Models of Müller Glial Cell Disruption and the Consequences on Retinal Health and

Visual Function in the Zebrafish Retina

NICOLAS A. YANNUZZI
Department of Molecular and Cellular Biology, Harvard University,
Cambridge, Massachusetts 02138
 Statement of Research
This thesis was completed under the guidance of Dr. Pamela M. Kainz and Dr.
John E. Dowling at the Department of Molecular and Cellular Biology at Harvard
College. Research was conducted from September, 2005 - May, 2006 and from
September, 2006 - April, 2007.

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 ACKNOWLEDGEMENTS

I began working at the Dowling Lab with the intention of completing my single

semester research requirement. Within a short time, however, I found that the

opportunity to conduct my own research was not only a privilege, but also a chance for

me to witness the active pursuit of scientific discovery. The completion of this thesis was

the most meaningful, enjoyable, and exciting part of my academic experience at Harvard.

It revived my love for science and research, and I am thankful for the freedom I was

given to express my thoughts and ideas during the project.

I would like to thank Dr. Pamela Kainz for all of her help and support during the

process and for teaching me everything I know about scientific research, from

experimental design to thoughtful data interpretation. Dr. Kainz is not only a great

scientist, but also a great teacher. I would also like to thank the other members of the

Dowling Lab for helping me during the process. Finally, I would like to thank my

mother for her support and my father for instilling in me a passion for scientific thought

and discovery. His achievements in ophthalmology inspired me to pursue research on the

retina.

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 ABSTRACT

Müller cells are the primary glial cells of the vertebrate retina. They contact and ensheath
every retinal cell type and regulate neuronal activity. Recent studies have suggested that
Müller cells also provide specific supportive roles for the survival and function of
photoreceptor cells, but there are currently few models where this relationship can be
explored. The purpose of this study was to evaluate two candidate models of Müller cell
disruption and to observe the consequences on retinal health and visual function. In the
first chapter I investigated a potential pharmacological model of Müller cell stress. Using
the gliotoxin α-aminoadipic acid (α-AAA), I observed only a modest sign of Müller cell
stress in adult zebrafish, while the effect on larvae was not glial-specific indicated by the
presence of pyknotic nuclei among retinal neurons. Since α-AAA failed to produce clear
and reproducible signs of glial cell defects, I decided to discontinue my pursuit of this
model and instead investigate a potential genetic model of Müller cell disruption by
characterizing the rose mutant, an endothelin receptor B (ET-B) gene knockout. I found
that the morphology of the larval rose retina appeared normal. However, when exposed
to constant light, wild type larvae were unaffected while the rose larval retina suffered
rod outer segment disruption, loss of 10% of the cells in the inner nuclear layer, where
the Müller cell bodies lie, and a decrease in visual sensitivity. These defects are
consistent with the hypothesis that compromised Müller glial cells lead to a decrease in
photoreceptor cell resilience. Questions remain as to what extent Müller cells are
involved in the light exposed rose phenotype, but this study provides the groundwork for
continued exploration concerning how the absence of ET-B compromises the retina and
the ways Müller cells may be involved in the preservation of photoreceptor cells.

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 GENERAL INTRODUCTION

Glial cells provide physical support and protection for neurons and guide

migrating neurons to their destinations in the brain during development. Glia have also

been shown to act as a template for axonal migration in the neural retina (Silver and

Rutishauser, 1984). Furthermore, they regulate the formation of synapses that enable

neuronal correspondence and promote the survival of some neurons while playing a role

in the birth of others (Helmuth, 2001).

Once dismissed by neuroscientists as playing a minimal role in the nervous

system, glia have recently been suspected to be involved significantly in the pathogenesis

of certain diseases. Research has shown that glia are integral to the understanding and

causes of neuropathic pain, epilepsy, and neurodegenerative diseases (Miller, 2005).

Studies have also suggested that glia may offer a new range of therapeutic targets in a

variety of diseases including Multiple Sclerosis and some psychiatric disorders, where

post mortem studies have demonstrated that there are abnormal amounts of glia in certain

areas of the brain (Miller, 2005).

Research on glia has not been limited to their function in the brain, where they

outnumber neurons in a ratio of ten to one. Glia have also been studied in the context of

the vertebrate retina. Müller glia are the primary support cells in the retina. They contact

and ensheath every retinal cell type and regulate neuronal activity by controlling

extracellular ion concentration and by recycling excess neurotransmitters used during

signaling processes (Newman and Reichenbach, 1996; Sarthy and Ripps, 2001).

Specifically, they express gated channels and neurotransmitter receptors which can cause

depolarization and intracellular Ca2+ waves. In addition, they transport K+ and glutamate

5
and regulate retinal pH levels via carbonic anhydrase (Newman and Reichenbach, 1996).

Although there are more neurons than Müller cells, Müller cell processes contact and

ensheath the synaptic and nuclear region of every cell in the retina; their nuclei reside in

the inner nuclear layer, which also contains horizontal, amacrine and bipolar cell nuclei,

and their endfeet project from the ganglion cell layer (Fig. 1), allowing for extensions that

span the entire depth of the neural retina (Peterson et al., 2001). Their apical processes

also contact the inner segments of photoreceptors. This framework provides a basis for

interaction with every neuron.

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 Apical
Processes

Müller
Cell
Body

Müller
Endfeet

Fig. 1. The various layers of the retina are labeled for convenience (Source: The purple and white illustration, except
for the illustration of the Müller cell (right) and the photograph of the adult zebrafish retina (left), is courtesy of The
Washington University School of Medicine). Müller cells and their processes span the width of the neural retina.
Their cell bodies reside in the inner nuclear layer, and their endfeet project out of the ganglion cell layer, while their
apical processes reach the outer segments. Rod outer segments are also labeled and are located directly under the
pigment epithelium.

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 Both clinical and scientific investigators have suggested that Müller cells play a

vital role in several retinal diseases such as X-linked Retinoschisis (Reid et al., 1999),

Cystoid Macular Degeneration (Loeffler et al., 1992), Idiopathic Macular Holes, and

Foveomacular Schisis (Gass, 1999). There has even been some suspicion that Müller cell

disease may be a principal agent in certain forms of Age Related Macular Degeneration,

the leading cause of blindness (DiLoreto et al., 1995). Clinical researchers suggest that

Müller cells are not necessarily a cause of disease. Instead, they believe that Müller cells

become reactive and hypertrophic in response to photoreceptor damage and eventually

lead to scarring. Because the activation of Müller cells in response to preexisting

problems with the neural retina is so common, it has been the main focus in the research

of retinal glia.

Virtually no clinical studies and very few animal studies have explored how

Müller cells themselves could be the primary cause of failing retinal health or function

and not just a response to preexisting damage. Thus, while some of the functions of

Müller cells have been determined, the precise relationship between Müller cells and

retinal cell health and maintenance is not yet fully understood. The effect of Müller cell

disruption on photoreceptor cells was the specific relationship that I set out to explore in

this study. The discovery that stressing or eliminating Müller cells could lead to

photoreceptor degeneration (DiLoreto et al., 1995) provided in part, the inspiration for

this thesis.

The chosen model organism for studying the relationship between retinal glia and

neurons was the zebrafish. Zebrafish are small, approximately one inch long freshwater

fish that can be raised inexpensively and in large numbers. Embryos develop rapidly

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resulting in zebrafish eyes that exhibit light response in just 3 days post fertilization (dpf)

(Brokerhoff et al., 1995). The zebrafish retina has the same cell classes and architecture

as other vertebrates, including humans. However, unlike the mammalian retina, which

contains astrocytes, the zebrafish retina contains only Müller glia and sparse microglia.

An advantage of using the larval zebrafish is that it lacks scales; therefore, the absorption

of a drug can occur readily through its skin. Finally, the visual behavior of larvae can be

tested using the optokinetic response assay (OKR), in which their eyes track the

movement of vertical black and white stripes passing through their visual field. This

response is not only common to zebrafish but to all vertebrates. Using this assay, their

visual threshold can be quantified by determining the lowest level of light at which their

eyes consistently track the moving stripes.

The forefront of research concerning the role of Müller cells in the zebrafish

retina has focused on a genetic mutant called lazy eyes (lze). Larvae homozygous for the

lze mutation at 5 dpf respond much less robustly compared to wild type in the OKR

assay. Histological observations revealed that the mutation seemed to affect selectively

the Müller cells and photoreceptor cells. Some mutant retinas contained fewer Müller

cells than wild type retinas, while others contained Müller cells that appeared

hypertrophied or unhealthy. Most lze retinas had fewer rods and small cone outer

segments. The combined effects of light stress and genetic manipulation were also

studied, and constant light was found to accelerate drastically the Müller cell

degeneration and to accentuate the lze functional deficit (Kainz et al., 2003). The lze

mutant is thus a striking example of the special relationship between Müller cells and

photoreceptor cells. However, larvae homozygous for the lazy eyes mutation for some

9
unknown reason die by 10 dpf. Therefore, there has yet to be a method of studying the

effects of Müller glial cell inactivity on the photoreceptor cells of the adult zebrafish

retina.

The overarching goal of my study was to explore two other potential models of

Müller cell disruption, one pharmacological and one genetic, and to determine if either of

these models resulted in compromised retinal health or function with special attention to

photoreceptors. For each model, photoreceptor stress, in the form of constant light, was

introduced and a morphological examination of the photoreceptors and Müller cells was

performed as well as an assessment of the function of the retina. In chapter one of this

thesis, I investigated a pharmacological model of Müller cell disruption by characterizing

the effects of the gliotoxin α-aminoadipic acid (α-AAA) on the wild type adult and larval

zebrafish. In chapter two, I examined a second potential model: a zebrafish mutant

missing the gene encoding endothelin receptor B (ET-B), which has been shown to be

expressed highly on Müller cells and involved in the response to light-induced stress on

photoreceptors (Rattner and Nathans, 2005).

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 Chapter 1: The Pharmacological Model

INTRODUCTION

One of the methods that has been used to study the aftermath of glial cell

dysfunction or degeneration is the administration of the DL-isomer of α-aminoadipic acid

(α-AAA), a glutamate analogue known to be gliotoxic to the central nervous system,

including the retina (Olney et al., 197; Casper and Reif-Lehrer, 1983). The effects of α-

AAA have been studied in mice through subcutaneous injection. The regularity,

thickness, and number of Müller cell processes were reduced in the inner retina (Reid and

Farber, 2004). An electrophysiological and morphological study carried out in the carp

retina revealed swollen Müller glial cells after one to two months of intraocular injections

with α-AAA at varying doses (Sugawara et al., 1990). Another study found that after

treating embryonic Xenopus retinas in culture with α-AAA, photoreceptor outer segments

became shortened and dysmorphic, further demonstrating the relationship between

Müller cells and photoreceptor health (Jablonski and Iannaccone, 2000).

Although these studies demonstrate that compromised Müller cells can result in

declining retinal health, they all had pitfalls in their approaches. First, the study on

Xenopus retinas was done in culture, where specific aspects of photoreceptor integrity

may have been affected already due to the inherent disruption caused by the culture

preparation. Second, intraocular injection by itself, causes retinal damage and therefore

makes the data difficult to interpret. In addition, in this approach and the others

involving injections of the drug, the concentration of the drug fluctuates dramatically

based upon the time of injection. In my pharmacological model, I delivered the toxin to

the water where it could be absorbed by the gills of adult zebrafish and through the skin

11
of zebrafish larvae. This delivery method was advantageous because the concentration of

the drug and the exposure time could be controlled precisely.

I monitored Müller stress both immunologically and histologically. Principally, I

followed the expression of antibodies for glial fibrillary acidic protein (GFAP) and

glutamine synthetase (GS), known Müller cell markers that have been shown to be

upregulated in times of retinal and Müller specific stress (Uhlmann et al., 2003). In

addition, I examined the histology of the adult and larval retinas for signs of Müller cell

hypertrophy and photoreceptor stress or death since I suspect that the health of these two

cell types is linked.

MATERIAL AND METHODS

Zebrafish maintenance
Wild type and lazy eyes heterozygous zebrafish were maintained in accordance to

Harvard University and National Institute of Health-approved protocols. The fish were

kept on a 14/10-hour light-dark cycle in 28.5ºC fish water containing 2g of Instant Ocean

salts per gallon of distilled water supplemented with vitamins. The lze mutant was

obtained from a family that had been isolated in a mutagenesis screen in which N-ethyl-

nitrosurea was used to induce DNA point mutations. The lze mutation is homozygous

recessive (Kainz et al., 2003).

Wild type larvae were obtained by mating several wild type fish together in a

basket cross. Lze larvae were obtained by mating adult fish heterozygous for the lze

mutation. The resultant progeny of this cross was comprised of wild type larvae, lze

mutants, and lze heterozygotes in a ratio of 1:1:2. Mutant lze were identified on 5 dpf

based on their failure to respond strongly in the OKR visual behavioral assay. Wild type

larvae repeatedly move their eyes with a smooth pursuit motion followed by a saccade in

12
response to moving vertical black and white stripes. Mutant lze larvae respond with

weak and infrequent eye movements or fail completely to move their eyes (Kainz et al.,

2003). The adult zebrafish used in this experiment were genetically wild type and

approximately 30 months old.

Alpha-aminoadipic acid treatment of adult zebrafish and zebrafish larvae

DL-α-aminoadipic acid was mixed with PBS (pH 7.4) and then adjusted to a pH

of 7.3 using 1M NaOH. The resulting solution was mixed with fish water containing 2g

of Instant Ocean salts per gallon of distilled water supplemented with vitamins to various

concentrations.

Treated and untreated adult fish were kept in 400mL volume of liquid. Fish used

in experiments lasting longer than one day were fed once daily, and the water was

changed shortly after each feeding. Adult fish were exposed to concentrations of 1mM,

10mM, 25mM, and 50mM α-AAA and for an incubation period from one to four days. It

has been suggested that adult fish absorb pharmacological agents into the blood stream

via the gills.

Treated and untreated larvae were kept in an incubator and were not fed

throughout the experiment because they still obtain nutrients from the yolk at this time in

development. Approximately 15-35 larvae were treated with 80mL of the toxin in a petri

dish starting at 3 dpf (after they had hatched from their chorion) and continuing for 48

hours until 5 dpf. The water was not changed after 3 dpf. Larvae were exposed to

concentrations of 10mM, 25mM, 50mM, and 100mM α-AAA. Control larvae were

reared in normal fish water.

The larval exposure period of day 3 to day 5 was chosen for several reasons. On

the third day of life, retinal functions first begins in the zebrafish larvae, and on 5 dpf,

13
visual function is readily observable and has been well-characterized (Schmitt and

Dowling, 1999; Brokerhoff et al., 1995). Another advantage to using young zebrafish

larvae for a pharmacological based experiment is that the larvae have not developed

scales yet and their skin is known to absorb chemicals in the fish water. Finally, based on

the work of Sugawara et al. on carp, exposure periods as short as 4-8 hours show

observable effects on Müller cells. Thus, a 48 hour exposure period was deemed a long

enough period of time.

Constant light rearing

Adult fish treated in constant light were first raised in constant darkness for one

week and were then placed in a box lined with several fluorescent bulbs, with a fan used

to minimize heat generated by the light for the duration of the treatment. The light level

was approximately 15,000 lux, about 50 times brighter than average room light, and the

temperature was maintained between 23-25ºC.

Preparation of adult eyes for immunohistology

Adult zebrafish were euthanized by over anesthetizing them in a 500mg/L

Tricaine solution and then decapitated. Adult heads were immediately placed into cold

fixative containing 4% paraformaldehyde (PFA) in 0.06M phosphate buffer, 3% sucrose

(pH 7.4) with 0.15mM CaCl2. Forceps were then used to loosen connective tissue

surrounding the eye, and the optic nerve was cut using a surgical scissor. Each eye was

removed gently and the cornea was poked with an insect pin to increase the access of the

fixative to the retina. Eyes were then transferred into fresh cold fixative and stored for 4-

8 hours at 4ºC. Eyes were washed for 5 minutes 3X in 0.06M phosphate buffer, 3%

sucrose (pH 7.4) with 0.15mM CaCl2 and then placed into 0.06M phosphate buffer, 15%

sucrose (pH 7.4) with 0.15mM CaCl2 for one hour at 4ºC. Eyes were then transferred to

14
0.06M phosphate buffer, 30% sucrose (pH 7.4) with 0.15mM CaCl2 at 4ºC overnight.

Eyes were removed and were mixed in a 1:1 solution of 30% sucrose and OCT and then

transferred to a mixture completely comprised of OCT. Eyes were embedded in OCT

and then frozen using dry ice. Eyes were sectioned at 10µm in thickness and placed onto

gelatin-coated slides.

Immunohistological analysis
Sections were removed from the freezer and air-dried for 2 hours. Slides were

then washed in PBS (pH 7.4), 5 minutes 3X and blocked in 5% normal goat serum (NGS)

in PBS with 0.3% Triton X-100 for 20 minutes at room temperature. Sections were then

incubated overnight with primary antibody diluted in blocking solution at 4ºC for 12-18

hours. Sections were then brought to room temperature and washed in PBS 15 minutes

4X. Secondary antibody diluted in blocking solution including the Hoechst nuclear dye

was applied, and slides were placed in 37ºC for 30 minutes. Sections were then washed

in PBS for 10 minutes 3X. Slides were mounted with Vectashield mounting medium and

stored in the freezer. Slides were analyzed using confocal microscopy and images were

captured digitally by Pamela Kainz. The following list includes the antibodies used, the

working dilution, and the cell types which possess the respective antigens: GFAP, 1:200,

Müller glial cells (primarily the cell endfeet); GS, 1:500, Müller cells. Secondary

antibodies used were AlexaFluor-488 or -555 conjugates.

Histology
Adult eyes were obtained using the method described above; however, the

fixative for this analysis contained 2.5% glutaraldehyde, 1% PFA in 0.06M PBS (pH

7.4), 3% sucrose, 0.15mM CaCl2. Isolated eyes were immediately transferred into fresh

cold fixative for 30 minutes. After this period, the eyes were placed in fresh fixative for

15
2 hours. Eyes were then rinsed for 15 minutes 2X in 0.06M PBS (pH 7.4), 3% sucrose,

0.15mM CaCl2 and were then dehydrated in a graded series of ethanol in 0.06M PBS (pH

7.4), 3% sucrose, 0.15mM CaCl2 and infiltrated with propylene oxide and resin.

Transverse sections of 1µm thick were collected and heat-mounted onto a gelatin-coated

glass slide and stained with 1% Methylene Blue, 1% Azure in 1% borax. Slides were

cover-slipped with DPX.

Larvae preparation for histological retinal analysis

Five-day old larvae were anesthetized in ice-cold fish water and fixed with 2.5%

glutaraldehyde, 1% PFA in 0.06M phosphate buffer (PBS) (pH 7.4), 3% sucrose,

0.15mM CaCl2 for 1.5 hours at 4ºC. Larvae were then rinsed in 0.06M PBS (pH 7.4), 3%

sucrose, 0.15mM CaCl2 2X five minutes. Larvae were dehydrated in a graded series of

ethanol in 0.06M PBS (pH 7.4), 3% sucrose, 0.15mM CaCl2 and infiltrated with

propylene oxide and resin. Transverse sections of 1µm thick were collected and heat-

mounted onto a gelatin-coated glass slide and stained with 1% Methylene Blue, 1%

Azure in 1% borax. Slides were cover-slipped with DPX.

Visual behavioral analysis

The optokinetic reflex assay (OKR) was used to test the visual sensitivity of

larvae at 5 dpf. For testing, 4-5 subjects were transferred into small petri dishes

containing 5% methyl cellulose and placed within a drum lined with vertical black and

white stripes, 1cm in width. The drum was illuminated with a tungsten light source, 9.74

* 10-2 W/cm2 and a 2 minute trial was conducted, during which the direction of rotation

of the drum was switched 3-4 times. The criterion for a positive response was that each

larva either demonstrated a full smooth pursuit-saccade cycle or eye tracking movements

16
in both the clockwise and counterclockwise directions when the drum was rotated

accordingly.

RESULTS

Müller glial cell expression of GFAP and GS is altered by α-aminoadipic acid

To analyze the effects of α-AAA on the adult zebrafish Müller cells, I exposed

several fish to different concentrations of the gliotoxin: 1mM, 10mM, and 25mM, 50mM

for time periods of 24 and 48 hours. A concentration of 50mM compromised severely

the overall health of the zebrafish and resulted in violent spasms. These fish were thus

sacrificed due to the fact that they appeared distressed and unhealthy. Fish exposed to

concentrations less than or equal to 25mM showed no behavioral abnormalities when

treated. These adults were fixed for retinal immunological and histological analysis.

Retinal sections were obtained, labeled with GS and GFAP, and imaged using

confocal microscopy and digital photography. Fish treated with the lowest concentration

(1mM) had retinas that appeared highly similar to wild type. Adults treated with the

highest, non-lethal concentrations (10mM and 25mM) also had retinas that appeared to

be well intact. To examine specifically Müller cells, antibodies to two known cell

specific proteins, GS and GFAP, were utilized. Exposure to 10mM α-AAA for 24 hours

appeared to affect modestly the expression of GS and GFAP.

The retinal images shown in figure 2 were obtained from a specified region in the

dorsal part of the retina from treated and untreated animals. The expression of GS in

untreated retinas was evenly distributed throughout the Müller cell bodies and therefore,

the retina (Fig. 2a), while the expression of GS in treated retinas had a spoke-like pattern

(reminiscent of the Müller cell bodies) suggesting that the either the expression of this

protein increased in response to the gliotoxin or the size of the Müller cell processes

17
increased (Fig. 2b). In addition, there appeared to be less GS labeling in the inner

plexiform synaptic layer in the treated retina compared to controls.

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 a b

wt control wt 10mM
c d
PRL

INL

Endfeet wt control wt 10mM

Fig. 2. Zebrafish treated with 10mM α-aminoadipic acid for 24 hours exhibited differences in GS (red) and GFAP
(green) expression in comparison to controls. The photoreceptor layer (PRL) and inner-nuclear layer (INL) are labeled.
a: GS expression in untreated retinas was evenly distributed in each layer of the retina. b: GS expression in treated
retinas appeared denser or spoke-like. c: GFAP expression in untreated retinas was confined to the Müller cell
endfeet. d: GFAP expression in treated retinas was somewhat elevated in Müller cell bodies and in radial processes
reaching the outer-plexiform layer.

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 More substantial differences were found in the expression of GFAP. In the

controls, immunoreactivity was mostly confined to the Müller cell endfeet (Fig. 2c).

However, in the treated retinas, the GFAP immunoreactivity was present throughout the

Müller cell bodies in radial processes reaching the outer plexiform layer (Fig. 2d)

suggesting that the expression of GFAP increased in response to exposure to the

gliotoxin, consistent with what would be predicted.

The pattern of Hoechst nuclear dye labeling shown in blue in figure 2c and 2d

illustrates how well-preserved the 10mM and 25mM α-AAA treated retinas were

compared to untreated controls. No gaps were seen in the nuclear layers indicating that

α-AAA did not result in massive cell death, and three distinct nuclear layers were

observed implying that retinal organization was not disrupted. In case the mild effect on

Müller cells disrupted their ability to support photoreceptors, I examined closely the

integrity of the photoreceptors cells in the treated animals. No evidence of photoreceptor

cell disruption was observed.

Increased dosage and a longer incubation period yielded similar results

The 24 hour exposure of 10mM and 25mM Müller appeared to have no major

detrimental effect on retinal neurons but did affect mildly Müller glia. Since the goal was

to determine whether α-AAA could compromise significantly the Müller glia while

having no effect on retinal neurons, I decided to push the system. Since higher

concentrations were lethal, I chose to increase the length of exposure. For this, I exposed

adult zebrafish to a concentration of 25mM over a period of 48 hours and found that the

severity of the effect had not increased. Again, the expression of GS and GFAP were

mildly elevated (data not shown). Greatly increasing the exposure time may have

strengthened the gliotoxin affect; however, we did not feel this was a very practical

20
approach given the large amount of drug we would need to utilize in order to be able to

change the 400mL solution of α-AAA daily. Instead I set out to determine the effect α-

AAA had on larval zebrafish.

When combined with constant light, α-aminoadipic acid caused a similar phenotype
Knowing that constant and intense light exposure exacerbated the Müller cell

phenotype in the larval genetic model of Müller cell disruption, the lze mutant, I tested

the impact of combining pharmacological stress with light toxicity in the adult. Although

teleost retinas have shown more resistance to light damage than rodents (where the retina

is largely rod dominated), light toxicity has been studied in albino adult zebrafish where

it has caused rod and cone cell apoptosis (Vihtelic and Hyde, 2000). We chose to avoid

the use of albino zebrafish due to their low viability as larvae and extreme susceptibility

to light toxicity. Instead, I tested α-aminoadipic acid treated and untreated adult wild

type zebrafish in a light regiment of 15,000 lux, nearly twice the intensity necessary to

observe photoreceptor cell death in albinos.

My initial result suggested that the combination of the toxin (25mM α-AAA) and

constant light caused photoreceptor cell death in the adult zebrafish (Fig. 3). This was

apparent from observing a Hoechst nuclear dye that indicated that the dorsal portion of

the outer-nuclear layer was only one nucleus thick in α-AAA light treated fish but 2-3

nuclei thick in controls (unexposed to light or the drug). When repeated several times,

the experiment provided new data to suggest that my light exposure regiment alone could

sometimes cause photoreceptor disruption and death (Fig. 4).

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 a

ONL

control

b
ONL

light
c

ONL

light/AAA light/AAA
Fig. 3. Adult zebrafish treated with 25mM α-aminoadipic acid for three days under constant lighting conditions had
photoreceptor cell degeneration. a: The outer nuclear layer (ONL) in untreated retinas appears healthy and is
approximately three nuclei thick when viewed under a Hoechst stain. b: The ONL in the retina exposed to constant
light appears overall healthy to the control, with the layer spanned by a thickness of 2-3 nuclei. c: The ONL in the
retina exposed to constant light and 25mM α-aminoadipic acid is approximately one nucleus thick indicating that many
photoreceptors were lost. Pyknotic nuclei are indicated by arrows.

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 a b

OS

ONL

INL

GCL control light

Fig. 4. Adult zebrafish retinas exposed to constant c
light exhibit a similar phenotype to those exposed to
constant light and 25mM α-aminoadipic acid for
three days. a: The untreated retina exhibits healthy
outer segments (OS). b: The retina exposed to
constant lighting expresses unhealthy and missing
outer segments (arrow) and missing photoreceptor
nuclei. c: The retina exposed to constant lighting
and treated with 25mM α-aminoadipic acid was
similar to the retina treated with light alone, having
photoreceptor cell loss and outer segment
disruption.
light/AAA

23
 Larval health was compromised at concentrations similar to the adult
Wild type larvae were treated with various concentrations of the toxin by adding

it to their water in petri dishes from 3 dpf to 5 dpf. Survival data is located in Table 1. I

found that 81% of larvae survived a two day exposure at concentrations of 10mM and

61% survived a two day exposure at a concentration of 25mM. Survival percentage was

0% at 50mM, although 1/15 treated fish survived a concentration of 100mM (7%). This

non-lethal dose pattern was thus similar to adults.

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 α-Aminoadipic Wild Type Larvae Wild Type Larvae Survival
Acid Treated Survived Percentage
Concentration
(48hr Exposure)
0mM (control) 45 45 100%
10mM 15 14 87%
25mM 36 22 61%
50mM 15 0 0%
100mM 15 1 7%
Table 1. Wild type larvae have similar dosage dependent survival as adult zebrafish. Larvae were able to tolerate an
exposure of 48 hours at concentrations up to 25mM α-aminoadipic acid before they displayed scant survival. Similarly,
the health of adults was severely compromised at concentrations of 50mM or higher.

25
 General characteristics of 5 dpf α-aminoadipic acid treated larvae
All of the treated larvae at 25mM appeared similar to untreated animals with

some exceptions. Treated larvae displayed a constant jaw twitching movement and

lacked an inflated swim bladder. Furthermore, they demonstrated little spontaneous

activity and had only a moderate response to touch. The yolk of treated larvae was also

observed to be partitioned. Heart rate was found to be the same between treated and

untreated wild type larvae. The jaw twitching, lethargy, and under-inflated swim bladder

were all characteristics in common with the lze Müller glia mutant.

Wild type larvae treated with α-aminoadipic acid demonstrate an OKR similar to controls
To test the vision of wild type larvae treated with the toxin, I utilized the

optokinetic reflex assay. I found that larvae treated at a concentration of 25mM

demonstrated an almost identical response to the assay as untreated wild type controls

(when the light level in the barrel was not attenuated). Both untreated and treated larvae

demonstrated a strong saccade and steady tracking of the black and white stripes.

Alpha-aminoadipic acid causes disruptions in the ganglion cell layer, inner nuclear
layer, horizontal cell layer, and marginal zone in zebrafish larvae
While the visual behavior of treated and untreated wild type larvae was similar,

histological findings showed that some retinal neurons were disrupted in larvae exposed

to 10mM α-AAA. Pyknotic nuclei, that appear darkened with a halo of empty space

surrounding them, were identified within the ganglion cell layer (GCL), indicating that

some of these neurons were degenerating (Fig. 5b). In addition, the toxin compromised

the marginal zone as indicated by gaps seen in this region where proliferative stem cells

are present in controls. At concentrations of 10mM, the toxin also appeared to have an

effect on the horizontal cell layer, causing large gaps, indicating the presence of fewer

horizontal cells compared to untreated larvae (Fig. 5b).

26
 Increasing the dosage of the drug proved to increase inner nuclear layer

disruption. At concentrations of 25mM, the toxin caused cell loss within GCL but also

caused death in INL (Fig. 5c). Large circular gaps were present in these treated retinas,

reminiscent of retinal locations where cell death had just occurred. Gaps in the brain

were observed in animals treated with 25mM α-aminoadipic-acid (Fig. 5d).

27
 a b

wt control wt 10mM
c d

wt 25mM wt 25mM
Fig. 5. Wild type zebrafish larvae retinas treated with α-aminoadipic acid at concentrations of 10mM and 25mM and
for an incubation period of 48 hours expressed disruptions in the ganglion cell layer, inner nuclear layer, horizontal cell
layer, and marginal zone. a: Control retinas showed no signs of cell death. b: Wild type retinas treated with
concentrations as low as 10mM expressed pyknotic cells in the ganglion cell layer (orange arrow), missing horizontal
cells (red arrow), and deficiencies in the marginal zone (green arrow). c: Increasing the dosage to 25mM resulted in
more inner nuclear layer deficiencies and large gaps. d: Concentrations of 25mM also caused larges gaps of missing
cells in the brain.

28
 Alpha-aminoadipic acid causes cell migration out of the retina through the optic
nerve in some wild type retinas
In two of the approximately 20 wild type treated retinas that were sectioned

(10%), I observed cell migration out of the retina through the optic nerve. I could not

ascertain definitively the direction of the migration, although it appears as if cells were

funneling out of the retina towards the brain (Fig. 6a). Cells were elongated, which is

indicative of migrating cells, and some seemed to be differentiated. A cell associated

with an outer segment (a presumed photoreceptor) can be seen within the migratory

stream of cells (Fig. 6b). This effect was seen in wild type treated larvae at a

concentration of 10mM and at a concentration of 100mM but never in wild type untreated

larvae (data not shown).

29
 a b

wt 10mM
wt 10mM wt 10mM
Fig. 6. In 10% of wild type treated retinas, α-aminoadipic acid caused retinal cell migration out of the retina through
the optic nerve. a: Differentiated and elongated cells appeared as though they were migrating out of the retina towards
the brain. b: A close up of the optic nerve region. The circle surrounds a cell with an outer segment, indicative of a
photoreceptor cell.

30
 Alpha-aminoadipic acid does not seem to worsen severely the lze phenotype
Since the zebrafish is a genetic model organism, mutants, such as lazy eyes, could

be used to investigate the combined effects of genetic and pharmacological manipulation

of glia on the neural retina. Knowing that lze mutants have compromised Müller glial

cells, I was curious whether α-AAA would increase the severity of the lze Müller cell

phenotype. Thus, the same experiments were carried out on lze larvae. Larvae from the

lze clutch that were treated with 10mM α-AAA exhibited subtle histological

abnormalities within the spectrum of what was observed in treated wild type larvae and in

lze untreated controls (Fig. 7).

31
 a b

lze 10mM lze control

Fig. 7. Lze larvae treated with α-aminoadipic acid exhibit a phenotype similar to lze untreated controls. a: Lze larvae
treated with 10mM α-aminoadipic acid appear to have some inner nuclear layer deficiencies (highlighted by the arrow)
but do not appear severely different from lze controls. b: A lze control is pictured.

32
 DISCUSSION

The goal of the work outlined in this chapter was to explore the gliotoxic effect of

α-AAA on the retina using the zebrafish animal model. Previous studies using α-AAA

have suggested that retinal glial cells were specifically affected; however, none have been

on the zebrafish nor have any been conducted without the use of invasive methods of

drug delivery such as intraocular injection. By delivering the drug via the fish water, the

concentration and thus dose of the drug at any one time is constant, unlike the

unavoidable fluctuation of drug dose with intraocular injection or subcutaneous injection

done in all previous in vivo studies. .

The investigation of an α-AAA-mediated pharmacological model of Müller cell

disruption in the adult zebrafish yielded tepid effects, considerably weaker than those

observed in other animal models treated with the toxin. While other studies have

observed Müller cell death, swelling, or hypertrophy, as well as photoreceptor death, I

did not. My sole finding was a increase in the expression of GFAP. Although the

increase was subtle, upregulation of GFAP is a classic indication of Müller cell stress

indicating that the drug was having the desired affect. Furthermore, labeling with the GS

antibody revealed that Müller processes appeared thicker in response to treatment

suggesting that some degree of hypertrophy may have occurred. Thus, I observed signs

of Müller cell stress although these indications were more subtle than predicted.

Unfortunately the effects could not be increased in severity by increasing concentration

or length of exposure, before lethality became a problem.

In the wild type larval retina, I observed evidence of modest neuronal

degeneration. In addition, I noticed cell migration of retinal cells out the optic nerve in a

33
few of the retinas. Both of these observations involved retinal neurons and indicated a

lack of glial-specificity. It is known that when α-AAA is used at too high of a

concentration, the affects can be neurotoxic (Sugawara et al., 1990) perhaps indicating

that my dosage was too high. This is possible but unfortunately neither neurons nor glia

were affected by lower doses.

In summary, the effect of the gliotoxin varied substantially from fish to fish and

was highly dependent on age, as the effects were very subtle in the adult retina and

detrimental to retinal neurons in the larval zebrafish. Although more variables in

treatment strategy could be explored, the neurotoxic effects observed in the larvae

discouraged me from further pursuing this direction for the present time. As a result, I

focused on a different model of Müller cell stress mediated by a genetic mutant.

34
 Chapter 2: The Genetic Mode

INTRODUCTION

The impetus for moving in this direction were the findings of Rattner and Nathans

(2006) in their study on the genes related to photoreceptor stress induced either by

detachment, genetic photoreceptor mutations, or light toxicity. Using microarray

technology, RNA blots, and in situ hybridization, they quantified the genomic responses

to both light damage and inherited photoreceptor degeneration and found that these

responses involve a relatively small number of overlapping genes (Rattner and Nathans,

2005). In their research, they discovered that the endothelin pathway is highly linked to

photoreceptor and Müller cell stress.

Endothelins are vasoactive peptides with various functions throughout vertebrates

including cardiovascular systems, pigmentation, and ocular homeostasis (Prasanna et al.,

2003). There are three isoforms of the peptide: endothelin 1, 2, and 3. Endothelin

receptors come in two subtypes, endothelin receptors A and B, both G-protein coupled

receptors (Sakurai et al., 1992). In the retina, endothelin receptor A (ET-A) is mainly

localized to the choroid and blood vessels, whereas endothelin receptor B (ET-B) has

been found mainly in the neural and glial components of the retina (Maccumber and

D’Anna, 1994) although the precise roles endothelins play in the retina are unknown.

Using the mouse model, Rattner and Nathans found that endothelin 2 is expressed

in photoreceptor cells and highly induced in all of their models of photoreceptor stress.

In addition, they found that ET-B localized to Müller cells and its expression was

upregulated >10 fold following phototoxic conditions. These data led the authors to the

hypothesis that the endothelin pathway plays a critical role in the Müller cell response to

35
stressed or dying photoreceptor cells and may be involved in the neuroprotective support

function Müller glia provide for photoreceptors.

Other studies have shown that the endothelin pathway is susceptible to

pharmacological manipulation. When administered to albino mice under phototoxic

conditions, Tezosentan, a mixed ET-A and ET-B antagonist, lowered the amount of

GFAP expression and also resulted in a lower amount of apoptotic cells throughout the

retina, as judged by a CC3 cell death assay. These findings imply that inhibition of

endothelinergic receptors could play a role in the preservation of vision by sparing

photoreceptors (Torbidoni et al., 2005). The investigators hypothesized that the

endothelin pathway triggers the Müller cells to upregulate GFAP expression resulting in a

scarring effect and that the prevention of the Müller cell processes could promote

neuronal survival and preserve vision.

Strong associations between the endothelin pathway, Müller cells, and

photoreceptor support mechanisms inspired my investigation of the ET-B knockout

zebrafish called rose. Using a genetic mutant allowed me to circumvent some of the

drawbacks with a pharmacological model, specifically the variability and fluctuation in

drug concentration caused by metabolism. Rose was initially discovered through its

abnormal body pigmentation and the initial study concluded that the only defect caused

by the absence of the ET-B was the lack of the production of a subset of the adult

melanocytes and iridiphores. This phenotype resulted in adults appearing reddish

compared to wild type (Fig. 8) (Parichy et al, 2000). Later studies provided data to

support that the ET-B gene is actually expressed in the zebrafish larval retina (Lister et

al., 2006).

36
Fig. 8. Wild type and rose mutant adult zebrafish (Source: Parichy et al., 2000). a: Wild type adults demonstrate
normal coloring. b: Rose mutants fail to develop the normal amount of melanocytes and iridophores during pigment
pattern metamorphosis, accounting for their reddish appearance.

37
 In the second chapter of this study, I characterized the homozygous ET-B mutant

rose zebrafish larval retina. Research on the links between phototoxicity and

photoreceptor stress in addition to lze’s increased Müller specific susceptibility to light

provided the impetus for exposing my genetic model to constant light. Rattner and

Nathans’ findings, that the endothelin pathway was involved in the response to

phototoxic condition, gave me further reasons to test how a retina missing ET-B would

react to constant lighting. I employed histological, behavioral, and quantitative

measurements to characterize the degree to which the health or function of the retina was

compromised. The OKR was used to measure the visual threshold of light-treated rose

mutants and control retinas, and these results were compared to wild type larvae under

the same two conditions: constant light (LL) and a normal light dark cycle (LD). Retinal

histology of rose was performed and cell counts on the inner and outer nuclear layers

were used to assess the presence or absence of cell death in the regions of the retina

containing Müller cells and photoreceptor cells. These experiments continue with the

theme of this thesis: exploring potential models where Müller cells are compromised and

observing the effects of this stress on photoreceptors and on vision in the zebrafish.

MATERIAL AND METHODS

Zebrafish maintenance
Wild type and rose homozygous recessive zebrafish were maintained as described

in Chapter 1.

Constant light rearing

Adult larvae were maintained in a standard 14/10-hour light-dark cycle until 2 dpf

when they were transferred to constant dark conditions because pretreatment with

constant darkness intensifies the effect of the light treatment to follow. Dark adaptation

38
from day 0 until day 2 was not necessary since larvae lack developed photoreceptors at

this point and opsin does not appear until 48hrs post fertilization (Schmitt et al., 1999).

At 4 dpf, larvae treated with constant light were transferred to a box lined with several

fluorescent bulbs and a fan used to minimize heat generated by the light. The light level

was approximately 15,000 lux, about 50 times brighter than average room light. Controls

were kept in standard light dark conditions from 2-6 dpf. Larvae were removed at 6 dpf

for visual testing and fixation.

Visual Threshold Assay

All OKR assays were run on 6 dpf larvae between the hours of 1 PM and 5 PM in a

completely darkened room. I used a dim, red head-lamp for visibility when needed. For

testing, 4-5 subjects were transferred into small petri dishes containing 5% methyl

cellulose and placed within a drum lined with vertical black and white stripes, 1cm in

width. The drum was illuminated with a tungsten light source, 9.74 * 10-2 W/cm2,

attenuated by 6.5 log units, and the drum was rotated at 10 rpm. A 2 minute trial was

conducted, during which the direction of rotation of the drum was switched 3-4 times.

The lowest light level that evoked an OKR response for each larva was determined. The

criterion for a positive response was that each larva either demonstrated a full smooth

pursuit-saccade cycle or eye tracking movements in both the clockwise and

counterclockwise directions when the drum was rotated accordingly. Fish that failed to

demonstrate a positive response were retested using 0.5 log unit brighter illumination.

Histology
Six-day old larvae were fixed using the same protocol as Chapter 1.

Cell Counts
Cell counts were completed in rose and wild type light treated and untreated

retinas in the inner and outer nuclear layers. Inner nuclear layer counts did not register

39
horizontal cells or nuclei in the marginal zone (Fig. 9). The marginal zone contains

proliferative stem cells, while the inner nuclear layer contains Müller, bipolar, and

amacrine nuclei.

40
Fig. 9. The region of the retina in which inner nuclear layer cell counts were performed is enclosed in red. Inner
nuclear layer nuclei were counted with careful attention not to include nuclei in the marginal zone (top arrow) or
horizontal cell nuclei (bottom arrow) in the count.

41
 RESULTS

Rose larvae have normal retinal morphology and visual function

Morphologically, rose and wild type larvae retinas were nearly indistinguishable

when the larvae were raised in a normal light dark cycle (Fig. 10a, 10c). Both had

healthy photoreceptors, a continuous span of nuclei in the outer and inner nuclear layers,

and healthy, dense, and organized rod outer segments. Furthermore, without dark

adaptation and light attenuation, rose larvae responded equally robustly to wild type in

response to the OKR.

42
 a b

wt LD wt LL
c d

rose LD rose LL
Fig. 10. Wild type and rose larvae are strikingly similar when raised in a normal light dark cycle or with constant light.
a: Wild type larvae raised in normal light dark cycle. Encircled is the rod dense ventral portion of the retina. Large
rod outer segments span the region containing melanin from the pigmented epithelial cells. b: Wild type larvae treated
with light. c: Rose mutant larvae kept in normal lighting conditions appear nearly identical to wild type larvae treated
in the same conditions. d: Rose mutant larvae treated in constant light show significant loss of rod outer segment
material in the ventral retina.

43
 When raised in constant light, rose showed higher susceptibility to rod outer
segment damage
Histological analysis of retinas from rose larvae exposed to constant light

revealed that there were limited but visible differences between rose and wild type retinas

(Fig. 10b, 10d). Both retinas appeared grossly normal and neither displayed indications

that massive cell death had occurred, in the form of pyknotic nuclei or gaps in the nuclear

layers. There were also no obvious signs of disruption within Müller cells. Their cell

bodies did not appear hypertrophied.

There was, however, one clear difference between retinas from constant light

reared wild type and rose larvae: the integrity of their rod outer segments. Rose light

treated retinas showed several degrees of rod outer segment health. Some appeared

slightly swollen in comparison to light dark treated rose larvae (Fig. 11). Most, however,

were disorganized, and many retinas were missing rod outer segments. Furthermore,

vacuoles within the RPE were very common. In contrast, the rod outer segments of wild

type light treated retinas almost always appeared equally healthy to their light-dark

counterparts. In some wild type retinas, there were slightly swollen or disorganized rod

outer segments; however, in only one of sixteen retinas were there actually fewer rod

outer segments.

44
 a

rose LL
b

rose LL
c

rose LL
Fig. 11. Rose mutant larvae treated with constant light showed varying degrees of rod outer segment health. a: Rod
outer segments appear healthy and organized yet a few small vacuoles appear in the RPE (red arrow). b: Rod outer
segments appear substantially disorganized, shortened, and dysmorphic (orange arrow), and the RPE contains vacuoles.
c: Significant loss of rod outer segments from the ventral retina has taken place.

45
 When treated with constant light, rose larvae had a 10% reduction in inner nuclear layer
nuclei, while the amount of inner nuclear layer nuclei in wild type remained constant
The lack of gaps in the nuclear layers and the absence of pyknotic nuclei suggest

that cell death in the rose retina did not occur, at least not on day 6 when the larvae were

sacrificed. To ascertain whether any cell death had occurred prior to day 6, the number

of nuclei were counted in the nuclear layer that contains the photoreceptor nuclei and the

nuclear layer that contains Müller cells. Average numbers were compared among light-

dark (LD) and light-light (LL) treated rose and wild type animals. Neither rose nor wild

type showed a decrease in the number of outer nuclear layer (ONL) nuclei upon the

introduction of constant light; however, wild type had on average had more ONL nuclei

than rose in both treatment groups. For instance, rose light treated larvae had an average

of 156 ONL nuclei, while wild type had an average of 183, 20% higher with a p-value of

0.00022 (Fig. 12, Table 2).

46
 Rose and Wild Type Outer Nuclear Layer
200
**
Average Number of Nuclei in ONL 190

180

170

160

150

140

130

120

110
rose LD rose LL wt LD wt LL
100

Fig. 12. Neither rose nor wild type larvae lost photoreceptor nuclei upon treatment with constant light. Although the
average number of nuclei in the ONL in rose LD and LL was different than wild type, no difference was observed
between rose LD and LL. Error bars represent 95% confidence intervals, and starred bars connect treatment groups for
which there was a statistically significant difference in outer nuclear layer.

47
 rose LD rose LL wt LD wt LL
INL ONL INL ONL INL ONL INL ONL
Average
Number of
Nuclei 385 161 349 156 339 179 358 183
Standard
Deviation 50 24 36 12 34 19 31 18

Number
Observations 20 8 38 17 14 17 13 13

95% Confidence [363, [144, [338, [151, [321, [170, [342, [174,
Interval 407] 178] 361] 162] 357] 188] 375] 193]
Table 2. Average inner and outer nuclear layer nuclei for rose and wild type larvae across the two treatment groups.

48
 Untreated rose larvae had an average of 385 nuclei in their INL in contrast to light

treated rose which had an average of 349 nuclei, roughly a 10% decrease. This

difference was found to be significant to a p-value of 0.0092 (Fig. 13, Table 2). No such

effect was observed in wild type light treated larvae. Another finding was that untreated

rose larvae had nearly 14% more INL nuclei than their untreated wild type counterparts,

which had an average of 339 nuclei. This difference was significant to a p-value of

0.0037 (Fig. 13, Table 2).

49
 Rose and Wild Type Inner Nuclear Layer
410 **
Average Number of Nuclei in the INL **
390

370

350

330

310

290

270
rose LD rose LL wt LD wt LL
250
Fig. 13. When treated with constant light, rose larvae lose 10% of their inner nuclear layer nuclei. Error bars represent
95% confidence intervals, and starred bars connect treatment groups for which there was a statistically significant
difference in inner nuclear layer nuclei. Rose untreated larvae had an average of 385 inner nuclear layer nuclei, while
rose light treated larvae had an average of 349 inner nuclear layer nuclei (p-value 0.0092). There was also a
statistically significant difference between rose untreated larvae and wild type untreated larvae which had an average of
339 nuclei (p-value 0.0037).

50
Untreated rose larvae exhibit a lower visual threshold than rose larvae treated with
constant light; wild type shows no loss in visual function after light treatment
While I had observed that untreated rose and wild type exhibited equally robust

responses to the OKR without dark adaptation, I realized that this was only a qualitative

observation. To obtain a quantitative assessment of visual behavior, I chose to measure

visual thresholds. Thresholds were found by determining the lowest light level that

evoked an OKR response for each larva. Larvae that failed to demonstrate a positive

response were retested using 0.5 log unit brighter illumination.

In light-dark conditions, rose and wild type exhibited an average threshold of -6.0

log units of light attenuation similar to wild type (Fig. 14, Table 3). When reared in light-

light, rose larvae had a significantly higher threshold than rose larvae raised in a normal

light dark cycle and than wild type larvae treated in constant light. Rose light treated

larvae had an average threshold of -4.5, while untreated rose larvae had an average

threshold of -6.0 (Fig. 14, Table 3). This difference was highly significant to a p-value of

2.8 * 10-7. In addition, this drop in visual sensitivity was not seen in wild type suggesting

that the light-light treatment used had no measurable effect on fish having an intact ET-B

gene. Light treated wild type larvae had an average threshold of -5.8. The difference

between rose and wild type light treated larvae was significant to a p-value of 2.7 * 10-6.

51
 Rose and Wild Type Visual Threshold

6.5 ** **
Average Threshold (Units of Negative Log Attenuation)

6.0

5.5

5.0

4.5

4.0

3.5

rose LD rose LL wt LD wt LL
3.0

Fig. 14. When treated with constant light, the visual threshold of rose larvae increases. In contrast, constant light does
not affect the visual sensitivity of wild type larvae. Error bars represent 95% confidence intervals, and starred bars
connect treatment groups for which there was a statistically significant difference in visual threshold. Rose light treated
larvae had an average threshold of -4.5, while untreated rose larvae had an average threshold of -6.0 (p-value of 2.8 *
10-7). There was also a statistically significant difference between rose light treated larvae and wild type light treated
larvae, which had an average threshold of -5.8 (p-value of 2.7 * 10-6).

52
 rose LD rose LL wt LD wt LL

Average Threshold -6 -4.5 -6 -5.8

Standard Deviation 0.5 1.5 0.5 0.5

Number Observations 29 42 30 33

95% Confidence Interval [-5.8, -6.2] [-4.1, -5.0] [-5.8, -6.2] [-5.7, -6.0]
Number Thresholds -5 or Lower 28 19 30 31

% Thresholds -5 or Lower 97% 45% 100% 94%

Number Thresholds Higher Than -5 1 23 0 2

% Thresholds Higher Than -5 3% 55% 0% 6%

Table 3. Average visual threshold for rose and wild type larvae across the two treatment groups.

53
 Rose light treated larvae in general varied substantially in their thresholds. Some

had thresholds equivalent to wild type, while others had significant drops in their visual

sensitivity. To illustrate this phenomenon, I calculated the percent of larvae in each

group that had a threshold of -5 or lower. I used -5 as cutoff because this is the highest

threshold any light-dark wild type larva ever demonstrated. While rose untreated larvae,

wild type untreated larvae, and wild type light treated larvae had thresholds of -5 or lower

in 97%, 100%, and 94% of the data points respectively, rose light treated larvae only had

a threshold of -5 or lower 45% of the time (Fig. 15, Table 3). The difference between

rose light treated and rose untreated larvae in this case was significant to a p-value of 1.4

* 10-7, a highly robust result.

54
 Rose and Wild Type Visual Threshold Binomial Data
100.0%

80.0%
Percent of Thresholds -5 or Lower

60.0%

40.0%

20.0%

rose LD rose LL wt LD wt LL
0.0%

Fig. 15. The percentage of larvae in each treatment group that had a threshold of -5 or lower is depicted. Error bars
represent 95% confidence intervals. While 97% of untreated rose larvae had a threshold of -5 or lower, only 45% of
the light treated rose larvae had a threshold of -5 or lower (p-value 1.4* 10-7).

55
Inner nuclear layer count and visual threshold are not correlated in rose light treated larvae
Curious whether there was relationship between the loss of cells in the INL and

high visual thresholds, I tested rose light treated larvae in the OKR and separated them

into two groups, those with a threshold of -5 or lower and those with a threshold higher

than -5. I then proceeded to calculate the average number of INL nuclei in each group.

There did not appear to be a correlation between INL count and visual threshold as both

groups expressed a nearly identical average number of nuclei (Fig. 16, Table 4).

56
 Inner Nuclear Layer Visual Threshold Correlation
390
Average Number of Nuclei in INL
370

350

330

310

290

270
Rose LL Low Rose LL High
Threshold Threshold
250

Fig. 16. When rose light treated larvae were separated into two groups, those with visual thresholds of -5 or lower
(Low Visual Threshold) and those with visual thresholds higher than -5 (High Visual Thresholds), there was no
significant difference in the average number of inner nuclear layer nuclei across the two groups. Error bars represent
95% confidence intervals.

57
 rose LL Low rose LL High
Visual Threshold Visual Threshold

Average Number of Nuclei in INL 356 358

Standard Deviation 35 31

Number Observations 7 15

95% Confidence Interval [330, 382] [342, 374]

Table 4. Average number of nuclei in the inner nuclear layer across rose light treated larvae with visual thresholds of
-5 or lower (Low Visual Threshold) or with visual thresholds higher than -5 (High Visual Threshold).

58
 DISCUSSION

In my evaluation of rose, I discovered several interesting characteristics of the

mutant. The first was its striking similarity to wild type. ET-B thus is apparently not

necessary for early retinal development. However, the absence of ET-B did result in

decreased photoreceptor resistance to light damage. Rose had compromised rod outer

segments and a higher visual threshold than wild type. The final observation about the

rose mutant was its INL vulnerability to constant light. While wild type showed no loss

of INL nuclei in response to light treatment, the average number of nuclei decreased by

10% in rose. Thus, the absence of ET-B does indeed compromise the retina’s resilience.

At this point however, I have not yet proven that this phenotype is related specifically to

Müller cells.

The nuclei that reside in the inner nuclear layer belong to bipolar, horizontal,

amacrine, and Müller cells. As a final experiment, I applied a Müller cell-specific

antibody, carbonic hydrase II, to retinal sections from rose treated and untreated retinas

to ascertain whether Müller cells were likely the cell type that was missing or partially

missing. While this experiment was attempted twice, I was unable to obtain any labeling

of Müller cells in rose retinas using this antibody, for reasons I do not understand.

The most logical explanation for a higher visual threshold is fewer

photoreceptors. Most research has implicated cones as the predominant contributor to

visual sensitivity at 6 dpf since they greatly outnumber rods at this stage in development

(Bilotta et al., 2001). However, if the visual sensitivity problems had been cone related, I

would have observed a difference in ONL nuclei, which I did not. The only differences I

observed were manifested in rods, specifically in the health of their outer segments. This

59
lead us to believe that although there were no pyknotic rod nuclei in the ONL, the

disorganization and scarcity of the rod outer segments in rose light treated retinas could

imply that the rods were significantly compromised and that rods may play a significant

role in visual function at this stage in development. Therefore, we believe that higher

visual threshold was likely a result of compromised rod outer segment health.

When assessing the reasons behind the rose mutant’s vulnerability to light

toxicity, we could not ignore that fact that rose has a deficiency in cells which are

pigmented: melanocytes and iridiphores. Thus, perhaps rose is more sensitive to light

damage merely because it is missing melanin in the retinal pigmented epithelial cells,

much like the albino model. I did not observe evidence in support of this possibility as

the melanin density in the retinal pigment epithelium in rose retinas did not appear

different from that which was observed in wild type.

A second hypothesis which we think holds more promise and that is consistent

with the literature is that ET-B is involved in a neuroprotective program, perhaps

mediated by Müller cells as implied by the results from mouse models. To follow this

idea, I would first need to confirm that Müller cells express ET-B in the zebrafish retina.

Then, I would want to explore how and whether this expression level changes as a result

of intense light exposure. If the expression of ET-B were specific to Müller cells and

increased in response to light exposure, more experiments would be needed to explore the

timing of rod photoreceptor cell disruption and the loss of INL nuclei to gain a better

understanding of how endothelins and Müller cells are involved in neuroprotection within

the retina.

60
 FUTURE EXPERIMENTS

As mentioned above, in situ hybridization experiments are needed to confirm the

expression of ET-B in Müller cells. The findings of Rattner and Nathans on the

association between ET-B and Müller cells, in addition to research on the pig retina,

which found ET- B to be expressed by the innermost retinal layers, ganglion cell somata,

and by Müller glial cells (Iandiev et al., 2005), have provided compelling circumstantial

evidence that ET-B would be expressed by zebrafish Müller cells. However, other

studies have demonstrated that ET-B is also expressed in other cells within the mouse

retina, including horizontal cells and the retinal pigment epithelium (Torbidoni et al.,

2005). Thus, I would need to investigate this possibility in the zebrafish. After obtaining

the pattern of ET-B expression in the wild type zebrafish retina, I would repeat the

analysis of ET-B expression on rose larvae that had been exposed to constant light to

determine whether Müller cells or any other ET-B positive cells had an appreciable

change in expression level. This would help to identify the cell type(s) involved in the

response to photoreceptor stress.

Next, I would like to determine the identity of the missing cells within the INL in

light-treated rose larvae. One possibility explaining the absence of these cells is that cell

death had occurred during the treatment, prior to day 5 when the animals were sacrificed.

To do this, I would use a TUNEL cell death assay on retinas from rose larvae exposed to

light for different periods of time and use cell-specific markers to determine the identity

of TUNEL positive cells.

Once I had more knowledge of the basis for the light-treated rose retinal

phenotype, I could combine this model with a morpholino “gene knockdown” approach,

61
targeting candidate genes thought to be involved in the endothelin pathway. Another

potentially interesting experiment would be to introduce the rose mutation to the lze

mutant to explore the effects of combining models involving Müller cells and light-

dependent degeneration.

Finally, since many of the genes in the zebrafish have more than one copy, I

might learn that there is more than one gene for ET-B. In this case, I would repeat the

experiment and analysis using rose larvae treated with a known ET-B inhibitor, called

BQ788, and assess the phenotype.

62
 GENERAL CONCLUSIONS

To examine how Müller cells may be involved in supporting photoreceptors,

reliable models where Müller cells are compromised are needed. In this thesis, I

examined two candidate models of Müller cell disruption in the zebrafish retina, one

pharmacological and one genetic. In my first model, I introduced a gliotoxin to adult and

larval zebrafish through the water and observed modest signs of Müller cell stress in the

adults but neuronal deficits in the larval retina. Because my approach did not yield the

desired glial-specific effects I had hoped to achieve, I chose to focus my attention on the

rose mutant model.

Upon the introduction of retinal stress via constant light, photoreceptor cells and

cells within the INL were compromised in the rose retina. The appearance of the failing

rod photoreceptor outer segments is similar to early stages of photoreceptor degeneration

observed in other animal models. Thus, even if I determine that the rose phenotype is not

mediated by Müller cell deficiencies, rose still serves as a model of photoreceptor

degeneration, where the same issues of neurotrophic support could be investigated.

Whether rose will be a useful model to study how Müller cells are involved in the

resilience of photoreceptors has yet to be determined; however, my finding that rod

photoreceptors were more vulnerable to phototoxic stress coupled with the Rattner and

Nathans result that light exposure led to the intense upregulation of ET-B in Müller cells

has inspired me to explore more about this mutant.

63
 REFERENCES
Brokerhoff SE, Hurley JB, Ulrike JB, Neuhauss SCF, Driever W, Dowling JE. 1995. A
behavioral screen for isolating zebrafish mutants with visual system defects. Neurobiol
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