 1983; Stockton and Slaughter, 1989).
The results of the PDA injection in control chicks is similar to that previously reported in
primates and other species (Sieving et al., 1994; Xu et al., 2003) in which a negative
component is removed, thus reducing the a-wave amplitude and increasing the b-wave
amplitude. The long flash results are similar to the short flash results in that the a-wave is
eliminated and the b-wave amplitude is increased. The d-wave is almost completely
eliminated by PDA, which is expected since it blocks the transmission from
photoreceptors to OFF bipolar cells.

The post-PDA injection long flash waveform is roughly similar in shape to that
which is eliminated by APB (i.e. the subtraction — compare Figure 3.6 and 3.4), which
would be anticipated because APB eliminates the ON response (leaving the OFF
response) and PDA eliminates the OFF response (leaving the ON response). Similarly,
the post-APB long flash waveform (i.e. the OFF response) is roughly similar in shape to
that which is eliminated by PDA (i.e. the OFF response).

Previous attempts to dissect the rge ERG with PDA by Montiani-Ferreira
revealed it had no effect on the waveform; however, a lower concentration (5 mM) was
used, whereas in this study, 7mM was used. The difference in concentrations may help to

explain the difference in effects of PDA on the ERG waveform. PDA had almost an

124

opposite effect on the rge ERG waveform compared to controls as it increased the
implicit time of the a-wave and greatly reduced the b-wave amplitude. Similarly the long
flash a-, b- and d-waves were decreased in amplitude and had increased implicit times.
These results suggest that the PDA sensitive components of the rge ERG may be
biphasic; there is an initial negative component that contributes to the a-wave generation
followed by a slower positive component that contributes a positive component to the b-
wave. And ultimately, this suggests that unlike the control birds, the OFF pathway in the
rge birds contributes a great deal to the ERG waveform.

Aspartate blocks all post-receptoral responses, thus revealing the P111 response or
the fast component of the a-wave, which originates from the photoreceptors (Cervetto
and MacNichol, Jr., 1972; Murakami et al., 1975). This was found to be true for both the
control and the affected chicks in this study in that the b-wave was completely
eliminated, leaving only the P111 response. The larger post-injection a-wave amplitude in
dark-adapted compared to light-adapted ERG waveforms suggest that cones are not as
capable of responding to light stimulation as rods are in the rge chicken.

Evidence suggests that inactive transducin is loosely associated with the rod outer
segment disc membrane (and thus rhodopsin which is a transmembrane receptor)
(Phillips et al., 1992; Herrmann et al., 2006). If transducin in cones is similarly located,
then GNB3 might play an important role of physically positioning the a subunit where it
can interact with activated rhodopsin. Therefore if the mutant GNB3 does not bind with
the 01 subunit adequately, it might not be available to interact with metarhodopsin II.
This would suggest that cones would have a decreased efficiency of photoactivation.

This possibility is supported by the rge aspartate results.

125

Aspartate had similar effects on the both the control and affected chicks’ long
flash ERGs in that it eliminated the b-wave and in doing so revealed the P111 response.
At lights off, a d-wave appeared, but it had a larger implicit time and was wider than the
pre-injection d-wave, suggesting that the chicken d-wave originates from both pre- and
post-receptoral sources as suggested in primates (Ueno et al., 2006). What remained after
the aspartate injection is solely from the photoreceptors; at lights on, the photoreceptors
slowly hyperpolarize and at lights off, they slowly depolarize.

The control chicks’ dark-adapted b-wave amplitudes were significantly different
between night and day when all intensities were compared together; however, none of the
individual intensities were significant when compared between night and day. The rge
chicks did not have any statistically significant differences between day and night a- or b-
wave amplitudes. Only a few intensities had significant differences in implicit times for
a- and b-waves in the affected chicks, suggesting that neither group had an obvious or
robust circadian rhythm to their ERG responses in these experiments. This was an
unexpected result as ERGs in normal birds have been shown to have a circadian rhythm
in previous studies (Schaeffel et al., 1991; Manglapus et al., 1998; Wu et al., 2000). It
may be possible that a larger number of birds need to be used to demonstrate significant
differences between day and night.

Several of the previously reported circadian studies use much different ERG
protocols, making our results difficult to compare with theirs. For instance, Wu et a1 did
not dark adapt the birds before day-time ERGs, so the ERG responses would be mostly
cOne responses, whereas at night, the responses would be mostly rod responses.

Manglapus et a1 kept the quail used in the study dark adapted for 50 hours over which

126

time they recorded ERGs. This would confuse the results as well because not only would
the quails’ circadian rhythms begin to disintegrate over the 50 hours of darkness, but the
day-time ERGs would initially be a mixed rod/cone response but as the retina lost its

circadian rhythm and became dark adapted, the cones would no longer be as responsive.

127

CHAPTER 4:

CONCLUSION & FUTURE STUDIES

4.1. Conclusion

GNB3 was reported to be the causative gene and mutation of the rge chick
phenotype by another research group (Tummala et al., 2006) during the research for this
project. The GNB3 Asp153 deletion was confirmed in our rge chicks and is found in a
highly conserved region of the gene. Although it is difficult to absolutely prove that
GNB3 is the gene and the Asp153 deletion is the mutation (and only mutation)
responsible for rge phenotype and not just a rare polymorphism that happens to be in
linkage disequilibrium with the disease status, the finding by Tummala et a1 (2006) that
there was a 70% decrease in GNB3 immunoreactivity in the retina supports the idea that
GNB3 is responsible. The examination of the electroretinographic changes can be
partially explained by the known functions of GNB3, but there remain some unexplained
changes in the rge ERG.

GNB3 has been shown to be part of cone-transducin, which is the guanine
nucleotide binding protein involved in phototransduction in cones coupled to
phosphodiesterase (Peng et al., 1992). Thus GNB3 plays an important role in regulating
the response of the cone photoreceptors to light stimulation. When the mutation (aspartic
acid residue deletion present in rge birds) was modeled with GNBl, it was shown to
eliminate 13 sheets in propellers 1 and 5 of the GNB protein and was predicted to create
an unstable protein susceptible to premature proteolysis (Tummala et al., 2006). By

causing this amount of tertiary protein misfolding, this mutation is likely to interfere with

128

the [37 complex’s ability to bind to the or subunit, which in turn is likely to interfere with
the heterotrimer’s ability to interact with the ll-cis retinal/cone opsin and the facilitation
of the 01 subunit activation (Phillips et al., 1992; Yarfitz et al., 1994; Herrmann et al.,
2006). If the By complex were unable to bind to interact with either metarhodopsin II
(Rho*) or with the or subunit, it may affect the cone cell’s ability to respond to light
stimulation. Additionally, according to Tummala et a1, there is a 70% reduction in GNB3
immunoreactivity in the rge retina, which would further contribute to disrupting
phototransduction. These predicted consequences could potentially cause a reduction in
cone sensitivity and perhaps slowed termination of the phototransduction cascade.

In rods, it has been shown that one responsibility of the By complex is to anchor
the or subunit to the disc membrane in close proximity to activated rhodopsin (Phillips et
al., 1992; Herrmann et al., 2006). Because of the similarities between rods and cone, it
could be assumed that the By dimer in cones has similar responsibilities to that of rods.
Therefore, the mutation likely alters the By complex’s ability to bind to the or subunit and
anchor it close to opsin in the cone photoreceptors. Because of this, phototransduction
could be less efficient in the rge birds because the 01 subunit is not available to activated
opsin and photons of light would go “unnoticed” by the photoreceptor because visual
transduction is not taking place properly. These “oblivious” cone photoreceptors may
explain why the cones in rge chickens appear to be less sensitive. The cone responses
that are still present may be explained by a much less efficient phototransduction
pathway that is nonetheless still able to produce responses in affected cells.

The results of the aspartate injection on the rge ERG support the reduced cone

function hypothesis because the remaining PIII response exposed after aspartate

129

administration is smaller than that of the mixed rodzcone response (Figure 3.7) suggesting
that rge cones do not function as well as rge rods. This finding suggests that the rge
chicken’s cone photoreceptors have reduced sensitivity compared to rods. This may be
due to both a reduction in GNB3 protein and to an altered function of the remaining
mutant GNB3 protein.

The phototransduction cascade is terminated by the inherent GTP hydrolyzing
activity of the transducin or subunit; following GTP hydrolysis, the bound PDE y subunit
is released and is available to inhibit the active a/B PDE complex. The finding that

Drosophila mutants with GB mutations have slowed deactivation of phototransduction, as

well as reduced sensitivity (Dolph et al., 1994), suggest that the By complex plays a role
in transducin deactivation (Sagoo and Lagnado, 1997).

In addition to its role in cone phototransduction, GNB3 has been found to be
associated with dendrites of both rod and cone ON bipolar cells and the alpha subunit of a
heterotrimeric G-protein (G0) in the outer plexiform layer (Huang et al., 2003). The
metabotropic glutamate receptor (mGluR6) is also associated with this exact retinal
location (dendritic tips of ON bipolar cells in the outer plexiform layer) and has also been
shown to interact with G001 (Nomura et al., 1994; Vardi and Morigiwa, 1997; Weng et al.,
1997; Vardi et al., 2000) suggesting that GNB3 may be associated with the signal
transduction resulting from the activation of mGluR6.

mGluR6 is stimulated by glutamate release from the photoreceptors that occurs
when they are depolarized, which is the resting (dark) state of photoreceptors. The
stimulation from glutamate release causes closure of cation channels in the ON bipolar

cell dendrite. When the photoreceptor is stimulated by light, it becomes hyperpolarized.

130

This reduces the rate of photoreceptor glutamate release and thus reduces the tonic
stimulation of mGluR6 that occurs in the dark, leading to opening of the cation channels
in the ON bipolar cell dendrites and depolarization of the cell. G0 is thought to link
mGluR6 to an effector that closes the ON bipolar cell channel (Nawy and Jahr, 1990).
The finding that mice deficient in the G001 subunit lack the b-wave of the
electroretinogram showed that the light response of ON bipolar cells requires Go
(Dhingra et al., 2000; Dhingra et al., 2002). It is known that mGluR6 couples the
reduced glutamate release by photoreceptor cells in response to light, to the
depolarization of ON-bipolar cells, via an as-yet unidentified cation channel (Nawy,
1999). This could potentially mean that the mutated GNB3 in the rge chicken disrupts
the normal response of the ON bipolar cells by disallowing the normal interactions of
mGluR6 with the G001 subunit of the heterotrimeric G protein. And ultimately, this could
mean that the ON bipolar cells are unable to depolarize (as efficiently) in response to a
decrease in glutamate from the hyperpolarizing photoreceptors, which may result in a
lower amplitude ON response from the bipolar cells.

This hypothesis of altered ON bipolar cell response is supported by the results of
the APB injection. APB selectively stimulates the mGluR6 glutamate receptor located
solely in ON bipolar cells thus maintaining it in the hyperpolarized state; therefore, it
typically eliminates the b-wave (Slaughter and Miller, 1981). As predicted, APB
eliminated the b-wave of the control chicken ERG; however, it had almost no effect on
the rge ERG (Figures 3.3 and 3.4). These results suggest that the rge b-wave does not
originate from the ON bipolar cells or that they are unable to respond the way normal

ones do.

131

The bulk of the b-wave of other species has been shown to originate from the ON
bipolar cells; however, other retinal cells are also known to contribute to the b-wave as
well. The b-wave is currently accepted to be the summation of opposing inputs from
both ON and OFF pathways with modification of the waveform by contributions from
third order retinal neurons (Sieving et al., 1994; Kapousta-Bruneau, 2000; Dong and
Hare, 2000; Dong and Hare, 2002). The ON bipolar cells do not appear to make a major
contribution to the rge b-wave based on the lack of response to APB. Another possibility
is that it originates from the OFF pathway and third order neurons.

The PDA injection provides further insight into the rge phenotype. PDA is
known to block the photoreceptor to OFF bipolar cell (cone pathway only) as well as
photoreceptor to horizontal cells and bipolar cells to third order neuron connections. In
the normal chick, it unmasks a positive component (assumed to be the ON bipolar cell
contribution) thus greatly reducing the a-wave amplitude and eliminating the photopic
hill effect. PDA had the opposite effect in the rge birds in that it increased the a-wave
implicit time and decreased the b-wave amplitude (Figures 3.5 and 3.6). These results
suggest that the rge waveform may have two different PDA sensitive components; one
that contributes to the a-wave generation and one that later contributes to the b-wave.

It is possible that the lack of functional GNB3 protein in the rge retina has caused
the retinal cells to create connections to different cells. This phenomenon has been
reported in the CNGA3 knock-out mice, which lack functional cones. In this model, the
cone bipolar cells create ectopic connections with rod photoreceptors (Haverkamp et al.,

2006). Therefore, it is possible that the altered cone and ON bipolar cell fimction in the

132

rge chicken causes other retinal cells to create new connections, which may help to
explain the abnormal pharmacological dissection findings.

Although the focus thus far has been on altered cone function, previous work with
the rge chicken suggested that rod mediated vision is also affected. The rge chicks have
elevated scotopic (dark adapted or rod-mediated) ERG response threshold (require
brighter light stimuli to elicit a response) compared to controls, lower a-wave amplitudes,
reduced visual acuity in dim light, and mislocalization and disorganization of opsin-
positive rod 085 (Montiani-Ferreira and Petersen-Jones, 2003; Montiani-Ferreira et al.,
2005; Montiani-Ferreira et al., 2007). Because rod photoreceptors utilize GNBl in their
visual transduction cycle (and not GNB3), they should function normally; however, since
they synapse with (rod) ON bipolar cells, which have also been shown to contain G001
and GNB3 immunoreactivity, their signal would be abnormal, which may appear to
manifest as defective rod function.

Oscillatory potentials (Figure 1.8) are thought to be the result of inhibitory
feedback from amacrine cells to second order neurons. If ON bipolar cells’ signal was
affected in the rge chicken, the amacrine cells might not receive the appropriate signal,
thus preventing oscillatory potentials from occurring. ChiCks with induced form-
deprivation myopia lack oscillatory potentials as well as have reduced a- and b-wave
amplitudes (Fujikado et al., 1997). This suggests that the lack of OPs in rge chicks could
be secondary to changes resulting from altered visual processing in the retina.

The gross globe alterations that develop in the rge chicks include increased radial
globe diameter, corneal radius, vitreous chamber depth and globe weight (Figure 1.10).

These changes develop after alterations in visual processing are apparent, suggesting that

133

the globe changes are a result of the vision abnormalities, and are not a cause of abnormal
vision in the rge chicks. These morphological globe-changes can be induced in normal
chicks by constantly exposing them to light (Li et al., 1995). If the GNB3 mutation
causes the ON bipolar cells to be constantly depolarized by causing reduced signal
transduction resulting from mGluR6 stimulation by photoreceptor glutamate, the ON
bipolar cells would act as though they are constantly light exposed. This may help to
explain the globe enlargement as chicks that are constantly light exposed also develop
secondary globe enlargement (Li etal., 1995).

In addition to altered cone photoreceptor, rod and cone ON bipolar cell function,
the GNB3 immunohistochemistry suggests that it is expressed in more proximal cells in
the retina such as amacrine cells and ganglion cells. If it is actually expressed in these
cells, it would cause additional, as yet unspecified abnormalities in visual processing in
the rge chicks.

At this point, the origin of the rge b-wave, particularly the supemorrnal b-wave, is
still not known and requires further study.

As of yet, the rge chicken is the only reported retinopathy model involving
GNB3. Two studies have been done thus far to examine patients (both human and
canine) with inherited retinal diseases for mutations in the GNB3 gene, and neither study
found any abnormalities in the individuals studied (Akhmedov et al., 1996; Akhmedov et

al., 1997; Gao et al., 1998).

134

4.2. Future Studies

The research presented here furthered the knowledge of the rge chick phenotype,
but it has lead to many more questions that are yet unanswered.

Additional electroretinography could be performed to further evaluate the
abnormalities of the rge ERG. Additional circadian ERGs could be done, as the control
chicks should display some sort of electroretinographic circadian rhythm. Additional
drugs that could be used include NMDA (N-methyl-d-aspartate; blocks the response of
third order neurons including amacrine and ganglion cells), DNQX and CNQX (6,7-
dinitroquinoxaline-2,3-dione and 6-cyano-7-nitroquinoxalinc-2,3-dione; antagonists of
KA/QQ receptors in All amacrine cells and ganglion cells), TTX (tetrodotoxin; blocks
voltage gated sodium channels found in amacrine and ganglion cells), and various
combinations of bicuculline (GABAA antagonist), 3-APMPA (3-
aminopropyl(methyl)phosphinic acid hydrochloride; GABAC antagonist) and strychnine
(glycine receptor antagonist), which block feedback from inner retinal neurons to bipolar
cells.

Additional electroretinography that would provide valuable information would
specifically examine photoreceptor (especially cone) kinetics. One way to do this is to
use a paired flash in which the cones’ ability to recover from a bright flash would be
examined. Another method involves a detailed analysis of the P111 response using two
separate techniques: a-wave modeling and aspartate injections.

Because the specific function of GNB3 in the ON bipolar cells is not known,
single cell recordings from rge ON bipolar cells would provide additional information as

to whether and how the GNB3 mutation affects ON bipolar cell firnction.

135

The preliminary immunohistochemistry using anti-GNB3 antibodies in the retina
suggested that GNB3 is found in both outer and inner segments of the retina; however,
the antibody that was used produced a significant amount of background staining
suggesting that it is not very specific to chicken GNB3. As was previously mentioned,
the peptide sequence against which that particular antibody was raised to is only 73%
similar to chicken GNB3. Additionally, the peptide sequence is found to match closely
with collagen, which most likely contributed to the background staining.

Additional immunohistochemistry could be performed with frozen sections and
fluorescent antibodies to avoid the problems the natural pigment of the outer retina
causes. A different antibody to GNB3 from Abcam Inc. (Cambridge, MA) was raised to
a different amino acid sequence from human GNB3, to which chicken GNB3 is 91%
similar, is available. To definitely look at specificity for the antibody to chicken GNB3, a
western. blot could be done. This antibody may provide more specific staining, although
it hasn’t yet been tested in chicken retina. Double labeling with other primary antibodies
is feasible with fluorescent immunohistochemistry. Additional primary antibodies that

may help to further localize GNB3 in the chicken retina include PKCor (rod ON bipolar

cells), Goot(ON bipolar cells), mGluR6 (ON bipolar cells) and calbindin (cones and
horizontal cells).

Additional immunohistochemistry could be performed to study, in detail, the
synapses between photoreceptors and second order neurons using a battery of antibodies
that have already been tested in chicks (Wahlin & Adler 2006). Some of these antibodies
include anti-piccolo (located in the pre-synaptic active zone), anti-syntaxin 3 (SNARE

protein) and NR2A (N MDA receptor).

136

In additional to more immunohistochemistry, the expression of GNB3 could also
be examined. It is reportedly widely expressed throughout the body (Levine et al., 1990).
mRNA could be collected from the following tissues: cardiac muscle, skeletal muscle,
liver, kidney, brain, lens, aorta, small intestine and lung. Reverse transcriptase PCR
could be done to look for GNB3 expression in these tissues.

Although no abnormalities except those of the retina were detected in rge chicks,
a polymorphism in GNB3 in humans (C825T) has been implicated in a large number of
serious metabolic diseases including hypertension, diabetes, obesity and functional
gastrointestinal disorders (Siffert, 2000; Holtmann et al., 2004). Therefore, the
expression of GNB3 in tissues other than the retina in chicken could allow further study
of the effects of this GNB3 mutation in other tissues and organs. Previous histopathology
of rge liver, muscle and heart revealed no abnormalities, but perhaps a more detailed
study of these and additional organs will be warranted based on the tissue expression of
GNB3. In addition to expression in these tissues, it would also be interesting to measure
rge chickens’ blood pressure and heart rate to evaluate for any signs of hypertension or

other cardiovascular abnormalities.

137

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