Vision Research 122 (2016) 1–11

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Vision Research
journal homepage: www.elsevier.com/locate/visres

Opposing effects of atropine and timolol on the color and luminance

emmetropization mechanisms in chicks
Laura A. Goldberg ⇑, Frances J. Rucker
New England College of Optometry, 424 Beacon Street, Boston, MA 02115, United States

a r t i c l e i n f o a b s t r a c t

Article history: This study analyzed the luminance and color emmetropization response in chicks treated with the non-
Received 27 July 2015 selective parasympathetic antagonist atropine and the sympathetic b-receptor blocker timolol.
Received in revised form 4 March 2016 Chicks were binocularly exposed (8 h/day) for 4 days to one of three illumination conditions: 2 Hz sinu-
Accepted 8 March 2016
soidal luminance flicker, 2 Hz sinusoidal blue/yellow color flicker, or steady light (mean 680 lux).
Available online 19 March 2016
Number of Reviews = 2
Atropine experiments involved monocular daily injections of either 20 ll of atropine (18 nmol) or
20 ll of phosphate-buffered saline. Timolol experiments involved monocular daily applications of 2 drops
of 0.5% timolol or 2 drops of distilled H2O. Changes in the experimental eye were compared with those in
Keywords:
Emmetropization
the fellow eye after correction for the effects of saline/water treatments.
Myopia Atropine caused a reduction in axial length with both luminance flicker (
0.078 ± 0.021 mm) and color
Atropine flicker (
0.054 ± 0.017 mm), and a reduction in vitreous chamber depth with luminance flicker
Timolol (
0.095 ± 0.023 mm), evoking a hyperopic shift in refraction (3.40 ± 1.77 D). Timolol produced an
Luminance increase in axial length with luminance flicker (0.045 ± 0.030 mm) and a myopic shift in refraction
Color (
4.07 ± 0.92 D), while color flicker caused a significant decrease in axial length (
0.046 ± 0.017 mm)
that was associated with choroidal thinning (
0.046 ± 0.015 mm).
The opposing effects on growth and refraction seen with atropine and timolol suggest a balancing
mechanism between the parasympathetic and b-receptor mediated sympathetic system through stimu-
lation of the retina with luminance and color contrast.
Ó 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Hanowsky, 2015; Rucker & Wallman, 2008, 2009; Rucker &
Wallman, 2012) that may be involved. In the meantime, promising
The increasing occurrence of myopia in the population presents pharmacological interventions (e.g., atropine) can slow the devel-
an important public health issue because of an association with opment of myopia progression (Chia et al., 2014, 2015, 2012;
elevated risk of ocular diseases including cataract, glaucoma, reti- Bedrossian, 1971; Lee et al., 2006; Li et al., 2014; Morgan, Ohno-
nal detachment, and blindness (Saw, Gazzard, Shih-Yen, & Chua, Matsui, & Saw, 2012; Walline, 2016; Wu, Yang, & Fang, 2011),
2005). In humans, the environmental effect of time spent outdoors although the effects of these treatments under different environ-
has been implicated in a reduction in myopia development (Dirani mental conditions have not been studied.
et al., 2009; Guggenheim et al., 2012; Jones et al., 2007; Jones-
Jordan et al., 2012; Onal et al., 2007; Parssinen & Lyyra, 1993; 1.1. Color and luminance contrast affect emmetropization
Rose, Morgan, Ip, et al., 2008; Rose, Morgan, Smith, et al., 2008;
Wu, Tsai, Hu, & Yang, 2010), and recent work has helped to clarify As a result of dispersion, short-wavelength light has a shorter
the protective effects of factors such as high light levels (chicks: focal length than long-wavelength light, producing an effect called
Ashby, Ohlendorf, and Schaeffel (2009), Backhouse, Collins, and longitudinal chromatic aberration. The differences in focus of the
Phillips (2013), Cohen, Belkin, Yehezkel, Avni, and Polat (2008) different wavelengths produce changes in color of the retinal
and Cohen, Belkin, Yehezkel, Solomon, and Polat (2011); monkeys: image with defocus (Rucker & Wallman, 2012), which in turn is
Smith, Hung, and Huang (2012)) and spatial and temporal changes reflected in changes in the stimulation of the retinal cones and
in the retinal image (Rucker, 2013; Rucker, Britton, Spatcher, & the retinotectal color and luminance pathways (review: Rucker
(2013)). A theoretical analysis of the change in the retinal image
⇑ Corresponding author. with defocus has indicated that with myopic defocus, the retina
E-mail address: [email protected] (L.A. Goldberg). would experience changes in luminance contrast, whereas with

http://dx.doi.org/10.1016/j.visres.2016.03.001
0042-6989/Ó 2016 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11

hyperopic defocus the retina would also experience changes in and thus in chicks accommodation should not be affected by
color contrast (Rucker & Wallman, 2009). In the laboratory, flicker- atropine.
ing light of fixed frequency and waveform can be used to simulate In the human eye, the sympathetic nervous system innervates
the changes in luminance and color contrast of the retinal image the ciliary muscle, ciliary epithelium, iris dilator muscle and
that occur with changes in focus and changes in fixation in the nat- smooth muscle of the vasculature. Innervation occurs through
ural environment. Rucker and Wallman (2012) tested their the action of the neurotransmitter noradrenaline on two sub-
hypothesis by exposing chicks to 2 Hz, high contrast, sinusoidal classes of post-synaptic adrenergic receptor types: a- and b-
changes in either luminance or color contrast for 3 days and found adrenoceptors (review: Chen, Schmid, & Brown, 2003). Timolol
hyperopic shifts (2.01 D) with changes in luminance contrast and maleate, which has been used as a clinical treatment for glaucoma
myopic shifts with changes in color contrast. Rucker et al. (2015) since the late 70s, is a non-selective b-adrenoceptor antagonist
determined that the reduction in eye length was most pronounced (Airaksinen, Saari, Tiainen, & Jaanio, 1979). a-Adrenoceptors con-
with exposure to high temporal frequencies (5–10 Hz), confirming sist of two subtypes a1 and a2, which can be further subdivided
earlier research (Gottlieb & Wallman, 1987; Schwahn & Schaeffel, into a2A, a2B and a2C subtypes (Regan & Cotecchia, 1992). Stim-
1997), and that the more myopically defocused blue component ulation of a-adrenoceptors can regulate contraction of the iris dila-
of the light source provided protection against increases in eye tor muscle (mydriasis) (van Alphen, 1976) and relaxation of the
growth at low temporal frequencies with luminance flicker. These ciliary body (Garner, Brown, Baker, & Colgan, 1983; Zetterstrom,
results have confirmed that the eye utilizes signals arising from 1988). b-Adrenoceptors consist of two subtypes, b-1 and b-2. b-1
temporally-sensitive changes in luminance and color contrast to receptors are mainly found in cardiac tissues, but they also make
determine the emmetropization response. up 10% of the receptors in human iris and ciliary body (Wax &
Molinoff, 1987). Most of the receptors in the ciliary body are of
the b-2 receptor subtype (Wax & Molinoff, 1987) and stimulation
1.2. Parasympathetic and sympathetic control of color and luminance causes muscle relaxation. In addition, b-2 receptors control secre-
pathways tion from the non-pigmented ciliary epithelium, and blockade of
these receptors by timolol reduces aqueous production
The molecular pathways for these color and luminance signals (Zimmerman & Kaufman, 1977) and thus intraocular pressure
are unknown. One possibility is that these light signals activate (IOP). Many studies have reported that IOP readings are higher in
the parasympathetic and sympathetic nervous systems. The neuro- human myopes than emmetropes (David, Zangwill, Tessler, &
transmitter acetylcholine (ACh) is released from parasympathetic Yassur, 1985; Jensen, 1992; Maurice & Mushin, 1966; Parssinen,
axon terminals that innervate the ciliary body, iris, smooth muscle 1990; Quinn, Berlin, Young, Ziylan, & Stone, 1995), although the
in the vasculature, but also from intrinsic interneurons in the differences are small (2 mmHg) and not predictive of future myo-
retina. There are two categories of acetylcholine receptors: nico- pia development (Goss & Caffey, 1999).
tinic (ionotropic; nAChR) and muscarinic (metabotropic; mAChR), It is well established in the accommodation literature that dual
which are coupled to heterotrimeric G-proteins (review: excitatory parasympathetic and inhibitory sympathetic innerva-
Nathanson, 1987). Atropine, which has been proposed as a treat- tion to the ciliary muscle occurs (Toates, 1972; Tornqvist, 1967),
ment for myopia because of its effect in reducing eye growth, is though sympathetic innervation is much weaker (<
2D) and
a non-selective antimuscarinic. In mammals, five muscarinic slower (maximal effect after 10–40 s) (Tornqvist, 1966). McBrien
receptor subtypes, M1 through M5, are present in the human eye and Millodot (1986) suggested that late-onset myopes, with a
(review: Mitchelson, 2012). Of these receptor types, the M3 recep- reduced dioptric level of tonic accommodation, indicative of
tor is the most predominant receptor type in human iris sphincter, decreased parasympathetic tone, have a related decrease in inhibi-
ciliary body (causing accommodation), retina and sclera (Collison, tory sympathetic tone. Furthermore, Gilmartin & Bullimore found
Coleman, James, Carey, & Duncan, 2000; Gil, Krauss, Bogardus, & that sympathetic blockade increases the decay time for accommo-
WoldeMussie, 1997; Ishizaka et al., 1998; Matsumoto, Yorio, dation after periods of extended near work (Gilmartin & Bullimore,
DeSantis, & Pang, 1994; Pang, Matsumoto, Tamm, & DeSantis, 1987), particularly in late-onset myopes at high stimulus levels
1994) with small amounts of M1, M4 and M5 in the iris sphincter (5D) (Gilmartin & Bullimore, 1991). The authors’ hypothesis that
and ciliary body (also M2) Mitchelson, 2012. There are also reports late-onset myopia may result from a deficit of the sympathetic ner-
of mAChRs being expressed in the human RPE (Osborne, vous system has received considerable support (Chen et al., 2003;
FitzGibbon, & Schwartz, 1991) and lens (Williams, Duncan, Riach, Ciuffreda & Lee, 2002; Ciuffreda & Wallis, 1998; Culhane, Winn, &
& Webb, 1993) with mainly M1 receptors in native human lens Gilmartin, 1999).
epithelium (Collison et al., 2000) and acetylcholinesterase on the In this study, we analyzed the effect of the non-selective
lens surface (Michon & Kinoshita, 1968). Muscarinic receptors are parasympathetic antagonist atropine and the non-selective b-
found throughout the retina on amacrine, bipolar, horizontal and adrenergic receptor blocker timolol on the parasympathetic and
ganglion cells, though the only cholinergic cells in the adult retina sympathetic nervous systems’ emmetropization responses to color
are the starburst amacrine cells (Fischer, McKinnon, Nathanson, & and luminance flicker. We predicted that luminance and color
Stell, 1998; McBrien, Jobling, Truong, Cottriall, & Gentle, 2009; stimulation may preferentially stimulate one or other of the auto-
Strang, Renna, Amthor, & Keyser, 2010; Townes-Anderson & nomic nerve pathways, since exposure to high-frequency lumi-
Vogt, 1989; Yamada et al., 2003). nance flicker has been associated with a reduction in eye growth
With regard to the chick animal model of myopia, four avian similar to that found with atropine.
mAChR subtypes have been characterized: cm2 (Tietje &
Nathanson, 1991), cm3 (Gadbut & Galper, 1994) cm4 (Tietje,
Goldman, & Nathanson, 1990) and cm5 (Creason, Tietje, & 2. Methods
Nathanson, 2000). Fischer et al. (1998) reported localization of
three of the different isoforms of mAChRs (cm2–cm4) subtypes 2.1. Subjects
in the chick eye, in the retina, choroid, retina pigment epithelium
(RPE) and ciliary body. It is important to note that chicks differ Subjects were white leghorn chicks (Gallus gallus domesticus)
from mammals in that only nicotinic receptors are involved in Cornell K strain (Cornell University, Ithaca, NY), hatched in an
accommodation (McBrien, Moghaddam, New, & Williams, 1993) incubator and raised in temperature-controlled brooders. Upon
 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11 3

Table 1 needle that was inserted through the lid on the temporal side of
Number of chicks used in data analysis for each experimental condition. one eye (based on protocols by McBrien, Moghaddam, and
Treatment LUM Color Steady Reeder (1993), Schmid and Wildsoet (2004)).
Atropine 8 9 8 In the timolol experiment, drug-treated chicks received two
Saline 8 15 8 drops of 0.5% timolol, while control chicks received two drops of dis-
Timolol 12 11 8 tilled H2O in one eye. The treatment was administered twice daily
Water 6 7 7 (10 am and 4 pm) under anesthesia (isofluorane 1.5–2%) (as
described by Schmid, Abbott, Humphries, Pyne, and Wildsoet
hatching, the chicks were housed in 12 h light/12 h dark cycles (2000)).
under approximately 300 lux illumination fluorescent bulbs. Food
and water were supplied ad libitum. The experiments were 2.4. Measurements
performed on chicks that were 5–7 days old at the start of
the experiment. Care and use of the animals conformed to the Chicks were exposed (both eyes) to their assigned illumination
Association for Research in Vision and Ophthalmology Resolution condition for 4 days, 8 h/day (9 am to 5 pm). They were otherwise
for the Care and Use of Animal Research. kept in the dark in a sound- and light-proof chamber. Measure-
ments of the axial dimensions of the ocular components were per-
2.2. Illumination conditions formed with A-scan ultrasonography using a 30 MHz transducer
sampled at 100 Hz and gain of 59 dB on Olympus NDT equipment
Three illumination conditions were used: luminance flicker (Nickla, Wildsoet, & Wallman, 1998). Measurements were taken
(LUM), blue/yellow color flicker (Color), or steady light (Steady), prior to and immediately following the experimental period (after
maintaining the same mean RGB lighting components in each con- 4 days). Axial length was measured as the distance from the ante-
dition. Chicks were free roaming in a 32  20 in. wire cage that was rior cornea to the posterior sclera. In the timolol experiment, pupil
illuminated with a computer-controlled, sinusoidally modulated reactions, accommodation fluctuations, and refraction were
light source (mean 680 lux) using light-emitting diodes that con- observed with an infrared photorefractor (Schaeffel, Farkas, &
sisted of independently controlled red, green, and blue compo- Howland, 1987), while in the atropine experiment, refractions
nents (Lamina Ceramics, Westhampton, NJ: Titan Light Engine; were measured with a modified Hartinger refractometer (Zeiss,
peak wavelengths: 619 nm, 515 nm, 460 nm; beam spread of Jena, Germany). All measurements were carried out under anesthe-
45°). The illuminants were driven by an eight-channel, 12-bit sia (1.5% isofluorane in oxygen, 2 L/min oxygen flow rate). Mea-
Access I/O, USB-DA12-8A digital-to-analog converter with surements of right and left eyes were randomized.
waveform-generator functionality connected to a BuckPuck driver
(LuxDrive: 3021 D-E-500) that provided a linear current output 2.5. Data analysis
over a range of 1.6–4.3 V. Light output was calibrated and a sinu-
soidal pattern was digitally generated using lookup tables and con- ‘‘Treatment Effect”: Within each illumination condition, the
firmed by measurement of illuminance (Newport Model 818-SL change during the course of the experiment in the fellow eye
serial number: 6915). Luminance flicker was produced with in- (DN) was subtracted from the change in the treated eye (DX) in
phase sinusoidal modulation (2 Hz) of the red (615 nm, half- both the drug and saline/water control conditions to provide a
bandwidth 20 nm), green (515 nm, half-bandwidth 35 nm), and measure of the Treatment Effect.
blue (465 nm, half-bandwidth 25 nm) light-emitting diodes. Blue/ ‘‘Drug Effect”: To separate the effect of administering the injec-
yellow color flicker was created with counterphase modulation tion or drop from the effect of the drug itself, the mean Treatment
of the red and green components with the blue component. Steady Effect in the saline/H2O treated eyes was then subtracted from that
light was produced by combining the red, green, and blue compo- of the drug-treated eyes. Means and standard errors were calcu-
nents without modulation. The mean irradiances of the individual lated from these values:
components of the light source were set to 50 lW cm
2 for red,
green and blue, which is equivalent to 214 ‘‘chick lux” for red, Drug Effect ¼ ½DrugðDX
DNÞ
Mean Saline=H2 OðDX
DNÞ
191 ‘‘chick lux” for green, and 64 ‘‘chick lux” for blue. Illuminance ð1Þ
was controlled with neutral density filters to maintain a mean illu-
minance equivalent to 680 human lux (Center Lightmeter 337). Averaging of the saline/water control reduces variability in
these data sets, possibly incurring a Type I error. Therefore, the
2.3. Experimental groups and procedures (atropine and timolol Treatment Effect was compared by two-way analysis of variance
experiments) (ANOVA) in Data Desk (Data Description, Inc.; Version 6) before
correction for saline injection/H2O drop effects in order to confirm
Chicks were randomly assigned to each of the four experimental the presence of a Treatment Effect.
groups (atropine, saline control, timolol, or distilled-water control) The Drug Effect was compared to zero with student t-tests to
under each illumination condition (LUM, Color, or Steady). The determine the effects of atropine and timolol on emmetropization,
number of chicks in each experimental condition is listed in Table 1. within each of the lighting conditions. Differences in the Drug
One eye was randomly selected and treated, while the fellow eye Effect between drug or illumination conditions and their interac-
remained untreated and acted as a control for the illumination con- tions were examined with ANOVA. t-Tests were performed if the
dition. Chicks with even-numbered tags received injections/drops F value was found to be significant.
in the right eye; chicks with odd-numbered tags were treated in
their left eye (chicks were tagged at random). Treated chicks were 3. Results
randomly divided into the three lighting conditions.
In the atropine experiment, drug-injected chicks received 20 ll 3.1. Effects of atropine
of atropine (18 nmol), while the saline-injected control chicks
received 20 ll of phosphate-buffered saline in one eye. Injections The effects of atropine injections and its saline control are seen
were administered around mid-morning under anesthesia (isoflu- in Fig. 1. Results of the effect of saline injections alone are
orane 1.5–2%) using a sterile Hamilton syringe with a sterile 30 G described in Section 3.4, Fig. 1A and Table 4.
4 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11

Fig. 1. (A) Treatment Effect of saline injections on ocular components over a four-day period. Saline-injected eyes demonstrated a myopic shift in refraction (Ref. Error)
despite a decrease in axial length (AL). There was no difference in the injection effect between illumination conditions. (B) Drug Effect on ocular components with atropine
over a four-day period. Eyes injected with atropine showed a significant reduction in axial length (AL) as well as vitreous chamber depth (Vit), resulting in small hyperopic
shifts. An increase in lens thickness (Lens), and reduction in anterior chamber depth (AC) was also seen with luminance flicker, while color flicker produced choroidal thinning
(Choroid), preventing a change in vitreous-chamber depth. (C) Diagrammatic representation of (B). Abbreviations as described in (A) and (B), N.S. = Non-Significant. *p < 0.05,
**
p < 0.01, ***p < 0.001.

3.1.1. Anterior eye – Treatment Effect 3.1.3. Posterior eye – Treatment Effect
Atropine exerted one of its biggest effects on interocular lens Atropine induced a hyperopic shift in refraction through a
changes. Our data demonstrate that lens thickness was strongly reduction in eye growth when compared to saline [ANOVA:
influenced by an interaction between the drug (atropine vs. saline) F = 5.06 (df = 1), p = 0.03].
and illumination condition [ANOVA: F = 3.56 (df = 2), p = 0.04]
(Fig. 1). 3.1.4. Posterior eye – Drug Effect
The Drug Effect in atropine-treated eyes exposed to LUM was
3.1.2. Anterior eye – Drug Effect about three times more hyperopic (3.40 D) than that in eyes
With LUM, chicks with atropine injections had significant thick- exposed to color flicker (1.23 D) or steady light (0.85 D) (p < 0.05)
ening of the lens in experimental eyes (0.140 ± 0.049 mm, (Fig. 1B, C).
p = 0.02), which corresponded with a decrease in the anterior- Refractive changes under LUM were associated with
chamber depth (
0.063 ± 0.034 mm, p = 0.1). In contrast, blue/ changes in vitreous chamber depth and axial length, while
yellow color flicker and steady light had a slight increase in under Color only axial length changed. Atropine-injected eyes
anterior-chamber length (Color: 0.057 ± 0.029 mm, p = 0.8; Steady: under LUM conditions exhibited a significant decrease in
0.051 ± 0.040 mm, p = 0.25) along with a decrease in lens thickness both the vitreous (
0.095 ± 0.023 mm, p = 0.005) and axial
(Color:
0.085 ± 0.049 mm, p = 0.11; Steady:
0.177 ± 0.076 mm, length (
0.078 ± 0.021 mm, p = 0.007). Atropine-injected eyes
p = 0.59) (Fig. 1B, C; Table 2). under color-flicker conditions also exhibited a decrease in
 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11 5

Table 2 eye growth (
0.054 ± 0.017 mm, p = 0.13), but there were no
Drug Effect on ocular components in atropine-injected chick eyes under luminance vitreal or refraction changes, partially because of choroidal
flicker, color flicker, and steady light conditions. Significant values are indicated in
bold (p < 0.05). All units are expressed in either mm or D (for RE). p Values and
compensation (Fig. 1B, C; Table 2). Exposure to color flicker
standard errors are shown. Abbreviations: AC = anterior chamber, Vit = vitreous, in atropine-injected eyes produced a marked thinning in
AL = axial length, RE = refractive error. choroidal thickness [ANOVA: F = 4.85 (df = 2), p = 0.02]. With
Atropine
color flicker, choroids thinned by
0.048 ± 0.019 mm
(p = 0.03), slightly more than with LUM (
0.034 ± 0.016 mm,
AC Lens Vit Choroid AL RE
p = 0.067) and steady light (0.029 ± 0.017 mm, p = 0.20)
LUM
0.063 0.140
0.095
0.034
0.078 3.40 (Table 2).
±0.034 ±0.049 ±0.023 ±0.016 ±0.021 ±1.77
p = 0.1 p = 0.02 p = 0.005 p = 0.067 p = 0.007 p = 0.20
Color 0.057
0.085 0.026
0.048
0.054 1.23
±0.029 ±0.049 ±0.039 ±0.019 ±0.017 ±0.71 3.2. Effects of timolol
p = 0.8 p = 0.11 p = 0.53 p = 0.03 p = 0.13 p = 0.12
Steady 0.051
0.177 0.029 0.029
0.059 0.85 The effects of timolol and distilled water drops are seen in Fig. 2.
±0.040 ±0.076 ±0.072 ±0.017 ±0.039 ±1.14 Results of the effect of water drops alone are described in
p = 0.25 p = 0.59 p = 0.77 p = 0.20 p = 0.16 p = 0.48
Section 3.5, Fig. 2A and Table 5.

Fig. 2. (A) Treatment Effect of distilled water drops on ocular components over a four-day period. In steady light, a small increase in axial length combined with choroidal
thinning produced an unexplained increase in vitreous chamber depth. There was no difference in the drop effect between illumination conditions. (B) Drug Effect on ocular
components with timolol over a four-day period. Timolol-injected eyes exposed to luminance flicker produced an increase in axial length and a myopic shift in refraction.
Color flicker and steady light caused a reduction in axial length that was compensated for by choroidal thinning in the color condition. (C) Diagrammatic representation of (B).
Abbreviations as in Fig. 1.
6 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11

Table 3 Table 4
Drug Effect on ocular components in timolol-injected chick eyes under luminance Interocular changes in ocular components (DX–DN) in saline-injected chick eyes
flicker, color flicker, and steady light conditions. Significant values are indicated in under luminance flicker, color flicker, and steady light conditions. Significant values
bold (p < 0.05). Units and abbreviations as in Table 2. are indicated in bold (p < 0.05). Units and abbreviations as in Table 2.

Timolol Saline
AC Lens Vit Choroid AL RE AC Lens Vit Choroid AL RE
LUM 0.030 0.006 0.045
0.014 0.045
4.07 LUM
0.034
0.046
0.019 0.015
0.060
2.68
±0.031 ±0.036 ±0.031 ±0.020 ±0.030 ±0.92 ±0.075 ±0.087 ±0.041 ±0.016 ±0.038 ±0.89
p = 0.35 p = 0.88 p = 0.18 p = 0.48 p = 0.17 p = 0.001 p = 0.67 p = 0.61 p = 0.67 p = 0.40 p = 0.15 p = 0.02
Color 0.009
0.053 0.041
0.046
0.046
0.57 Color
0.045
0.012
0.069 0.003
0.125
1.46
±0.041 ±0.059 ±0.035 ±0.015 ±0.017 ±0.84 ±0.024 ±0.040 ±0.019 ±0.018 ±0.027 ±0.87
p = 0.83 p = 0.39 p = 0.27 p = 0.01 p = 0.02 p = 0.51 p = 0.132 p = 0.742 p = 0.002 p = 0.75 p = 0.000 p = 0.56
Steady 0.043
0.055
0.036 0.000
0.047
0.94 Steady
0.038 0.016
0.047
0.029
0.107 1.12
±0.037 ±0.040 ±0.018 ±0.017 ±0.014 ±1.03 ±0.027 ±0.041 ±0.029 ±0.029 ±0.061 ±1.71
p = 0.28 p = 0.18 p = 0.07 p = 0.98 p = 0.01 p = 0.39 p = 0.17 p = 0.69 p = 0.13 p = 0.32 p = 0.10 p = 0.51

3.2.1. Anterior eye – Treatment Effect

Atropine-treated eyes showed more lens thickening with LUM
Timolol demonstrated no significant Treatment Effect of the
than color flicker [ANOVA: F = 6.82 (df = 2), p = 0.0024; difference:
drug or illumination condition on lens thickness nor anterior
0.225 mm, p = 0.046] or steady light (difference: 0.317 mm,
chamber [ANOVA: F = 0.93 (df = 2) and 0.35 (df = 1); p = 0.55 and
p = 0.027).
0.40, respectively] nor any significant interactions [F = 0.004
(df = 2), p = 0.99] (Fig. 2).
3.4. Saline injections (atropine experiment)
3.2.2. Anterior eye – Drug Effect
With timolol treatment, there was no significant change of the Saline injections produced a minimal effect on all ocular com-
anterior segment of the eye for any of the illumination conditions. ponents in all three illumination conditions. Neither refractive
Anterior chamber depth increased slightly with LUM nor axial changes were significantly different in any illumination
(0.030 ± 0.031 mm, p = 0.35) and steady light (0.043 ± 0.037 mm, condition [Rx ANOVA: F = 2.48 (df = 2), p = 0.10; Axial ANOVA:
p = 0.28), but no change occurred with color flicker F = 0.74 (df = 2), p = 0.49] (Fig. 1A). Injected eyes exposed to the
(0.009 ± 0.041 mm, p = 0.83) (Table 3). LUM condition became more myopic compared with fellow eyes
(
2.68 ± 0.89 D) but without an increase in axial length. In Color
and Steady, the injected eye was smaller than the fellow eye, par-
3.2.3. Posterior eye – Treatment Effect
ticularly in eyes exposed to color flicker (AL:
0.125 ± 0.027 mm;
Timolol, when compared to water, caused an increase in axial
Vit:
0.069 ± 0.019 mm). The relative myopic shift in LUM and
length that was dependent on the exposure to the LUM illumina-
the relative decrease in eye size after exposure to color flicker
tion condition [ANOVA: F = 4.08 (df = 2), p = 0.02].
can be attributed to a combined injection and illumination effect
(Table 4).
3.2.4. Posterior eye – Drug Effect
While there was a myopic shift with LUM that was associated
with an increase in axial length, there was no refractive change 3.5. Distilled water drops (timolol experiment)
with Color since there was no change in vitreous chamber depth.
A significant myopic shift in refraction was observed with LUM Treatment with water drops also led to minimal effects on the
(
4.07 ± 0.92 D, p = 0.001), which coincided with an increase in ocular components. Neither refractive nor axial changes were sig-
axial length (0.045 ± 0.030 mm, p = 0.17) (Fig. 2B, C; Table 3). In nificantly different in any illumination condition [Rx ANOVA:
contrast, color flicker and steady light caused no significant change F = 1.15 (df = 2), p = 0.35; Axial ANOVA: F = 2.3 (df = 2), p = 0.14]
in refraction (Color:
0.57 ± 0.84 D, p = 0.51; Steady: (Fig. 2A). In steady-light conditions, the vitreous of the experimen-

0.94 ± 1.03 D, p = 0.39) despite a significant decrease in eye tal eye was longer than that of the control eye (0.051 ± 0.027 mm),
growth (Color:
0.046 ± 0.017 mm, p = 0.02; Steady: probably as a result of the small increase in axial length combined

0.047 ± 0.014 mm, p = 0.01). This lack of refractive change with choroidal thinning. No injection was given in this experiment,
occurred because the vitreous chamber depth remained constant and there is no obvious explanation why greater-than-normal vit-
with choroidal thinning in the color-flicker condition (Vitreous:
0.041 ± 0.035 mm, p = 0.27) (Table 3). Table 5
Interocular changes in ocular components (DX–DN) in distilled water-treated chick
3.3. Interactive effects of drugs and lighting eyes under luminance flicker, color flicker, and steady light conditions. Significant
values are indicated in bold (p < 0.05). Units and abbreviations as in Table 2.

The Drug Effect on refraction (Rx), vitreous (Vit) and eye-length Distilled
(AL) changes revealed an interaction between the lighting and drug water
conditions [ANOVA (df = 2): Rx: F = 4.71, p = 0.0133; AL: F = 3.33, AC Lens Vit Choroid AL RE
p = 0.04; Vit: F = 3.16, p = 0.0504] (Tables 2 and 3). LUM
0.008
0.041
0.024 0.010
0.046 2.57
When chicks were exposed to LUM, timolol rendered the eyes ±0.041 ±0.055 ±0.037 ±0.019 ±0.034 ±1.77
more myopic than atropine (difference:
7.47 D; p < 0.001), with p = 0.86 p = 0.49 p = 0.54 p = 0.60 p = 0.24 p = 0.21
more growth (difference: 0.123 mm, p = 0.024) and a greater Color
0.050 0.059
0.034 0.032 0.003
0.33
increase in vitreous chamber depth (difference: 0.140 mm, ±0.061 ±0.076 ±0.046 ±0.015 ±0.026 ±2.49
p = 0.04). Timolol-treated eyes grew much more with LUM than p = 0.443 p = 0.47 p = 0.50 p = 0.08 p = 0.91 p = 0.90
with color flicker (difference: 0.91 mm; p = 0.02), making the Steady 0.020
0.047 0.051
0.019
0.004
1.23
LUM-treated eyes more myopic (difference:
3.50 D, p = 0.045) ±0.053 ±0.068 ±0.027 ±0.037 ±0.061 ±1.93
p = 0.39 p = 0.47 p = 0.045 p = 0.77 p = 0.24 p = 0.30
than Color-treated eyes.
 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11 7

Fig. 3. Comparison of the results to the initial hypothesis. Atropine caused a reduction in eye growth with both luminance and color flicker, suggesting that parasympathetic
stimulation increases eye growth and is independent of the environmental stimulus. Timolol caused an increase in eye growth with luminance flicker but a decrease with
color flicker, suggesting that the effect of sympathetic stimulation on eye growth is dependent on the environmental stimulus.

reous chamber depth was observed without flicker in the steady- length. Studies have investigated the role of non-selective
light condition (Fig. 2A, Table 5). parasympathetic antagonists atropine (Luft, Ming, & Stell, 2003;
McBrien et al., 1993; Raviola & Wiesel, 1985; Schmid & Wildsoet,
3.6. Correlation between choroid and axial length, and refractive error 2004; Schwahn, Kaymak, & Schaeffel, 2000; Stone, Lin, & Laties,
and axial length 1991; Young, 1965) and oxyphenonium (Luft et al., 2003). In addi-
tion, studies have investigated pirenzepine (an M1 receptor antag-
No significant correlation was found between the change in onist that corresponds to cm2 and cm4 in chicks (Ellis & Seidenberg,
choroidal thickness and the change in axial length for LUM and 2000)) (Cottriall & McBrien, 1996; Leech, Cottriall, & McBrien,
color flicker in the atropine (LUM: r2 = 0.04, p = 0.64; Color: 1995; Luft et al., 2003; Stone et al., 1991), and himbacine and
r2 = 0.21, p = 0.21) and timolol (LUM r2 = 0.33, p = 0.051; Color: MT3 (M4 receptor antagonists: Cottriall, Truong, & McBrien,
r2 = 0.075, p = 0.41) experiments. Furthermore, there was no signif- 2001; McBrien, Arumugam, Gentle, Chow, & Sahebjada, 2011), in
icant correlation between the change in refractive error and the effective reduction of form deprivation induced experimental myo-
change in axial length for luminance and color flicker in the atro- pia. Parasympathetic antagonists are also effective in reducing neg-
pine (LUM: r2 = 0.02, p = 0.49; Color: r2 = 0.1, p = 0.29) and timolol ative lens induced myopia (atropine: Diether, Schaeffel, Lambrou,
(LUM r2 = 0.08, p = 0.65; Color: r2 = 0.16, p = 0.34) experiments. Fritsch, & Trendelenburg, 2007; atropine, pirenzepine, but not
MT3: Nickla, Zhu, & Wallman, 2013) in chicks.
It has been suggested that atropine acts in the retina by stimu-
4. Discussion lating retinal dopamine release via its actions on dopaminergic
amacrine cells (Schwahn et al., 2000). Retinal involvement is sug-
4.1. Summary of results gested through evidence that the highly selective muscarinic
antagonist MT3 (M4 receptor antagonist) and MT7 (M1 receptor
The experiments support our hypothesis that color and lumi- antagonist) can still inhibit myopia development even at concen-
nance changes in visual stimulation influence the activity of the trations close to their receptor affinity constants (Arumugam &
parasympathetic and sympathetic nervous systems and affect McBrien, 2012). Dopamine has been associated with myopia devel-
emmetropization. Earlier experiments in untreated chicks showed opment since the ratio of retinal dopamine to DOPAC levels
that exposure to luminance flicker simulates myopic defocus and changes with form deprivation (Schaeffel, Bartmann, Hagel, &
causes a reduction in eye length and hyperopia, while exposure to Zrenner, 1995), and eye growth is reduced in form deprivation
color flicker simulates hyperopic defocus causing an increase in myopia with dopaminergic agonists (Cohen, Peleg, Belkin, Polat,
eye growth and a myopic shift in refraction. In this experiment, & Solomon, 2012; Iuvone, Tigges, Stone, Lambert, & Laties, 1991;
inhibition of the parasympathetic nervous system with atropine Rohrer, Spira, & Stell, 1993; Schmid & Wildsoet, 2004; Stone, Lin,
resulted in further growth inhibition with both luminance and Laties, & Iuvone, 1989). Form deprivation myopia is also reduced
blue/yellow color flicker (Fig. 3). In contrast, timolol reversed the with exposure to 10 Hz stroboscopic flicker (Rohrer, Iuvone, &
findings in untreated chicks, causing an increase in eye growth with Stell, 1995; Schwahn & Schaeffel, 1997), causing upregulation of
luminance flicker and a reduction in eye growth with color flicker. the expression of the c-fos gene in the retina. This gene promotes
These results suggest that with exposure to luminance contrast expression of tyrosine hydroxylase, the rate-limiting enzyme for
(myopic defocus) growth activity depends on the relative innerva- dopamine synthesis. Nevertheless, it is important to note that atro-
tion of the parasympathetic and sympathetic nervous systems. On pine still inhibits form deprivation in retinas in which most of the
the other hand, with exposure to color contrast (hyperopic defocus) NChAT+ (choline acetyltransferase-positive: acetylcholine-
an increase in eye growth occurs through stimulation of the sympa- synthesizing) amacrine cells have been ablated (Fischer, Miethke,
thetic nervous system and parasympathetic nervous system. Morgan, & Stell, 1998) suggesting a non-retinal pathway, possibly
through action on the M1 receptors in the sclera (Lind, Chew,
4.2. Atropine-induced changes Marzani, & Wallman, 1998; Luft et al., 2003), occurring as a result
of the high concentrations used (Gallego et al., 2012; Lind et al.,
Current results agree with those of previous experiments in that 1998). The effect is not thought to occur through an accommoda-
they link atropine’s anti-myopia effects with a reduction in axial tive mechanism (McBrien et al., 1993).
8 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11

In the current experiment, atropine-injected eyes showed a sig- system and noradrenaline via the sympathetic system (Fischer,
nificant increase in lens thickness under luminance conditions McGuire, Schaeffel, & Stell, 1999; Fischer & Stell, 1999; Fujikado
compared with fellow eyes, as well as a decrease in anterior cham- et al., 1997; Nickla & Wildsoet, 2004; Nickla, Wilken, Lytle, Yom,
ber depth. Lens thickening has also been observed in a previous & Mertz, 2006), causing vasodilation and vasoconstriction, respec-
experiment with atropine, in binocularly open chick eyes tively. A second possibility is that light-stimulus-driven changes in
(McBrien et al., 1993). Although lens thinning was found in early the ionic (potassium, sodium, chloride, and calcium) environment
biometric measures in a human atropine study at 4 months, this alter the distribution of ions across the retina, choroid, and sclera.
was followed by lens thickening at subsequent time points The movement of these ions directly controls the rate and direction
(Kumaran, Htoon, Tan, & Chia, 2015). Lens thickening causes light of transretinal fluid flow through changes in osmotic pressure and
rays to converge in front of the retina, bringing the image into thus choroidal thickness (Crewther, Liang, Junghans, & Crewther,
focus for a smaller eye, but also potentially creating a myopic defo- 2006; Crewther, Murphy, & Crewther, 2008; Goodyear, Crewther,
cus that may slow eye growth and produce hyperopia. On the other & Junghans, 2009; Goodyear et al., 2008). In support of the first
hand, activation of muscarinic M1 receptors present in the mam- theory relating to vascular changes, Lovasik, Kergoat, and
malian lens causes the release of intracellular [Ca2+] ions and a Wajszilber (2005) found that blue flicker, which stimulated rod
calcium-induced blockade of lens K+ channels (Williams et al., activity, led to an attenuated sub-foveal choroidal blood flow via
1993). It remains to be determined whether lens thickening pre- vasoconstriction of choroidal blood vessels. In support of the latter,
ceded the changes in axial length, whether they occurred in Liang, Crewther, Crewther, and Junghans (2004) demonstrated a
response to the relaxation of the lens zonules in a smaller eye, or significant difference in relative concentrations of Na+ and Cl
ions
if they occurred through changes in osmotic pressure of the lens, in the outer retina, retinal pigment epithelium, and choroid
or as a result of cell proliferation. between form-deprived myopic eyes and fellow non-deprived
eyes. It remains unclear whether one or both mechanisms are
4.3. Timolol-induced changes involved in the choroidal thinning observed in this experiment
with color flicker.
The most remarkable result from this experiment was that With respect to timolol, b2-adrenergic receptors have been
timolol produced an increase in eye length when the eyes were identified in choroidal and retinal blood vessels (Grajewski,
exposed to luminance flicker, an opposite effect to atropine, and Ferrari-Dileo, Feuer, & Anderson, 1991) and blockade of these
a decrease in eye length when eyes were exposed to color flicker. receptors can cause vasodilation (Van Buskirk, Bacon, &
One possible explanation is that luminance flicker stimulates the Fahrenbach, 1990). Past studies have shown that b-adrenergic
sympathetic nervous system and induces the release of dopamine blockers such as betaxolol and levobunolol exert vasodilatory
from dopaminergic amacrine cells (Rohrer et al., 1995), an effect effects on retinal vessels and increase pulsatile ocular blood flow
that is blocked by timolol. In support of this hypothesis, it was in ocular hypertensive patients (Krakau, 1992; Langham, 1987;
shown that similar reductions in eye growth and refraction were Morsman, Bosem, Lusky, & Weinreb, 1995; Silver, Farrell,
seen with luminance flicker in both atropine-injected and Langham, O’Brien, & Schilder, 1989). In regard to timolol, however,
apomorphine-injected eyes (Chuang & Rucker, 2013; Schmid & findings are inconsistent. Although some researchers noted
Wildsoet, 2004). A correlation for the action of a b-adrenergic increases in retinal vein diameter (Schwartz, Takamoto, & Lavin,
blocker on dopamine release is seen in the role of dopamine in cog- 1995), most studies have found that timolol treatment causes a
nitive flexibility; propanolol (a b-adrenergic blocker) improves decrease in retinal vessel diameter (vasoconstriction) Martin &
cognitive flexibility under stress (Zabelina, Colzato, Beeman, & Rabineau, 1989; Yoshida et al., 1991 as well as a reduction in chor-
Hommel, 2016). On the other hand, when the eye is exposed to oidal blood flow (Schwartz et al., 1995) that we would expect to
color flicker or steady light, timolol application reduced eye lead to choroidal thinning.
growth. These results suggest that timolol is causing a small
decrease in eye growth potentially through an IOP-reducing 4.5. Clinical relevance
mechanism.
It has been suggested that increased intraocular pressure could Clinically, the results of these experiments present evidence
lead to myopia if the scleral walls of such eyes were equal to or that the effects of atropine on refraction can be enhanced by
more susceptible than emmetropic eyes to stretch under the influ- changes in visual stimulation with luminance contrast, induced
ence of IOP increase. Therefore, it follows that IOP-lowering drugs, with myopic defocus and fixation changes, potentially increasing
such as timolol, should reduce or prevent eye enlargement and the protective effect of atropine alone on myopia development.
thus myopia development and/or progression. However, signifi- However, because luminance contrast also enhances axial length
cant IOP reductions have been shown to have little effect on the with timolol, the results suggest that an imbalance of autonomic
development of form deprivation or lens-induced myopia in chicks stimulation may increase the risk of myopia, as previously sug-
(Schmid et al., 2000). The reduction in eye growth seen in this gested (Charman, 1982; Gilmartin & Bullimore, 1987; McBrien &
experiment in the color and steady-light conditions may be appar- Millodot, 1986). On the other hand, protective effects of timolol
ent because of the smaller eye. A form deprived eye is grossly over on refraction can be enhanced by exposure to changes in color con-
extended and the tissues are likely to be less susceptible to con- trast or steady light, possibly through a reduction in IOP.
traction under the influence of small IOP reductions.
Acknowledgments
4.4. Choroidal changes with atropine and timolol
The authors wish to thank Dr. Deborah Nickla, Dr. Xiaoying Zhu,
In this study choroidal thinning was found with exposure to and Dr. Steven Koevary for valuable discussions. Ms. Kristin Toto-
color flicker, with both drug types, but not with exposure to steady nelly is gratefully acknowledged for her technical contributions.
light or luminance flicker. Two general theories exist regarding the We would also like to thank Dr. Li Deng for expert assistance with
mechanisms behind the defocus-induced choroidal changes. One the statistical analysis and Marek Jacisin for help with preparing
possibility is that signaling proceeds with paracrine molecule mes- the figures. This research was funded in part by a T35 Summer
sengers (Morgan, 2003; Rymer & Wildsoet, 2005; Wallman & Research Fellowship and a Student Research Grant from Beta
Winawer, 2004) such as nitric oxide via the parasympathetic Sigma Kappa. Research reported in this publication was also sup-
 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11 9

ported by the National Eye Institute of the National Institutes of Li, S. M., Wu, S. S., Kang, M. T., Liu, Y., Jia, S. M., Li, S. Y., et al. (2014). Atropine slows
myopia progression more in Asian than white children by meta-analysis.
Health under Award Number R01EY023281. The content is solely
Optometry and Vision Science, 91(3), 342–350.
the responsibility of the authors and does not necessarily represent Wu, P. C., Yang, Y. H., & Fang, P. C. (2011). The long-term results of using low-
the official views of the National Institutes of Health. concentration atropine eye drops for controlling myopia progression in
schoolchildren. Journal of Ocular Pharmacology and Therapeutics, 27(5), 461–466.
Gottlieb, M. D., & Wallman, J. (1987). Retinal activity modulates eye growth:
References Evidence from rearing in stroboscopic illumination. Society of Neuroscience
Abstracts, 13, 1297.
Schwahn, H. N., & Schaeffel, F. (1997). Flicker parameters are different for
Saw, S. M., Gazzard, G., Shih-Yen, E. C., & Chua, W. H. (2005). Myopia and associated
suppression of myopia and hyperopia. Vision Research, 37(19), 2661–2673.
pathological complications. Ophthalmic and Physiological Optics, 25(5), 381–391.
Nathanson, N. M. (1987). Molecular properties of the muscarinic acetylcholine
Parssinen, O., & Lyyra, A. L. (1993). Myopia and myopic progression among
receptor. Annual Review of Neuroscience, 10, 195–236.
schoolchildren: A three-year follow-up study. Investigative Ophthalmology &
Mitchelson, F. (2012). Muscarinic receptor agonists and antagonists: Effects on
Visual Science, 34(9), 2794–2802.
ocular function. Handbook of Experimental Pharmacology, 208, 263–298.
Rose, K. A., Morgan, I. G., Ip, J., Kifley, A., Huynh, S., Smith, W., et al. (2008a). Outdoor
Collison, D. J., Coleman, R. A., James, R. S., Carey, J., & Duncan, G. (2000).
activity reduces the prevalence of myopia in children. Ophthalmology, 115(8),
Characterization of muscarinic receptors in human lens cells by
1279–1285.
pharmacologic and molecular techniques. Investigative Ophthalmology & Visual
Rose, K. A., Morgan, I. G., Smith, W., Burlutsky, G., Mitchell, P., & Saw, S. M. (2008b).
Science, 41(9), 2633–2641.
Myopia, lifestyle, and schooling in students of Chinese ethnicity in Singapore
Gil, D. W., Krauss, H. A., Bogardus, A. M., & WoldeMussie, E. (1997). Muscarinic
and Sydney. Archives of Ophthalmology, 126(4), 527–530.
receptor subtypes in human iris-ciliary body measured by
Jones, L. A., Sinnott, L. T., Mutti, D. O., Mitchell, G. L., Moeschberger, M. L., & Zadnik,
immunoprecipitation. Investigative Ophthalmology & Visual Science, 38(7),
K. (2007). Parental history of myopia, sports and outdoor activities, and future
1434–1442.
myopia. Investigative Ophthalmology & Visual Science, 48(8), 3524–3532.
Ishizaka, N., Noda, M., Yokoyama, S., Kawasaki, K., Yamamoto, M., & Higashida, H.
Jones-Jordan, L. A., Sinnott, L. T., Cotter, S. A., Kleinstein, R. N., Manny, R. E., Mutti, D.
(1998). Muscarinic acetylcholine receptor subtypes in the human iris. Brain
O., et al. (2012). Time outdoors, visual activity, and myopia progression in
Research, 787(2), 344–347.
juvenile-onset myopes. Investigative Ophthalmology & Visual Science, 53(11),
Pang, I. H., Matsumoto, S., Tamm, E., & DeSantis, L. (1994). Characterization of
7169–7175.
muscarinic receptor involvement in human ciliary muscle cell function. Journal
Guggenheim, J. A., Northstone, K., McMahon, G., Ness, A. R., Deere, K., Mattocks, C.,
of Ocular Pharmacology, 10(1), 125–136.
et al. (2012). Time outdoors and physical activity as predictors of incident
Matsumoto, S., Yorio, T., DeSantis, L., & Pang, I. H. (1994). Muscarinic effects on
myopia in childhood: A prospective cohort study. Investigative Ophthalmology &
cellular functions in cultured human ciliary muscle cells. Investigative
Visual Science, 53(6), 2856–2865.
Ophthalmology & Visual Science, 35(10), 3732–3738.
Onal, S., Toker, E., Akingol, Z., Arslan, G., Ertan, S., Turan, C., et al. (2007). Refractive
Osborne, N. N., FitzGibbon, F., & Schwartz, G. (1991). Muscarinic acetylcholine
errors of medical students in Turkey: One year follow-up of refraction and
receptor-mediated phosphoinositide turnover in cultured human retinal
biometry. Optometry and Vision Science, 84(3), 175–180.
pigment epithelium cells. Vision Research, 31(7–8), 1119–1127.
Dirani, M., Tong, L., Gazzard, G., Zhang, X., Chia, A., Young, T. L., et al. (2009). Outdoor
Williams, M. R., Duncan, G., Riach, R. A., & Webb, S. F. (1993). Acetylcholine
activity and myopia in Singapore teenage children. British Journal of
receptors are coupled to mobilization of intracellular calcium cultured human
Ophthalmology, 93(8), 997–1000.
lens cells. Experimental Eye Research, 57(3), 381–384.
Wu, P. C., Tsai, C. L., Hu, C. H., & Yang, Y. H. (2010). Effects of outdoor activities on
Michon, J., Jr., & Kinoshita, J. H. (1968). Experimental miotic cataract. I. Effects of
myopia among rural school children in Taiwan. Ophthalmic Epidemiology, 17(5),
miotics on lens structure, cation content, and hydration. Archives of
338–342.
Ophthalmology, 79(1), 79–86.
Ashby, R., Ohlendorf, A., & Schaeffel, F. (2009). The effect of ambient illuminance on
McBrien, N. A., Jobling, A. I., Truong, H. T., Cottriall, C. L., & Gentle, A. (2009).
the development of deprivation myopia in chicks. Investigative Ophthalmology &
Expression of muscarinic receptor subtypes in tree shrew ocular tissues and
Visual Science, 50(11), 5348–5354.
their regulation during the development of myopia. Molecular Vision, 15,
Cohen, Y., Belkin, M., Yehezkel, O., Solomon, A. S., & Polat, U. (2011). Dependency
464–475.
between light intensity and refractive development under light-dark cycles.
Fischer, A. J., McKinnon, L. A., Nathanson, N. M., & Stell, W. K. (1998). Identification
Experimental Eye Research, 92(1), 40–46.
and localization of muscarinic acetylcholine receptors in the ocular tissues of
Backhouse, S., Collins, A. V., & Phillips, J. R. (2013). Influence of periodic vs
the chick. Journal of Comparative Neurology, 392(3), 273–284.
continuous daily bright light exposure on development of experimental myopia
Strang, C. E., Renna, J. M., Amthor, F. R., & Keyser, K. T. (2010). Muscarinic
in the chick. Ophthalmic and Physiological Optics, 33(5), 563–572.
acetylcholine receptor localization and activation effects on ganglion response
Cohen, Y., Belkin, M., Yehezkel, O., Avni, I., & Polat, U. (2008). Light intensity
properties. Investigative Ophthalmology & Visual Science, 51(5), 2778–2789.
modulates corneal power and refraction in the chick eye exposed to continuous
Townes-Anderson, E., & Vogt, B. A. (1989). Distribution of muscarinic acetylcholine
light. Vision Research, 48(21), 2329–2335.
receptors on processes of isolated retinal cells. Journal of Comparative Neurology,
Smith, E. L., Hung, L. F., & Huang, J. (2012). Protective effects of high ambient
290(3), 369–383.
lighting on the development of form-deprivation myopia in rhesus monkeys.
Yamada, E. S., Dmitrieva, N., Keyser, K. T., Lindstrom, J. M., Hersh, L. B., & Marshak, D.
Investigative Ophthalmology & Visual Science, 53(1), 421–428.
W. (2003). Synaptic connections of starburst amacrine cells and localization of
Rucker, F., & Wallman, J. (2012). Chicks use changes in luminance and chromatic
acetylcholine receptors in primate retinas. Journal of Comparative Neurology, 461
contrast as indicators of the sign of defocus. Journal of Visualization, 12(6), 23.
(1), 76–90.
Rucker, F. J., & Wallman, J. (2009). Chick eyes compensate for chromatic simulations
Tietje, K. M., & Nathanson, N. M. (1991). Embryonic chick heart expresses multiple
of hyperopic and myopic defocus: Evidence that the eye uses longitudinal
muscarinic acetylcholine receptor subtypes. Isolation and characterization of a
chromatic aberration to guide eye-growth. Vision Research, 49(14), 1775–1783.
gene encoding a novel m2 muscarinic acetylcholine receptor with high affinity
Rucker, F. J., & Wallman, J. (2008). Cone signals for spectacle-lens compensation:
for pirenzepine. The Journal of Biological Chemistry, 266(26), 17382–17387.
Differential responses to short and long wavelengths. Vision Research, 48(19),
Gadbut, A. P., & Galper, J. B. (1994). A novel M3 muscarinic acetylcholine receptor is
1980–1991.
expressed in chick atrium and ventricle. The Journal of Biological Chemistry, 269
Rucker, F. J. (2013). The role of luminance and chromatic cues in emmetropisation.
(41), 25823–25829.
Ophthalmic and Physiological Optics, 33(3), 196–214.
Tietje, K. M., Goldman, P. S., & Nathanson, N. M. (1990). Cloning and functional
Rucker, F., Britton, S., Spatcher, M., & Hanowsky, S. (2015). Blue light protects
analysis of a gene encoding a novel muscarinic acetylcholine receptor
against temporal frequency sensitive refractive changes. Investigative
expressed in chick heart and brain. The Journal of Biological Chemistry, 265(5),
Ophthalmology & Visual Science, 56(10), 6121–6131.
2828–2834.
Bedrossian, R. H. (1971). The effect of atropine on myopia. Annals of Ophthalmology,
Creason, S., Tietje, K. M., & Nathanson, N. M. (2000). Isolation and functional
3(8), 891–897.
characterization of the chick M5 muscarinic acetylcholine receptor gene. Journal
Chia, A., Chua, W. H., Cheung, Y. B., Wong, W. L., Lingham, A., Fong, A., et al. (2012).
of Neurochemistry, 74(2), 882–885.
Atropine for the treatment of childhood myopia: Safety and efficacy of 0.5%,
McBrien, N. A., Moghaddam, H. O., New, R., & Williams, L. R. (1993). Experimental
0.1%, and 0.01% doses (atropine for the treatment of myopia 2). Ophthalmology,
myopia in a diurnal mammal (Sciurus carolinensis) with no accommodative
119(2), 347–354.
ability. Journal of Physiology, 469, 427–441.
Chia, A, Chua, WH, Wen, L, Fong, A, Goon, YY, & Tan, D (2014). Atropine for the
Chen, J. C., Schmid, K. L., & Brown, B. (2003). The autonomic control of
treatment of childhood myopia: changes after stopping atropine 0.01%, 0.1%
accommodation and implications for human myopia development: A review.
and 0.5%. American Journal of Ophthalmology, 157 (2). 451-7e1.
Ophthalmic and Physiological Optics, 23(5), 401–422.
Chia, A., Lu, Q. S., & Tan, D. (2015). Five-year clinical trial on atropine for the
Airaksinen, P. J., Saari, K. M., Tiainen, T. J., & Jaanio, E. A. (1979). Management of
treatment of myopia 2: Myopia control with atropine 0.01% eyedrops.
acute closed-angle glaucoma with miotics and timolol. British Journal of
Ophthalmology.
Ophthalmology, 63(12), 822–825.
Morgan, I. G., Ohno-Matsui, K., & Saw, S. M. (2012). Myopia. Lancet, 379(9827),
Regan, J. W., & Cotecchia, S. (1992). The a-adrenergic receptors: New subtypes,
1739–1748.
pharmacology and coupling mechanism. In M. R. Brann (Ed.), Molecular Biology
Walline, J. J. (2016). Myopia control: A review. Eye & Contact Lens, 42(1), 3–8.
of G-Protein Coupled Receptors (pp. 76–112). Boston: Birkhauser.
Lee, J. J., Fang, P. C., Yang, I. H., Chen, C. H., Lin, P. W., Lin, S. A., et al. (2006).
van Alphen, G. W. (1976). The adrenergic receptors of the intraocular muscles of the
Prevention of myopia progression with 0.05% atropine solution. Journal of
human eye. Invest Ophthalmology, 15(6), 502–505.
Ocular Pharmacology and Therapeutics, 22(1), 41–46.
10 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11

Zetterstrom, C. (1988). Effects of adrenergic drugs on accommodation and distant Cottriall, C. L., Truong, H. T., & McBrien, N. A. (2001). Inhibition of myopia
refraction in daylight and darkness. A laseroptometric study. Acta development in chicks using himbacine: A role for M(4) receptors? NeuroReport,
Ophthalmology (Copenhagen), 66(1), 58–64. 12(11), 2453–2456.
Garner, L. F., Brown, B., Baker, R., & Colgan, M. (1983). The effect of phenylephrine McBrien, N. A., Arumugam, B., Gentle, A., Chow, A., & Sahebjada, S. (2011). The M4
hydrochloride on the resting point of accommodation. Investigative muscarinic antagonist MT-3 inhibits myopia in chick: Evidence for site of
Ophthalmology & Visual Science, 24(4), 393–395. action. Ophthalmic and Physiological Optics, 31(5), 529–539.
Wax, M. B., & Molinoff, P. B. (1987). Distribution and properties of beta-adrenergic Diether, S., Schaeffel, F., Lambrou, G. N., Fritsch, C., & Trendelenburg, A. U. (2007).
receptors in human iris-ciliary body. Investigative Ophthalmology & Visual Effects of intravitreally and intraperitoneally injected atropine on two types of
Science, 28(3), 420–430. experimental myopia in chicken. Experimental Eye Research, 84(2), 266–274.
Zimmerman, T. J., & Kaufman, H. E. (1977). Timolol a b-adrenergic blocking agent for Nickla, D. L., Zhu, X., & Wallman, J. (2013). Effects of muscarinic agents on chick
the treatment of glaucoma. Archives of Ophthalmology, 95(4), 601–604. choroids in intact eyes and eyecups: Evidence for a muscarinic mechanism in
Maurice, D. M., & Mushin, A. S. (1966). Production of myopia in rabbits by raised choroidal thinning. Ophthalmic and Physiological Optics, 33(3), 245–256. http://
body-temperature and increased intraocular pressure. Lancet, 2(7474), dx.doi.org/10.1111/opo.12054.
1160–1162. Arumugam, B., & McBrien, N. A. (2012). Muscarinic antagonist control of myopia:
Parssinen, O. (1990). Intraocular pressure in school myopia. Acta Ophthalmology Evidence for M4 and M1 receptor-based pathways in the inhibition of
(Copenhagen), 68(5), 559–563. experimentally-induced axial myopia in the tree shrew. Investigative
Jensen, H. (1992). Myopia progression in young school children and intraocular Ophthalmology & Visual Science, 53(9), 5827–5837.
pressure. Documenta Ophthalmologica, 82(3), 249–255. Schaeffel, F., Bartmann, M., Hagel, G., & Zrenner, E. (1995). Studies on the role of the
Quinn, G. E., Berlin, J. A., Young, T. L., Ziylan, S., & Stone, R. A. (1995). Association of retinal dopamine/melatonin system in experimental refractive errors in
intraocular pressure and myopia in children. Ophthalmology, 102(2), 180–185. chickens. Vision Research, 35(9), 1247–1264.
David, R., Zangwill, L. M., Tessler, Z., & Yassur, Y. (1985). The correlation between Rohrer, B., Spira, A. W., & Stell, W. K. (1993). Apomorphine blocks form-deprivation
intraocular pressure and refractive status. Archives of Ophthalmology, 103(12), myopia in chickens by a dopamine D2-receptor mechanism acting in retina or
1812–1815. pigmented epithelium. Visual Neuroscience, 10(3), 447–453.
Goss, D. A., & Caffey, T. W. (1999). Clinical findings before the onset of myopia in Iuvone, P. M., Tigges, M., Stone, R. A., Lambert, S., & Laties, A. M. (1991). Effects of
youth: 5. Intraocular pressure. Optometry and Vision Science, 76(5), 286–291. apomorphine, a dopamine receptor agonist, on ocular refraction and axial
Tornqvist, G. (1967). The relative importance of the parasympathetic and elongation in a primate model of myopia. Investigative Ophthalmology & Visual
sympathetic nervous systems for accommodation in monkeys. Invest Science, 32(5), 1674–1677.
Ophthalmology, 6(6), 612–617. Cohen, Y., Peleg, E., Belkin, M., Polat, U., & Solomon, A. S. (2012). Ambient
Toates, F. M. (1972). Accommodation function of the human eye. Physiological illuminance, retinal dopamine release and refractive development in chicks.
Reviews, 52(4), 828–863. Experimental Eye Research, 103, 33–40.
Tornqvist, G. (1966). Effect of cervical sympathetic stimulation on accommodation Stone, R. A., Lin, T., Laties, A. M., & Iuvone, P. M. (1989). Retinal dopamine and form-
in monkeys. An example of a beta-adrenergic, inhibitory effect. Acta Physiologica deprivation myopia. Proc. Natl. Acad. Sci. U.S.A., 86(2), 704–706.
Scandinavica, 67(3), 363–372. Rohrer, B., Iuvone, P. M., & Stell, W. K. (1995). Stimulation of dopaminergic amacrine
McBrien, N. A., & Millodot, M. (1986). Amplitude of accommodation and refractive cells by stroboscopic illumination or fibroblast growth factor (bFGF, FGF-2)
error. Investigative Ophthalmology & Visual Science, 27(7), 1187–1190. injections: Possible roles in prevention of form-deprivation myopia in the chick.
Gilmartin, B., & Bullimore, M. A. (1987). Sustained near vision augments Brain Research, 686(2), 169–181.
inhibitory sympathetic innervation of the ciliary muscle. Clinical Vision Fischer, A. J., Miethke, P., Morgan, I. G., & Stell, W. K. (1998). Cholinergic amacrine
Science, 1, 197–208. cells are not required for the progression and atropine-mediated suppression of
Gilmartin, B., & Bullimore, M. A. (1991). Adaptation of tonic accommodation to form-deprivation myopia. Brain Research, 794(1), 48–60.
sustained visual tasks in emmetropia and late-onset myopia. Optometry and Lind, G. J., Chew, S. J., Marzani, D., & Wallman, J. (1998). Muscarinic acetylcholine
Vision Science, 68(1), 22–26. receptor antagonists inhibit chick scleral chondrocytes. Investigative
Ciuffreda, K. J., & Lee, M. (2002). Differential refractive susceptibility to sustained Ophthalmology & Visual Science, 39(12), 2217–2231.
nearwork. Ophthalmic and Physiological Optics, 22(5), 372–379. Gallego, P., Martinez-Garcia, C., Perez-Merino, P., Ibares-Frias, L., Mayo-Iscar, A., &
Ciuffreda, K. J., & Wallis, D. M. (1998). Myopes show increased susceptibility to Merayo-Lloves, J. (2012). Scleral changes induced by atropine in chicks as an
nearwork aftereffects. Investigative Ophthalmology & Visual Science, 39(10), experimental model of myopia. Ophthalmic and Physiological Optics, 32(6),
1797–1803. 478–484.
Culhane, H. M., Winn, B., & Gilmartin, B. (1999). Human dynamic closed-loop Kumaran, A., Htoon, H. M., Tan, D., & Chia, A. (2015). Analysis of changes in
accommodation augmented by sympathetic inhibition. Investigative refraction and biometry of atropine- and placebo-treated eyes. Investigative
Ophthalmology & Visual Science, 40(6), 1137–1143. Ophthalmology & Visual Science, 56(9), 5650–5655.
McBrien, N. A., Moghaddam, H. O., & Reeder, A. P. (1993). Atropine reduces Chuang, K, & Rucker, F (2013). The Role of Dopamine in Eye Growth Responses to
experimental myopia and eye enlargement via a nonaccommodative Color and Luminance Flicker in Chicks International Myopia Conference.
mechanism. Investigative Ophthalmology & Visual Science, 34(1), 205–215. Asilomar CA. .
Schmid, K. L., & Wildsoet, C. F. (2004). Inhibitory effects of apomorphine and Zabelina, D. L., Colzato, L., Beeman, M., & Hommel, B. (2016). Dopamine and the
atropine and their combination on myopia in chicks. Optometry and Vision creative mind: Individual differences in creativity are predicted by interactions
Science, 81(2), 137–147. between dopamine genes DAT and COMT. PLoS One, 11(1), e0146768.
Schmid, K. L., Abbott, M., Humphries, M., Pyne, K., & Wildsoet, C. F. (2000). Timolol Morgan, I. G. (2003). The biological basis of myopic refractive error. Clinical and
lowers intraocular pressure but does not inhibit the development of Experimental Optometry, 86(5), 276–288.
experimental myopia in chick. Experimental Eye Research, 70(5), Rymer, J., & Wildsoet, C. F. (2005). The role of the retinal pigment epithelium in eye
659–666. growth regulation and myopia: A review. Visual Neuroscience, 22(3), 251–261.
Nickla, D. L., Wildsoet, C., & Wallman, J. (1998). Visual influences on diurnal Wallman, J., & Winawer, J. (2004). Homeostasis of eye growth and the question of
rhythms in ocular length and choroidal thickness in chick eyes. Experimental Eye myopia. Neuron, 43(4), 447–468.
Research, 66(2), 163–181. Fischer, A. J., McGuire, J. J., Schaeffel, F., & Stell, W. K. (1999). Light- and focus-
Schaeffel, F., Farkas, L., & Howland, H. C. (1987). Infrared photoretinoscope. Applied dependent expression of the transcription factor ZENK in the chick retina.
Optics, 26(8), 1505–1509. Nature Neuroscience, 2(8), 706–712.
Stone, R. A., Lin, T., & Laties, A. M. (1991). Muscarinic antagonist effects on Fischer, A. J., & Stell, W. K. (1999). Nitric oxide synthase-containing cells in the
experimental chick myopia. Experimental Eye Research, 52(6), 755–758. retina, pigmented epithelium, choroid, and sclera of the chick eye. Journal of
Luft, W. A., Ming, Y., & Stell, W. K. (2003). Variable effects of previously untested Comparative Neurology, 405(1), 1–14.
muscarinic receptor antagonists on experimental myopia. Investigative Fujikado, T., Kawasaki, Y., Fujii, J., Taniguchi, N., Okada, M., Suzuki, A., et al. (1997).
Ophthalmology & Visual Science, 44(3), 1330–1338. The effect of nitric oxide synthase inhibitor on form-deprivation myopia.
Schwahn, H. N., Kaymak, H., & Schaeffel, F. (2000). Effects of atropine on refractive Current Eye Research, 16(10), 992–996.
development, dopamine release, and slow retinal potentials in the chick. Visual Nickla, D. L., & Wildsoet, C. F. (2004). The effect of the nonspecific nitric oxide
Neuroscience, 17(2), 165–176. synthase inhibitor NG-nitro-L-arginine methyl ester on the choroidal
Raviola, E., & Wiesel, T. N. (1985). An animal model of myopia. New England Journal compensatory response to myopic defocus in chickens. Optometry and Vision
of Medicine, 312(25), 1609–1615. Science, 81(2), 111–118.
Young, F. A. (1965). The effect of atropine on the development of myopia in Nickla, D. L., Wilken, E., Lytle, G., Yom, S., & Mertz, J. (2006). Inhibiting the transient
monkeys. American Journal of Optometry and Archives of American Academy of choroidal thickening response using the nitric oxide synthase inhibitor l-NAME
Optometry, 42, 439–449. prevents the ameliorative effects of visual experience on ocular growth in two
Ellis, J., & Seidenberg, M. (2000). Site-directed mutagenesis implicates a threonine different visual paradigms. Experimental Eye Research, 83(2), 456–464.
residue in TM6 in the subtype selectivities of UH-AH 37 and pirenzepine at Crewther, S. G., Liang, H., Junghans, B. M., & Crewther, D. P. (2006). Ionic control of
muscarinic receptors. Pharmacology, 61(2), 62–69. ocular growth and refractive change. Proc. Natl. Acad. Sci. U.S.A., 103(42),
Cottriall, C. L., & McBrien, N. A. (1996). The M1 muscarinic antagonist pirenzepine 15663–15668.
reduces myopia and eye enlargement in the tree shrew. Investigative Crewther, S. G., Murphy, M. J., & Crewther, D. P. (2008). Potassium channel and
Ophthalmology & Visual Science, 37(7), 1368–1379. NKCC cotransporter involvement in ocular refractive control mechanisms. PLoS
Leech, E. M., Cottriall, C. L., & McBrien, N. A. (1995). Pirenzepine prevents form One, 3(7), e2839.
deprivation myopia in a dose dependent manner. Ophthalmic and Physiological Goodyear, M. J., Crewther, S. G., & Junghans, B. M. (2009). A role for aquaporin-4 in
Optics, 15(5), 351–356. fluid regulation in the inner retina. Visual Neuroscience, 26(2), 159–165.
 L.A. Goldberg, F.J. Rucker / Vision Research 122 (2016) 1–11 11

Goodyear, M. J., Junghans, B. M., Giummarra, L., Murphy, M. J., Crewther, D. P., & Morsman, C. D., Bosem, M. E., Lusky, M., & Weinreb, R. N. (1995). The effect of
Crewther, S. G. (2008). A role for aquaporin-4 during induction of form topical beta-adrenoceptor blocking agents on pulsatile ocular blood flow. Eye
deprivation myopia in chick. Molecular Vision, 14, 298–307. (London), 9(Pt. 3), 344–347.
Lovasik, J. V., Kergoat, H., & Wajszilber, M. A. (2005). Blue flicker modifies the Silver, D. M., Farrell, R. A., Langham, M. E., O’Brien, V., & Schilder, P. (1989).
subfoveal choroidal blood flow in the human eye. American Journal of Physiology Estimation of pulsatile ocular blood flow from intraocular pressure. Acta
Heart and Circulatory Physiology, 289(2), H683–H691. Ophthalmology Supplement, 191, 25–29.
Liang, H., Crewther, S. G., Crewther, D. P., & Junghans, B. M. (2004). Structural and Schwartz, B., Takamoto, T., & Lavin, P. (1995). Increase of retinal vessel width in
elemental evidence for edema in the retina, retinal pigment epithelium, and ocular hypertensives with timolol therapy. Acta Ophthalmologica Scandinavica.
choroid during recovery from experimentally induced myopia. Investigative Supplement, 215, 41–53.
Ophthalmology & Visual Science, 45(8), 2463–2474. Martin, X. D., & Rabineau, P. A. (1989). Vasoconstrictive effect of topical timolol on
Grajewski, A. L., Ferrari-Dileo, G., Feuer, W. J., & Anderson, D. R. (1991). Beta- human retinal arteries. Graefe’s Archive for Clinical and Experimental
adrenergic responsiveness of choroidal vasculature. Ophthalmology, 98(6), Ophthalmology = Albrecht von Graefes Archiv fur klinische und
989–995. experimentelle Ophthalmologie, 227 (6), 526–530.
Van Buskirk, E. M., Bacon, D. R., & Fahrenbach, W. H. (1990). Ciliary vasoconstriction Yoshida, A., Feke, G. T., Ogasawara, H., Goger, D. G., Murray, D. L., & McMeel, J. W.
after topical adrenergic drugs. American Journal of Ophthalmology, 109(5), (1991). Effect of timolol on human retinal, choroidal and optic nerve head
511–517. circulation. Ophthalmic Research, 23(3), 162–170.
Krakau, C. E. (1992). Calculation of the pulsatile ocular blood flow. Investigative Charman, W. N. (1982). The accommodative resting point and refractive error.
Ophthalmology & Visual Science, 33(9), 2754–2756. Ophthalmic Optics, 21, 469.
Langham, M. E. (1987). Ocular blood flow and visual loss in glaucomatous eyes. In G.
K. Kreiglestein (Ed.). Springer.