C H A P T E R
21
Zebrafish in Biomedical Research: the Retina
and Vision
Whitney M. Cleghorn, Susan E. Brockerhoff
Biochemistry, University of Washington, Seattle, WA, United States of America
Introduction
The retina is a highly organized tissue with limited
cell types and easy access, making it one of the most
informative neuronal tissues to study. The orderly
arrangement of the retina allows us to directly modulate
neural transmission and measure functional output,
map neuronal circuitry and synaptic connections, and
test how neurons form support systems with different
neuronal classes and Müller glia to maintain
homeostasis and cell health.
With the aid of pharmacological agents, zebrafish can
remain transparent well after larval retina function is
established, making them an ideal model for visual
studies. Imaging experiments using fluorescent probes
(dyes, genetically encoded sensors) can be used to
monitor function in a living eye in its native environ-
ment. Zebrafish genetics are sophisticated and quick to
manipulate using a variety of methods, including Tol2-
kit (Don et al., 2017; Kwan et al., 2007) and CRISPR/
Cas9 (Ablain, Durand, Yang, Zhou, & Zon, 2015; Li,
Zhao, Page-McCaw, & Chen, 2016), allowing the expres-
sion of a variety of fluorescent and mutant proteins.
Zebrafish are cone-dominant similar to the human
macula and mimic a variety of blindness pathologies
discovered to affect humans, such as cone dystrophy
(Stearns, Evangelista, Fadool, & Brockerhoff, 2007),
and Leber congenital amaurosis (Iribarne et al., 2017).
Disease and degeneration phenotypes can be monitored
in real-time (Lewis, Williams, Lawrence, Wong, & Brock-
erhoff, 2010; Ma et al., 2013), and the permeability of
larval fish allows for measuring effects of pharmacolog-
ical treatment (Wang et al., 2015). Zebrafish retinas can
regenerate their neurons, whereas human retinas do
not, making them informative in gene therapy and
genetic approaches to curing blindness (Gallina, Todd,
The Zebrafish in Biomedical Research
https://doi.org/10.1016/B978-0-12-812431-4.00021-X
& Fischer, 2014; Rao, Didiano, & Patton, 2017). There
are a large number of complementary behavioral assays
and techniques, discussed in this chapter, used to
monitor retinal function and disease.
The Organization and Function of the Zebrafish
Retina Organization
Both human and zebrafish retinas are polarized
tissues that contain the same major cell classes in three
nuclear layers: outer nuclear layer (ONL), which
contains light-sensing photoreceptors, inner nuclear
layer (INL), made up of bipolar, amacrine, and horizon-
tal cells, and the ganglion nuclear layer (GCL) nearest to
the lens, which contains ganglion cell bodies. Two
plexiform layers, outer (OPL) and inner (IPL), contain
synaptic connections between cells in different nuclear
layers (Fig. 21.1).
Light Detection and Signal Transmission
Photoreceptors are polarized neurons with an opsin
rich outer segment, an inner segment that contains the
nucleus, a dense mitochondrial cluster, which regulates
cell health and homeostasis, and a synapse, which trans-
forms cell membrane potential into the controlled
release of glutamate (Dowling, 1987). In the dark photo-
receptors are in a depolarized state. Absorption of light
causes a conformational change in opsin receptors
located in the outer segment that initiate a signal trans-
duction cascade that hyperpolarizes the neuron, thus
reducing the release of the neurotransmitter glutamate
at the synapse. Unlike other neurons, photoreceptors
communicate via graded potentials rather than action
potentials. Bipolar cells form synapses with either rods
237 © 2020 Elsevier Inc. All rights reserved.
238 21. Zebrafish in Biomedical Research: the Retina and Vision
FIGURE 21.1 The retina. (Left) This schematic of the vertebrate
retina highlights the cellular layering and different cell classes. The
retinal pigment epithelium (RPE; dark green) is at the back of the eye
where it extends processes that surround both rod and cone outer
segments. The outer nuclear layer (ONL) contains rod (gray) and cone
(blue) photoreceptors (PRs). PRs make synaptic connections to hori-
zontal (yellow) and bipolar (light purple) cells within the outer plexi-
form layer (OPL). Amacrine cells (pink) reside at the vitreal side of the
inner nuclear layer (INL). Cells within the INL form synapses with the
ganglion cells (light green) in the ganglion cell layer (GCL) at the inner
plexiform layer (IPL). Muller glia (purple) extend through much of the
retina. (Right) This micrograph shows the larval zebrafish retina at six
dpf. Already at this age, the retina is laminated with differentiated
photoreceptors and robust visual behavioral responses (see text).
or cones in the outer plexiform layer, where they receive
signals via glutamate released from photoreceptors
(Ayoub & Copenhagen, 1991). Activated bipolar cells
transmit information onto ganglion cells, the innermost
layer of retinal neurons. Ganglion cells are the only cell
type in the retina that project their axons directly into the
brain for signal integration and interpretation.
Regulation and Maintenance of Retinal Neurons
There are two types of regulatory roles maintained by
the additional retinal neurons. First, horizontal and
amacrine cells directly modulate the light response by
influencing photoreceptor-bipolar or bipolar-ganglion
cell interactions, respectively. Horizontal cells laterally
inhibit photoreceptor outputs from producing a
controlled input to bipolar neurons, and the amacrine
cells integrate and modulate the output message from
the bipolar cells to ganglion cells. These interneurons
allow for neuronal cross-talk between layers and are
active within the IPL (Masland, 2012). Second, Muller
glia act as support structures and span nearly the entire
length of the retina, starting in the INL and reaching just
II. Biology
within the ONL to photoreceptor nuclei. Muller glia
have a range of functions that are vital to the health of
all retinal neurons, including removal of toxic waste
and neuronal debris created by excited or dying neurons
(Bejarano-Escobar, Sanchez-Calderon, Otero-Arenas,
Martin-Partido, & Francisco-Morcillo, 2017), uptake of
excess glutamate from postsynaptic connections to pre-
vent cell toxicity (Derouiche & Rauen, 1995), supply of
critical nutrients to fuel neurons, and replenishment of
neurotransmitters by providing glutamate precursor
glutamine to neurons (Lindsay et al., 2014).
Neuronal Classification in the Zebrafish Retina
While the overall retina organization and presence of
major cell types are conserved in vertebrates, there are
species-specific features that reflect the unique visual re-
quirements of different animals. For example, whereas
mice contain primarily rod photoreceptors because
they are nocturnal animals, zebrafish are cone dominant
with a sophisticated color vision adapted for detecting a
broad range of wavelengths extending from approxi-
mately 350 to 560 nm. As described in the following par-
agraphs, several studies report the subclasses of cells
within the zebrafish retina. This information sets the
stage for studies aimed at dissecting determinants of cir-
cuitry formation underlying visual behavioral
specializations.
Photoreceptors
Zebrafish photoreceptors located in the ONL have
four distinct cone subtypes, UV-sensitive, blue, and
red-green double cones, which are spatially organized
into a strictly arranged mosaic pattern (Allison et al.,
2010). The photoreceptor population within the zebra-
fish retina contains further subdivisions of duplicated
opsins, totaling 10 different opsin genes, and eight of
these define eight unique cone subtypes. Zebrafish
have four green-sensitive opsin genes with peak absorp-
tion maxima (lmax) each slightly shifted in wavelength
sensitivity: RH2e1 (467 nm), RH2e2 (476 nm), RH2e3
(488 nm), and RH2e4 (505 nm), with RH2-2 being the
most predominant form in the adult retina (Chinen,
Hamaoka, Yamada, & Kawamura, 2003). Zebrafish also
have two different red opsins that are spectrally distinct:
LWS-1, 558 nm, the most abundant form, and LWS-2,
with a peak absorbance at 548 nm. These subtype vari-
ants have unique spatial distributions in both larvae
and adults: shorter wavelength sensitive opsin subtypes
LWS-2, RH2-1, and RH2-2, are expressed in the central to
dorsal retina, whereas longer wavelength subtypes
LWS-1, RH2-3, and RH2-4 are found in the ventral retina
(RH2-3 surrounds the central retina, and RH2-4
 Behavioral Assays
circumscribes RH2-3 in the ventral region (Takechi &
Kawamura, 2005; Tsujimura, Chinen, & Kawamura,
2007). SWS1 (355 nm), or UV cones, and SWS2 (416), or
blue cones, are each single-copy shorter wavelength op-
sins expressed throughout the retina mosaic (Chinen
et al., 2003).
Rods in the zebrafish retina are arranged in regularly
spaced rows. The rod cell bodies extend through the
cone mosaic in a predictable pattern (Fadool, 2003).
In most vertebrates, rhodopsin is a highly conserved
single-copy gene. However, zebrafish express two
different rod opsins with nearly identical peak sensi-
tivity: RH1-1 (501 nm) and slightly blue-shifted RH1-2
(496 nm) (Morrow, Lazic, & Chang, 2011, 2017). While
the functional characteristics of RH1-2 appear to mimic
those of RH1-1, RH1-2 releases retinal at a rate similar
to rhodopsin, RH1-1 is the predominant form with
RH1-2 expression restricted to the ventral peripheral
retina, a defined spatial distribution similar to what is
seen for the other opsin duplicates. This defined
distribution is not uncommon in teleosts, as specific
expression patterns of precisely spectrally tuned opsin
variants may be an advantage for detecting
downwelling light (Morrow et al., 2011; Temple, 2011).
Diversity and Connectivity of Retinal Circuits
Downstream of photoreceptor neurons, distinctions
in cell composition and circuitry also vary across spe-
cies. The zebrafish retina contains 17 different types of
bipolar neurons (Connaughton, Graham, & Nelson,
2004) (compared to the mouse, with 12 bipolar cells
(Wassle, Puller, Muller, & Haverkamp, 2009), at least
11 ganglion neurons (Mangrum, Dowling, & Cohen,
2002; Ott, Walz, Paulsen, Mack, & Wagner, 2007) (22 gan-
glion cells, mouse (Volgyi, Chheda, & Bloomfield, 2009)),
at least 28 types of amacrine cells (Jusuf & Harris, 2009),
and four types of horizontal cells (Li, Matsui, & Dow-
ling, 2009) (compared to the macaque, which has two
(Dacey, 1999), or the mouse, with only one (Peichl &
Gonzalez-Soriano, 1994)). The connectivity maps of
neuronal circuits within the retina are highly complex.
Each class of bipolar cell synapses with multiple photo-
receptors to create and ON- OFF- center-surround array,
while ganglion neurons, characterized by dense asym-
metric dendritic branching patterns, make most connec-
tions with respective bipolar neurons in the IPL.
However, the subclass of every neuronal type in the
retina is defined by many factors: the unique number
and pattern of dendrites; dendritic and synaptic projec-
tions into the retinal sublaminar; cell shape and size; and
the classes of other retinal neurons with which they form
synaptic connections. For example, of the four zebrafish
horizontal cells, three subtypes connect with specific
II. Biology
239
cones: H1 forms contacts with L-, M-, and S-cones, H2
sample M-, S-, and UV-cones, and H3 horizontal cells
only receive input from S and UV cones (Li et al.,
2009). The last horizontal cell subtype, H4, exclusively
receives input from rods (Li et al., 2009). While circuitry
and function of the retina are generally conserved
between vertebrates, the zebrafish retina contains one
notable difference: rod bipolar cells receive input from
not only rod photoreceptors, but also from L-type cones
(Li, Tsujimura, Kawamura, & Dowling, 2012). Therefore,
unlike mice and monkeys, zebrafish do not have a
neuronal pathway that selectively only represents the
rod population.
Function
One of the properties that make zebrafish an excellent
experimental system for vision studies is the rapid
development of the visual function. Zebrafish develop
ex utero and are free swimming with highly sensitive
and accurate visual function already at 5 days postferti-
lization (dpf). The first visual responses are detected at
3dpf (Branchek, 1984; Easter & Nicola, 1996). Zebrafish
larvae rely on vision, particularly cone photoreceptor
function, to efficiently capture prey. By five dpf, larvae
have depleted their endogenous food supply, and prey
capture is required for survival. Larvae with impaired
cone function have difficulty surviving without
extra food provided to make foraging more successful
(Brockerhoff et al., 2003). This rapid development of
the visual system has been exploited by investigators
interested in identifying genes critical for visual func-
tion: genetic screens analyzing visual behavior can be
conducted early in development reducing the cost and
labor associated with maintaining fish for long periods
of time. To this end, many different behavioral screening
strategies have been established for quantifying zebra-
fish (particularly larval zebrafish) visual function
(Fleisch & Neuhauss, 2006; Neuhauss, 2003). Because
many of the visually mediated behavioral responses
are so robust and reproducible, they have recently
been used to begin to define the circuits that are used
to transform sensory input into action (Bianco & Engert,
2015; Dunn et al., 2016; Naumann et al., 2016).
Behavioral Assays
The Optokinetic Response (OKR)
The OKR is a reflex in which the eye moves in
response to the movement of a visual stimulus. There
are two components to the OKR, a smooth pursuit
tracking movement in the same direction as the moving
240 21. Zebrafish in Biomedical Research: the Retina and Vision
stimulus and a subsequent rapid saccade back to the
starting position. The zebrafish OKR is a very robust
response that develops early and rapidly; 25% of larvae
display an OKR at 72hpf, and 100% respond at 80hpf
(Easter & Nicola, 1996). For eliciting the OKR, larvae
are immobilized by being placed in a viscous aerated
aqueous media. A moving grate is then provided as a
stimulus, and eye movements are measured. Because
this assay is robust and responses are easy to detect
and measure, forward genetic screens have used this
strategy to identify novel mutants with visual function
defects (Brockerhoff et al., 1995; Muto et al., 2005).
Many different OKR tracking systems have been
reported. In the simplest versions, stripes are manually
placed inside a drum whose rotational speed and direc-
tion can be mechanically controlled (Brockerhoff, 2006;
Neuhauss et al., 1999). Other versions use computer-
generated moving gratings that are projected onto the
drum using a digital light projector that is placed either
on the plane of the subject (linear projection) or below
the subject (Huber-Reggi, Mueller, & Neuhauss, 2013).
Eye movements are recorded and then quantified.
In addition to leading to the successful identification of
visually impaired zebrafish, the OKR has been used to
analyze the development of visual acuity (Haug, Biehl-
maier, Mueller, & Neuhauss, 2010; Rinner, Rick, &
Neuhauss, 2005).
The initial analyses of zebrafish vision using the OKR
were done using larvae. The OKR systems used for these
studies were not designed for analyzing adults. The
main difference is that adult zebrafish are strong swim-
mers and so they must be held tightly in place, as well as
kept wet to ensure that oxygen is exchanged across the
gills. Larvae can be easily restrained in viscous
solutions, and oxygen readily diffuses across the skin.
Adults require underwater restraint systems that keep
their bodies from moving and their heads visible so
that eye movements can be recorded and analyzed.
Several groups have now developed OKR systems for
quantifying adult vision (Cameron et al., 2013; Mueller
& Neuhauss, 2010; Tappeiner et al., 2012).
Phototaxis
While the OKR is perhaps the most robust vision-
dependent behavioral response, it is also relatively
slow to perform since eye movements are analyzed on
individual fish. The optomotor and phototactic
responses, in contrast, can be measured on groups of
larval zebrafish. Phototaxis describes the movement of
an organism either toward (positive phototaxis) or
away (negative phototaxis) from a light stimulus. Early
studies reported that zebrafish larvae display both pos-
itive and negative phototactic behavior but it appeared
II. Biology
highly variable (Brockerhoff et al., 1995; Gerlai, Lahav,
Guo, & Rosenthal, 2000; Orger & Baier, 2005; Serra, Med-
alha, & Mattioli, 1999). Conditions establishing the rela-
tionship between the robustness and rate of phototaxis
and relative light intensity were reported in 2010
(Burgess, Schoch, & Granato, 2010). Both the maximal
number of responding larvae and the speed of move-
ment toward a spot of light occur when the target illumi-
nation is 10-fold less intense than the uniform
background light. Pharmacological manipulations and
visually impaired mutants were used to demonstrate
that the visual pathway responding to light on (the
ON pathway) controls the rate of approach, while the vi-
sual pathway responding to light decrements (the OFF
pathway) elicits turns that enable steering (Burgess
et al., 2010).
The Optomotor Response (OMR)
The OMR is a reflexive swimming behavior that can
be elicited in response to moving visual stimuli, such
as rotating stripes. This behavior is robust in both larvae
and adults. For larvae, the response is strong at seven
dpf and can be evaluated on groups of different clutches
in parallel (Neuhauss et al., 1999; Orger, Smear, Anstis,
& Baier, 2000). Larvae are placed in a swimming
container and moving sinusoidal gratings are presented
to them either from below or from the side. Detection of
the “stripes” causes a majority of larvae to swim in the
same direction as the apparent motion. These properties
have made this assay useful for screening for recessive
mutations that cause blindness (Muto et al., 2005). This
assay has also been used to measure the dominant chro-
matic inputs for motion detection, which varies with age
and species (Orger & Baier, 2005).
In adults, the OMR is measured on individual fish
since schooling can interfere with this behavioral
response. Single fish are placed in a circular drum
with a rod in the center to prevent swimming across
the apparatus. The gratings are projected onto the
drum and swimming is evoked in the apparent direction
of rotation. This assay has been used to measure how
spatial and temporal properties of repetitive stimuli
influence the OMR and to measure the color contribu-
tion to motion detection (Krauss & Neumeyer, 2003;
Maaswinkel & Li, 2003).
The Adult Escape Response
The adult escape response is initiated by the appear-
ance of a visual stimulus that elicits a reverse in swim-
ming direction (i.e., an escape). The experimental
setup is the same as for the OMR except the stimulus
is a single “threatening” stripe. The adult escape
 Fluorescent Strategies
response has been used to identify fish with visual
defects (Li & Dowling, 1997; Maaswinkel, Mason, & Li,
2003a, Maaswinkel, Ren, & Li, 2003b).
Physiological Assays
Electroretinogram (ERG)
The ERG is a diagnostic test used to measure the func-
tion of the outer retina. It is a noninvasive procedure that
records the light-induced changes in electrical potential
across the eye measured at the corneal surface. These
changes are the result of altered sodium and potassium
fluxes occurring in both neuronal and nonneuronal
retinal cell types. Vertebrate ERG recordings have four
principal components: the a-wave, a cornea-negative
potential derived from phototransduction within the
photoreceptor cell; the b-wave, a cornea-positive poten-
tial derived from the ON bipolar cell activity; the
c-wave, which appears more slowly and is thought to
originate from the RPE and; the d-wave, which origi-
nates from OFF bipolar cell activity and can be separated
from the b-wave by long-duration flashes.
Early functional studies indicated that only cones
contribute to zebrafish vision until approximately
2 weeks of development (Bilotta, Saszik, & Sutherland,
2001; Branchek, 1984). The larval ERG has a photopic
response dominated by the b-wave. The photoreceptor
component (a-wave) can be selectively isolated using
the pharmacological agent L-(þ)-2-amino-4-
phosphonobutyric acid (L-AP4), an agonist for the
metabotropic glutamate (mGluR6) receptor expressed
by the ON bipolar cells. Combinations of drugs and sub-
tractions of waveforms have been used to isolate many
ERG elements that reflect the retinal organization and
summation of multiple cone signals (Nelson & Singla,
2009). This has made the ERG useful for identifying
zebrafish mutants that selectively lack all cone photore-
ceptor function (Brockerhoff et al., 2003; Stearns et al.,
2007) and also for characterizing and defining defects
in mutants with subtle circuitry defects (Lewis et al.,
2011).
ERGs can be recorded using an isolated eye or eyecup
dissected from a larval or adult zebrafish, or they can be
done on the intact eye in a living anesthetized animal.
Using either strategy, an electrode, placed either on the
cornea or on the vitreal side of the retina, transmits the
electrical signal through an amplifier, an analog to digi-
tal converter, then to a computer for analysis and pro-
cessing. The original zebrafish ERGs were done on
whole animals (Brockerhoff et al., 1995). Larvae are
kept moist but are not submerged in water. Adults
have water continuously flushed through the gills by
placing a tube with flowing water into the mouth.
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241
Adults also require treatment with a muscle stabilizer
gallamine triethiodide (Li & Dowling, 1997). Recordings
using whole animals are still routine (Korenbrot, Mehta,
Tserentsoodol, Postlethwait, & Rebrik, 2013; Lin et al.,
2016), but studies using isolated eyes or eyecups result
in robust recordings for significantly longer times than
with whole animals, and drugs permeate isolated eyes
more readily (Wong, Gray, Hayward, Adolph, & Dow-
ling, 2004).
Single-cell Recordings
While the ERG is a powerful strategy for analyzing
retinal function, it is a summed response and does not
reflect the activity of individual cells. Two general types
of cell preparations are used to record activity from indi-
vidual cells. Cells either are identified in the context of
the retina (in an eyecup or retinal slice), or they are
dissociated and isolated from neighboring cells. Several
groups have reported physiological recordings of indi-
vidual retinal neurons (Aquila, Benedusi, Fasoli, & Ris-
poli, 2015; Connaughton, Nelson, & Bender, 2008;
Connaughton & Nelson, 2000; Fan & Yazulla, 1997;
Klaassen et al., 2011). These strategies have been used
on adults and juveniles, but not on larvae due to the
small size of their neurons. The retinal slice preparation
has several advantages (Connaughton, 2003). Since cells
are not dissociated, the cellular arrangement and synap-
tic connections present in vivo are maintained. Further,
since the slice is a tangentially cut section of the retina,
all neurons in the different retinal layers are visible
and accessible.
Fluorescent Strategies
Genetically Encoded Indicators
Another strategy commonly used to measure the
functional activity of single cells or cellular circuits is
to make transgenic zebrafish expressing genetically
encoded indicators. Retinal slices from transgenic fish
can be placed in a perfusion chamber, and then specific
cellular responses due to changing the media can be
measured in real-time at subcellular resolution (Giar-
marco, Cleghorn, Sloat, Hurley, & Brockerhoff, 2017;
Giarmarco, Cleghorn, Hurley, & Brockerhoff, 2018)
(Fig. 21.2). A more common strategy is to image the
entire eye and recently the entire fish brain; there are
genetic and pharmacological ways to maintain zebrafish
transparently, and thus, it is possible to image fluores-
cent sensors in real-time in the living animal. In the
retina these strategies have been used to image events,
such as dying photoreceptors (Lewis et al., 2010;
Ma et al., 2013), formation of synaptic connections
242 21. Zebrafish in Biomedical Research: the Retina and Vision

Obtain eye, dissect Move retina to filter Remove Apply desired

retina, cut into s. paper. Flatten with RPE. drug / stain(s) to
gentle suction. retina & wash.

Slice with
tissue Rotate slices 90° & bury edges in Image on confocal
slicer. strips of wax on a coverslip. microscope.

Retinal Slice Preparation: Retinas are isolated, quartered, mounted on filter paper in cold oxygenated media,
stained with BODIPY (membrane stain), and washed. Mounted, stained retinas are cut into 400 m slices using a
tissue slicer. Slices were rotated 90°, and edges buried in strips of wax on a coverslip for confocal imaging with a
dipping lens.

FIGURE 21.2 The retinal slice preparation. Retinal slices are useful for many different experiments involving adult retinas (see text).

(Williams et al., 2010), vesicular trafficking (George, Summary

Hayden, Stanton, & Brockerhoff, 2016) and dedifferenti-
ation of Mueller glia (Bernardos, Barthel, Meyers, &
Raymond, 2007). Due to the small size of the zebrafish
larva, it is possible to image the vast majority of neurons
in the brain at the same time (Feierstein, Portugues, &
Orger, 2015) in the intact whole animal, while it is per-
forming a stereotyped behavior in response to visual
stimuli. This is an amazing achievement and a very
powerful approach for dissecting the complete circuitry
underlying visual behavior. Calcium sensors have often
been used for these studies since Ca2þ dynamics reflect
neuronal activity, and there are many varieties of genet-
ically encoded Ca2þ indicators with different sensitiv-
ities and kinetics (Lin & Schnitzer, 2016). These types
of experiments are putting zebrafish at the forefront of
research aimed at defining the neuronal sequence from
sensory input to behavioral output for visual, as well
as other stimuli (Kawashima, Zwart, Yang, Mensh, &
Ahrens, 2016; Naumann et al., 2016; Oteiza, Odstrcil,
Lauder, Portugues, & Engert, 2017; Randlett et al., 2015).
This review describes the structural organization of
the zebrafish retina and the many behavioral, physiolog-
ical, and fluorescent approaches that can be used to mea-
sure visual function. This provides the foundation for
dissecting mechanisms underlying disease and for
developing therapeutic strategies to treat blindness.
The sophisticated genetic, cell biological, behavioral,
physiological, and biochemical approaches available
combined with rapid development and high fecundity
promise to ensure the continued use of the zebrafish
model for important discoveries in vision research.
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II. Biology
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