April 2017
Volume 58, Issue 4
Open Access
Eye Movements, Strabismus, Amblyopia and Neuro-ophthalmology  |   April 2017
Bright Light Suppresses Form-Deprivation Myopia Development With Activation of Dopamine D1 Receptor Signaling in the ON Pathway in Retina
Author Affiliations & Notes
  • Si Chen
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Zhina Zhi
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Qingqing Ruan
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Qingxia Liu
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Fen Li
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Fen Wan
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Peter S. Reinach
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Jiangfan Chen
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Jia Qu
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Xiangtian Zhou
    School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health P. R. China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China
  • Correspondence: Xiangtian Zhou, School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, 270 West Xueyuan Road, Wenzhou, Zhejiang, 325003, People's Republic of China; zxt-dr@wz.zj.cn
  • Jia Qu, School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, 270 West Xueyuan Road, Wenzhou, Zhejiang, 325003, People's Republic of China; jia.qu@163.com
  • Footnotes
     SC and ZZ contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science April 2017, Vol.58, 2306-2316. doi:10.1167/iovs.16-20402
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      Si Chen, Zhina Zhi, Qingqing Ruan, Qingxia Liu, Fen Li, Fen Wan, Peter S. Reinach, Jiangfan Chen, Jia Qu, Xiangtian Zhou; Bright Light Suppresses Form-Deprivation Myopia Development With Activation of Dopamine D1 Receptor Signaling in the ON Pathway in Retina. Invest. Ophthalmol. Vis. Sci. 2017;58(4):2306-2316. doi: 10.1167/iovs.16-20402.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To determine whether dopamine receptor D1 (D1R) signaling pathway activation by bright light (BL) in specific retinal neuronal cell types contributes to inhibiting form-deprivation myopia (FDM) in mice.

Methods: Mice (3-weeks old) were raised under either normal light (NL: 100–200 lux) or BL (2500–5000 lux) conditions with or without form deprivation. Refraction changes were evaluated with an eccentric infrared photorefractor, and ocular axial components with optical coherence tomography. The D1R antagonist, SCH39166, was intraperitoneally injected daily to evaluate if BL mediates declines in FDM development through D1R activation. Six different biomarkers of retinal neuronal types delineated differential distribution of D1R expression. c-Fos and phosphorylated tyrosine hydroxylase (p-TH) immunofluorescent staining evaluated D1R receptor activation and dopamine synthesis, respectively.

Results: Bright light exposure for 4 weeks (6 hours per day) inhibited FDM development by reducing ocular elongation and shifting refraction toward hyperopia compared with changes occurring in NL. SCH39166 injections completely reversed the inhibitory effects of BL on both refraction and ocular elongation. Bright light increased the number of cells expressing p-TH and c-fos. Increases in c-fos+ cells occurred mainly in D1R+ bipolar cells (BCs), especially D1R+ ON-BCs.

Conclusions: Bright light increases D1R activity in the BCs of the ON pathway, which is associated with less myopic shift and ocular elongation than those occurring in NL. These declines suggest that increased D1R activity in the ON pathway contributes to the BL suppression of FDM development in mice.

Myopia prevalence has dramatically increased in recent years,1 and in cases in which the refractive error is greater than −6.00 diopters (D) such rises can lead to severe visual impairment as well as even blindness.2,3 Recent studies indicate that increases in time spent outdoors are an important modifiable environmental factor that has a protective effect on the risk of developing myopia in children.48 Such an association is evident because extending outdoor exposure time led to declines from 23% to 50% in the onset of myopia and inhibited myopia progression in elementary school children.4,5 Light intensity is a variable affecting this process because on a clear day it can be ×1000 greater than artificial indoor light. Schaeffel et al.9 first showed that exposure to bright light inhibited form-deprivation myopia (FDM) in chickens. Other animal studies further indicated that laboratory bright light (BL) is a factor contributing to myopia inhibition in chicks,1012 guinea pigs,13 and primates.14,15 However, the underlying receptor-linked signaling mechanisms mediating such suppression require clarification. 
Dopamine (DA) is a retinal neurotransmitter released by dopaminergic amacrine cells (ACs). This neurotransmitter appears to play an important role in vision-guided eye growth because its synthesis and release are positively associated with the light intensity impinging on the retina, and BL partially rescued the drop in retinal DA.16 Another indication of DA involvement in mediating light inhibitory effects on FDM is that the nonselective DA receptor agonist apomorphine inhibited FDM in chicks,17 mice,18 guinea pigs,19 and rhesus monkeys,20 which indicates that activation by BL of DA receptor-linked signaling pathways contributes to controlling myopia progression by BL.21 The two DA receptor subtypes expressed in the retina are the D1-like (including D1/D5R) and D2-like receptors (including D2/D4R). Both D1R and D2R are implicated in refractive development.22,23 A large body of experimental evidence supports the specific involvement of D2R in myopia development.2426 However, it is less clear whether or not D1R is involved in myopia development. Previously identified D1R involvement is based on pharmacology studies with chicken models of myopia.24,25,27 For example, a D1R agonist SKF38393 inhibited FDM,28 while a D1R antagonist, SCH 23390, enhanced this response22 and inhibited the ameliorating effects of periods of clear vision on lens-induced myopia in chicks.29 Similarly, a D1R antagonist SCH23390 enhanced progression of naturally occurring myopia while a D1R agonist SKF38393 inhibited it in albino guinea pigs.30 Moreover, preliminary studies indicate that D1R-like agonists inhibit FDM while D1R-like antagonists enhance FDM in C57BL/6 mice. These results support the notion of D1R involvement in myopia development at least in chicks, mice, and guinea pigs. However, there are no studies describing whether or not D1R activation contributes to mediating light-induced suppression of myopia. 
Another uncertainty pertains to the identity of the specific retinal cell types on which DA activation elicits signaling pathway events modulating refractory development. A recent study using Drd1a-tdTomato mice detected the transgenic expression of D1R in the bipolar cells (BCs), horizontal cells (HCs), and ACs in the inner nuclear layer (INL).31 GABAergic ACs have been implicated in myopia development in chicks32 and guinea pigs.33 Genetic knockout studies indicate that nob mice, which have an ON pathway defect are more susceptible to form deprivation (FD).34,35 However, there are no reports describing which retinal cell types expressing D1R are activated by BL. Such insight is needed to delineate the signaling pathways mediating D1R control of refraction development. 
In this study, we first confirmed that BL (2500–5000 lux) increases DA synthesis, and inhibits FDM development as well as optical axis elongation. Importantly, we identified the critical role of D1R signaling in mediating BL inhibition of FDM by abrogating the inhibitory effect of BL on FDM with a D1R antagonist, SCH39166. Furthermore, by determining the expression of c-fos in D1R+ cells (tdTomato+ cells) and in specific subtypes of D1R+ cells, we demonstrated that while normal light (NL) mainly activates retinal D1R activity (i.e., c-fos+/D1R+) in ACs, BL triggers larger rises in D1R activity in ON-BCs compared with NL. Taken together, our findings suggest that BL-induced D1R signaling activation in the retinal ON pathway contributes to suppressing FDM development. 
Materials and Methods
Animals
The Animal Care and Ethics Committee at Wenzhou Medical University, Wenzhou, China, approved the research in this study. Treatment and care of the animals were conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Three-week-old male wild-type C57BL/6 mice (n = 282) and Drd1a-tdTomato mice (strain name: B6.Cg-Tg [Drd1a-tdTomato] 6Calak/J) (n = 8) were obtained from the Animal Breeding Unit at Wenzhou Medical University. All animals were raised in standard transparent mouse cages (24 × 18 × 13 cm) with a 12-hour light/12-hour dark cycle (light from 8 AM to 8 PM). The rooms were kept at 22 ± 2°C, and mice received an unlimited supply of food and water. 
Experimental Design
Two hundred sixty-two C57BL/6 mice (3-weeks old) were randomly assigned to two different groups each of which were exposed to either NL or BL. Each group was further divided into subgroups based on a variety of treatment protocols (normal vision or FD, SCH39166 or vehicle dimethylsulfoxide [DMSO] injection). All of the treatments were continued for 4 weeks. Ocular biometry including refraction and ocular axial components were measured at the outset and on day 28. 
Another 20 C57BL/6 mice and 8 Drd1a-tdTomato mice (3-weeks old) were randomly divided into NL and BL groups for 2 days of light exposure followed by retinal immunofluorescent staining. Phosphorylated tyrosine hydroxylase (p-TH), the rate limiting synthetase of DA, was probed for by immunostaining C57BL/6 mice to determine the effect of BL on retinal DA synthesis (n = 10 for each group). Various types of retinal neurons, including ACs, HCs, and BCs, were double labeled with c-fos and neuron type-specific markers in Drd1a-tdTomato mice to characterize the response of different D1R+ retinal neurons types to BL (n = 4 for each group). The neuron type-specific markers included PAX6 (a pan-AC marker), GAD 67 (a GABAergic AC marker), Parvalbumin (PV, a subset of amacrine [PV+ ACs] and HC marker), CHX 10 (a pan-BC marker), Goα (an ON-BC marker), and recoverin (an OFF-BC marker). Table 1 provides details regarding antibody sources and suppliers. 
Table 1
 
Primary Antibody Details
Table 1
 
Primary Antibody Details
Lighting Conditions
The NL and BL luminance was approximately 100–200 lux and 2500–5000 lux, respectively, on the cage floor provided by fluorescent bulbs. During the light-on cycle, the mice in the NL were exposed to NL for 12 hours, while the BL groups were exposed to NL for 3 hours, followed by 6 hours of BL, and then another 3 hours of NL. 
Induction of FDM and Drug Administration
Form-deprivation myopia was induced by the placement of a translucent occluder onto a randomly selected eye for 4 weeks, as described previously by Schaeffel et al.36 
D1R antagonist, SCH39166 solution (0.04 μg/μL; Tocris bioscience, Glasgow, UK) was freshly prepared by dissolving it in dimethyl sulfoxide (DMSO; 0.11 μg/μL; Sigma, Buchs, Switzerland). SCH39166 (0.4 μg/g body weight), or DMSO (1.1 μg/g bodyweight) were intraperitoneally injected daily without anesthesia to drug-injection groups. 
Biometric Measurements
All measurements were performed by a research optometrist with help from an assistant in which the identity of the different groups were masked. 
The refraction was measured as described in a dark room using an eccentric infrared photorefractor.36 
The axial components of the eye parameters included anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL). They were measured as described using a custom-made optical coherence tomography (OCT).37 Mice were intraperitoneally anesthetized with a mixture of 0.12 g/mL ketamine hydrochloride and 1.8 mg/mL xylazine hydrochloride before the measurement. The injection volume was 8 mL/kg of body weight. Pupils were dilated with tropicamide phenylephrine eye drops (0.5% tropicamide and 0.5% phenylephrine; Santen, Tokyo, Japan). 
Immunofluorescence
Both flat-mount and transverse-sectioned retinas were preincubated in a blocking solution of 6% normal donkey serum, 1% bovine serum albumin, and 0.3% Triton X-100 in 0.1 M PBS for 2 hours. The specimens were then incubated with the primary antibodies diluted in the 3% normal donkey serum, 0.5% bovine serum albumin, and 0.3% Triton X-100 in 0.1 M PBS in 4°C (overnight for transverse section; 72 hours for flat-mount retina). The secondary antibodies were applied for 2 hours at room temperature for transverse section, and for 24 hours at 4°C for flat-mount retinas. Finally specimens were placed in mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Lot No.: H-1200; Vector Laboratories, Burlingame, CA, USA). 
Photography and Cell Number Measurement
The flat-mount retina and transverse sections were observed and photographed with a Zeiss LSM 710 confocal microscope (ZEISS, Gottingen, Germany) with ×20 magnification. The number of retinal cells expressing p-TH (Np-TH+) was counted throughout the whole retina in the flat-mount retina. The number of different types of retinal neurons in transverse sections coexpressing D1R and/or c-fos in INL was also determined in the central section with the optic disc as a reference. For example, in PAX6 and c-fos double-labeled retinas, the number of PAX+ cells (NPAX+), PAX6+ cells coexpressing D1R or/and c-fos (ND1R+PAX+, Nc-fos+PAX+, Nc-fos+D1R+PAX+), and the percentage of D1R+ cells and c-fos+ cells in PAX6+cells (P[D1R+PAX+/PAX+], P[c-fos+PAX+/PAX+]), the percentage of c-fos+ cells in D1R+PAX6+ cells (P[c-fos+D1R+PAX+/D1R+PAX+]) were separately determined. The cells labeled with the other five neuron type-specific markers were also calculated in an identical way. Parvalbumin immunoreactivity is localized on both HCs and a subset of ACs in the INL according to previous studies.38 Thus, the PV+ neurons in the inner border of INL were identified as PV+ ACs, while those in the outer border of INL were identified as HCs. Goα+ cell number located at both the inner and outer borders of INL, only those in the outer border were identified as ON-BCs according to the location of BCs. 
Statistics
All data are shown as the mean ± SEM. The data distributions were tested for normality. Independent student's t-test was used to compare the results between NL and BL groups. Baseline biometric parameters recorded from the various groups were analyzed by 1-way ANOVA. Two-way ANOVA with Bonferroni correction was used to compare biometric results among (1) NL, NL+FD, BL, and BL+FD groups, (2) NL, NL+DMSO, NL+SCH, NL+FD, NL+FD+DMSO, and NL+FD+SCH groups, and (3) BL, BL+DMSO, BL+SCH, BL+FD, BL+FD+DMSO, and BL+FD+SCH groups. Results were considered to be significant if P less than 0.05 and highly significant if P less than 0.01 (SPSS Version 19.0; Chicago, IL, USA). 
Results
BL Induces Hyperopia In Healthy Animals and Suppresses Myopia Development in FD Animals
Baseline measurements of all biometric parameters were similar among different groups (P > 0.05, 1-way ANOVA) and between eyes of individual animals in the same group (P > 0.05, paired t-test). There were also no changes in any parameter between the healthy control and the fellow eye of all groups (P > 0.05, independent t-test) at any time. Thus, the interocular difference was used to assess biometric changes in the experimental eyes. 
Bright light significantly shifted refraction toward hyperopia compared with NL group after 4 weeks of illumination (P = 0.031, 2-way ANOVA; Table 2; Fig. 1A). However, other ocular biometric parameters including ACD, LT, VCD, and AL were not different between the NL and BL groups (P ≥ 0.708, 2-way ANOVA; Table 2; Figs. 1C, 1D). This disconnect between refraction changes and biometric parameter invariance may be due to the limited resolving power of OCT to identify biometric changes if the refractive error difference between BL and NL is small. In this case, the BL induced hyperopic change from the NL condition was only 3.59 D. Moreover, FD for 4 weeks induced myopia in both NL and BL groups (P < 0.001, 2-way ANOVA; Table 2; Fig. 1B). Bright light significantly suppressed FDM by approximately 46% (P < 0.001, 2-way ANOVA; Table 2; Fig. 1B). The changes in VCD and AL elongation in these two groups were consistent with those in refraction. Form deprivation induced significant elongation of VCD and AL in the NL environment (NL versus NL+FD: VCD, P = 0.045; AL, P = 0.014, 2-way ANOVA), but not in the BL group (BL versus BL+FD: VCD, P = 0.088; AL, P = 0.084, 2-way ANOVA; Table 2; Figs. 1C, 1D). 
Table 2
 
Biometric Results of Each Treatment Group
Table 2
 
Biometric Results of Each Treatment Group
Figure 1
 
Effects of BL and FD on ocular biometry. (A) Refraction. The refraction of BL group was significantly shifted toward hyperopia after 4 weeks. (B) Interocular difference in refraction. The amount of FDM was significantly suppressed by BL. (C) Interocular difference of VCD. (D) Interocular difference of AL. Form deprivation increased VCD and AL elongation in NL environment, but not in BL environment. *P < 0.05, **P < 0.01, ***P < 0.004, 2-way ANOVA.
Figure 1
 
Effects of BL and FD on ocular biometry. (A) Refraction. The refraction of BL group was significantly shifted toward hyperopia after 4 weeks. (B) Interocular difference in refraction. The amount of FDM was significantly suppressed by BL. (C) Interocular difference of VCD. (D) Interocular difference of AL. Form deprivation increased VCD and AL elongation in NL environment, but not in BL environment. *P < 0.05, **P < 0.01, ***P < 0.004, 2-way ANOVA.
Reversal of BL-Induced Inhibition of Myopia Development in FD Animals by D1R Antagonist SCH 39166
To evaluate the role of the D1R in mediating BL control of refraction, we examined the ability of SCH39166, a D1R antagonist, to reverse BL-induced hyperopia shift and inhibition of FDM. SCH39166 did not alter refraction in either NL or BL illumination in animals with normal vision (NL+DMSO versus NL+SCH, BL+DMSO versus BL+SCH, P ≥ 0.507, independent t-test; Table 2). However, in FD animals, SCH39166 enhanced myopia development in both groups (NL+FD+DMSO versus NL+FD+SCH, P = 0.026; BL+FD+DMSO versus BL+FD+SCH, P < 0.001, 2-way ANOVA; Table 2; Fig. 2A). In particular, SCH39166 completely blocked the protective effect of BL on FDM. In agreement with changes in refraction, SCH39166 also reversed the protective effects of BL on the elongation of VCD and AL induced by FD (BL+FD+DMSO versus BL+FD+SCH: VCD, P = 0.018; AL, P = 0.001, 2-way ANOVA; Table 2; Figs. 2B, 2C). 
Figure 2
 
Effects of SCH39166 on ocular biometry under NL and BL environments. SCH39166 enhanced FDM in NL, and completely blocked the protective effect of BL on FDM development. The elongation of VCD and AL were generally consistent with the refractive error increases. (A) Changes in interocular difference of refraction. (B) Changes in interocular difference of VCD. (C) Changes in interocular difference of AL. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA.
Figure 2
 
Effects of SCH39166 on ocular biometry under NL and BL environments. SCH39166 enhanced FDM in NL, and completely blocked the protective effect of BL on FDM development. The elongation of VCD and AL were generally consistent with the refractive error increases. (A) Changes in interocular difference of refraction. (B) Changes in interocular difference of VCD. (C) Changes in interocular difference of AL. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA.
BL Increases the Number of Activated Dopaminergic ACs
The effect of BL on DA synthesis was determined by identifying the cells expressing p-TH, an indicator of DA synthesis in the retina.39 Bright-light illumination for 2 days increased the number of p-TH+ cells compared with those exposed to NL (NL versus BL: 561 ± 12 vs. 605 ± 12, P = 0.017, independent t-test) suggesting that it stimulated DA synthesis (Fig. 3). 
Figure 3
 
The number of dopaminergic ACs (labeled by p-TH) in NL and BL. (A) A whole-mount retina stained with p-TH (green). (B) Bright light increased the number of activated retinal dopaminergic ACs. *P < 0.05, independent t-test.
Figure 3
 
The number of dopaminergic ACs (labeled by p-TH) in NL and BL. (A) A whole-mount retina stained with p-TH (green). (B) Bright light increased the number of activated retinal dopaminergic ACs. *P < 0.05, independent t-test.
Effects of Light Intensity on D1R Expression in Different Neuronal Types
Though D1R and c-fos were coexpressed in both INL and ganglion cell layer (GCL), their colocalization was mainly limited to the INL. Thus, we focused our analysis on the c-fos and D1R expression in INL in this part of the experiment. Irrespective of NL or BL exposure, INL D1R expression was detected in all tested neuronal cell types (Table 3; Fig. 4B). Using documented cellular biomarkers, we confirmed that approximately half of the BCs expressed D1R, which included approximately one-third of the ON-BCs and more than 90% of the recoverin+ OFF-BCs (Table 3; Figs. 4B, 4C). For ACs, approximately one-third of them expressed D1R, which included approximately two-thirds of the GABAergic ACs and almost all of the PV+ ACs. Almost all of the HCs expressed D1R as well (Table 3; Figs. 4B, 4C). There was no significant difference in the number of neuronal cell types (P ≥ 0.143, independent t-test) or number of D1R+ cells in each of these neuronal cell types (P ≥ 0.257, independent t-test) exposed to NL and BL (Table 3; Figs. 4B, 4C). 
Table 3
 
D1R and c-Fos Expression Patterns in Different Types of Retinal Neurons in NL and BL Environments
Table 3
 
D1R and c-Fos Expression Patterns in Different Types of Retinal Neurons in NL and BL Environments
Figure 4
 
c-Fos and D1R expression in different types of retinal neurons. (A) A schematic illustration for costaining of specific neuron type markers and c-fos in Drd1a-tdTomato mice. (B) The distribution of c-fos and D1R in different retinal neurons in NL and BL. (B1) The filled arrow indicates a cell colabeled with PAX6 (white), c-fos (green), and D1R (red); the empty arrow indicates a cell colabeled with PAX6 and c-fos; the oblique arrow indicates a cell colabeled with PAX6 and D1R. Labeling patterns in panels (B2B6) are identical to those in panel (B1). (B2) GAD67 staining. (B3) PV staining. The blue arrow indicates a HC, and the white arrows indicate PV+ ACs. (B4) CHX10 staining. (B5) GO αstaining. (B6) Recoverin staining. (C) The number (C1) and percentage (C2) of D1R+ cells in different types of retinal neurons in the INL. (D) The number (D1) and percentage (D2) of c-fos+ cells in different types of D1R+ retinal neurons in the INL. Bright light increased the c-fos expression in BCs, especially in the D1R+ ON-BCs. *P < 0.05, **P < 0.01, independent t-test.
Figure 4
 
c-Fos and D1R expression in different types of retinal neurons. (A) A schematic illustration for costaining of specific neuron type markers and c-fos in Drd1a-tdTomato mice. (B) The distribution of c-fos and D1R in different retinal neurons in NL and BL. (B1) The filled arrow indicates a cell colabeled with PAX6 (white), c-fos (green), and D1R (red); the empty arrow indicates a cell colabeled with PAX6 and c-fos; the oblique arrow indicates a cell colabeled with PAX6 and D1R. Labeling patterns in panels (B2B6) are identical to those in panel (B1). (B2) GAD67 staining. (B3) PV staining. The blue arrow indicates a HC, and the white arrows indicate PV+ ACs. (B4) CHX10 staining. (B5) GO αstaining. (B6) Recoverin staining. (C) The number (C1) and percentage (C2) of D1R+ cells in different types of retinal neurons in the INL. (D) The number (D1) and percentage (D2) of c-fos+ cells in different types of D1R+ retinal neurons in the INL. Bright light increased the c-fos expression in BCs, especially in the D1R+ ON-BCs. *P < 0.05, **P < 0.01, independent t-test.
D1R Activity in ACs is Insensitive to BL
Bright light altered the pattern of D1R activation based on changes in c-fos expression in specific cell types. For ACs, NL activated 14 ± 1% of pan-ACs (determined by expression of c-fos) and 27 ± 4% of D1R+ ACs (determined by coexpression of c-fos and D1R; Table 3). Bright light increased the percentage of activated ACs from 14 ± 1% to 19 ± 1% (P = 0.018, independent t-test); however, it did not alter the D1R activity in ACs (P = 0.232, independent t-test; Table 3; Fig. 4D). 
BL Selectively Activates/Recruits D1R Activity in Outer Retina, Including ON-BCs and HCs
First, in contrast to the NL environment, which mainly activated D1R of ACs in the inner retina (c-fos+ cells: 20.9 ± 0.6 in ACs vs. 6.0 ± 2.2 in BCs; Table 3), BL mainly activated D1R located in the outer retina BCs (c-fos+ cells: 26.6 ± 2.9 in BCs vs. 23.7 ± 0.4 in ACs; Table 3). For BCs, NL activated 19 ± 9% of pan-BCs and 7 ± 3% D1R+ BCs (Table 3), including 30 ± 12% of ON-BC and 21 ± 7% of D1R+ ON-BCs, as well as 12 ± 4% of recoverin+ OFF-BC and 12 ± 3% of D1R+ recoverin+ OFF-BCs (Table 3). Notably, BL almost doubled c-fos expression in BCs (NL versus BL: P[c-fos+CHX+/CHX+], P = 0.045, independent t-test; Table 3). In particular, BL increased c-fos expression in D1R+ BCs, approximately by 4-fold (NL versus BL: P[c-fos+D1R+CHX+/D1R+CHX+], P = 0.001, independent t-test; Table 3; Fig. 4D). However, BL did not alter the c-fos expression in ON-BCs (NL versus BL: P(c-fos+Goα+/Goα+), P = 0.067, independent t-test), but increased the c-fos expression in D1R+ ON-BCs (NL versus BL: P[c-fos+D1R+Goα+/D1R+Goα+], P = 0.008, independent t-test; Table 3; Fig. 4D), indicating that BL selectively activates ON-BCs containing D1R. Bright light also significantly increased the c-fos expression in recoverin+ OFF-BCs and HCs, which were mainly D1R+ (NL versus BL: P(c-fos+D1R+rec+/D1R+rec+), P = 0.041; Nc-fos+D1R+PV(HC)+, P = 0.016; P(c-fos+D1R+PV[HC]+/D1R+PV[HC]+), P = 0.056, independent t-test; Table 3; Fig. 4D). 
Discussion
The most important finding of this study is that BL attenuates FDM development (Fig. 1) via increases in the number of activated dopaminergic ACs and with activation of D1R in ON-BCs and HCs (Figs. 215524). Because D1R expression in different types of retinal neurons is diverse, it is technically challenging to define the specific D1R+ cell types that are activated by BL. To address this critical issue, we used mice coexpressing Drd1a-tdTomato and c-fos. In these mice, we pinpointed both D1R expression and activation.40,41 Bright light selectively activated the D1R+ cells in HCs and BCs, especially in D1R+ ON-BCs, and effectively inhibited FDM development, whereas BL-induced inhibition of FDM development and ocular elongation was completely reversed by the D1R antagonist, SCH39166. Thus, activation of the D1R (likely D1R in BCs of the ON pathway) contributes to the inhibitory effect of BL on FDM. 
D1R (At Least Partially) Mediates BL Inhibitory Effects on Refraction Development and FDM
Consistent with the previous findings of the effects of BL across multiple species,10,14,4244 Figure 1 shows that BL (6 hours/day) shifted refraction toward hyperopia, and inhibited FDM development by at least 46% with corresponding changes in VCD and AL. Bright light exposure for less than 2 hours/day had no effect on FDM in chicks, and was only effective when exposure times were longer than 5 hours/day, suggesting that the duration of BL exposure is an important factor in myopia control.45 Consistent with this notion, BL exposure for 6 hours/day is sufficient for FDM prevention in mice. On the other hand, some small refractive changes shown in Figures 1 and 2 were not always associated with any significant changes in VCD and AL. An OCT insensitivity to resolve small changes in VCD and AL may contribute to this disconnect between the refractive changes and the invariance of these two biometric measurements. Encouraging outdoor activity during class recess for 1 year does not seem to have such a prominent effect (approximately inhibited 0.06 D and 0.13 D/year) on myopia prevention in children.4,5 However, Stone et al.46 also found that natural daylight exposure had only a minimal and temporal inhibitory effect on FDM in chicks. It is noteworthy that the light intensity in this study was lower (1500–3300 lux) than in the other studies (10,000–40,000 lux), but similar to the intensity employed in the current study (i.e., 2500–5000 lux).9,15,42,45 This difference may be a critical factor because there are numerous reports indicating that laboratory light intensity and duration are in fact contributing factors because they do affect FDM progression in animal studies.6,8,3638 Our results agree other reports indicating that the intensity and duration of light exposure are contributing factors to FDM in mice. 
In this study, D1R activation is involved in eliciting suppression of FDM development by BL because the D1R antagonist, SCH39166, completely reversed the protective effect of BL on FDM development. On the other hand, the involvement of the cell type expressing D2R that is activated by BL has not yet been addressed in this study. Such an effect is tenable because a critical role for the D2R was described in contributing to the BL inhibitory effect on myopia development.47 For example, intraocular injection of the D2R agonist quinpirole during a 3-hour dark period was sufficient to restore a light inhibitory effect on FDM,25 whereas the D2R antagonist spiperone prevented the ocular growth inhibitory effect resulting from a brief period of normal unobstructed vision.24 As was done to delineate the retinal cell type on which D1R activation by BL suppresses FDM, we are undertaking future studies to delineate the cell types on which D2R modulation by light in transgenic mouse strains. 
BL Selectively Activates/(Recruits) D1R Signaling in the ON Pathway
The distribution of D1R in BCs, HCs, and ACs in Drd1a-tdTomato mice is consistent with a recent study using the same Drd1a-tdTomato mice.48,49 c-Fos is a general activity marker and a downstream target of D1R activation.40,41 Thus, c-fos expression levels in these cell types provide readout of D1R activity. Indeed, c-fos expression has been shown to be activated by light in both excitable cells such as NOS+ AC and nonexcitable cholinergic ACs in rabbit cells.50 Bright light activates c-fos in specific D1R-containing cells in tdTomato+ cells. We found that more than half of the c-fos+ cells expressing D1R are largely localized in the inner layer of INL. This is consistent with the previous finding that flickering light (300 lux, equivalent intensity to NL in our study) induced c-fos expression in INL, which was mimicked by a D1R agonist SKF38393.51 Notably, the frequency of light stimulation seems to activate different retinal cells. With constant light stimulation, c-fos is mainly activated in the inner layer of INL, while flickering light stimulation (2 Hz) activates c-fos in the outer layer of the INL.52 Similarly, the D1R activation patterns induced by NL and BL were different from one another in the INL. In NL (100–200 lux), D1R was mainly activated on ACs in the inner border of INL, while D1R was activated by BL on BCs and HCs in the outer border of INL. Differential activation of D1R in the inner and outer borders of INL may be due to different levels of DA released by activation of dopaminergic ACs in bright- or flickering-light environment. Interestingly, we showed that BL increased the number of activated dopaminergic ACs (Np-TH+), indicating that BL elevated both DA synthesis and most likely retinal DA levels. These effects are consistent with reported light-stimulated DA release.53 Because dopaminergic ACs receive inputs mainly from ON-BCs,54 these findings lead us to propose a working hypothesis for a positive feed-forward mechanism underlying BL-induced DA release in the retina: BL activates D1R+ ON-BCs, which synapse with and activate dopaminergic ACs, leading to higher levels of DA release and further activation of D1Rs on ON-BCs, which in turn further triggers DA release. Thus, BL-induced release of DA may be further stimulated by the activation of ON-BCs via D1R until a new homeostatic state of retinal neural activity and DA level is achieved during BL exposure. Because not all ON-BCs synapse with dopaminergic AC, future studies are still needed to identify specific types of ON-BCs that synapse with dopaminergic ACs. Such insight will provide a cellular basis for this postulated feed-forward mechanism for DA release by light stimulation. 
The selective activation of D1R in the outer border of INL by BL indicates that BL may enhance the signaling transmission in the outer retina via the D1R pathway. Previous studies indicate that DA and a D1R agonist, ADTN, potentiate the voltage-dependent calcium current in presynaptic terminals of both ON- and OFF-BCs in fish retina.55 Dopamine modulates the electrical coupling between adjacent HCs, which in turn enhances the spatial retinal contrast sensitivity.56 Thus, the activation of D1R in BCs and HCs during BL exposure may result in improving visual quality by enhancing spatial retinal contrast sensitivity and promoting signaling between BCs and retinal ganglion cells (RGCs), especially those in the ON signaling pathway. This enhancement of ON pathway signaling and spatial contrast sensitivity may play a critical role in eliciting the BL preventive effect on FDM development as they compensate for declines in retinal signaling induced by FD. Previous studies reported that the ON pathway mutation in mGluR6−/− and nob mice is associated with myopic shift in refractive development, and leads to an increased susceptibility to FD.34,35 As recoverin+ OFF-BCs constitute a part of the OFF-BCs, further investigation is needed to determine if changes in the OFF pathway also contribute to myopia development. 
In summary, both biological and biometric evidence suggests that BL prevents the development of myopia via the D1R signaling pathway because blocking D1R activity effectively eliminated the protective effect of BL on FDM. Furthermore, BL stimulated the synthesis of DA and selectively activated ON-BCs expressing D1R, indicating that D1R activation in the ON-pathway contributes to myopia suppression. 
Acknowledgments
Supported by grants from the National Natural Science Foundation of China (Grants 81400411 and 81371047; Beijing, China), Natural Science Foundation of Zhejiang Province (Grants LZ14H120001 and LQ12H12001; Hangzhou, China), the National Science Foundation for Excellent Young Scholars of China (Grant 81422007; Beijing, China), The Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents (Hangzhou, China), and The National Young Excellent Talents Support Program (Beijing, China). 
Disclosure: S. Chen, None; Z. Zhi, None; Q. Ruan, None; Q. Liu, None; F. Li, None; F. Wan, None; P.S. Reinach, None; J. Chen, None; J. Qu, None; X. Zhou, None 
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Figure 1
 
Effects of BL and FD on ocular biometry. (A) Refraction. The refraction of BL group was significantly shifted toward hyperopia after 4 weeks. (B) Interocular difference in refraction. The amount of FDM was significantly suppressed by BL. (C) Interocular difference of VCD. (D) Interocular difference of AL. Form deprivation increased VCD and AL elongation in NL environment, but not in BL environment. *P < 0.05, **P < 0.01, ***P < 0.004, 2-way ANOVA.
Figure 1
 
Effects of BL and FD on ocular biometry. (A) Refraction. The refraction of BL group was significantly shifted toward hyperopia after 4 weeks. (B) Interocular difference in refraction. The amount of FDM was significantly suppressed by BL. (C) Interocular difference of VCD. (D) Interocular difference of AL. Form deprivation increased VCD and AL elongation in NL environment, but not in BL environment. *P < 0.05, **P < 0.01, ***P < 0.004, 2-way ANOVA.
Figure 2
 
Effects of SCH39166 on ocular biometry under NL and BL environments. SCH39166 enhanced FDM in NL, and completely blocked the protective effect of BL on FDM development. The elongation of VCD and AL were generally consistent with the refractive error increases. (A) Changes in interocular difference of refraction. (B) Changes in interocular difference of VCD. (C) Changes in interocular difference of AL. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA.
Figure 2
 
Effects of SCH39166 on ocular biometry under NL and BL environments. SCH39166 enhanced FDM in NL, and completely blocked the protective effect of BL on FDM development. The elongation of VCD and AL were generally consistent with the refractive error increases. (A) Changes in interocular difference of refraction. (B) Changes in interocular difference of VCD. (C) Changes in interocular difference of AL. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA.
Figure 3
 
The number of dopaminergic ACs (labeled by p-TH) in NL and BL. (A) A whole-mount retina stained with p-TH (green). (B) Bright light increased the number of activated retinal dopaminergic ACs. *P < 0.05, independent t-test.
Figure 3
 
The number of dopaminergic ACs (labeled by p-TH) in NL and BL. (A) A whole-mount retina stained with p-TH (green). (B) Bright light increased the number of activated retinal dopaminergic ACs. *P < 0.05, independent t-test.
Figure 4
 
c-Fos and D1R expression in different types of retinal neurons. (A) A schematic illustration for costaining of specific neuron type markers and c-fos in Drd1a-tdTomato mice. (B) The distribution of c-fos and D1R in different retinal neurons in NL and BL. (B1) The filled arrow indicates a cell colabeled with PAX6 (white), c-fos (green), and D1R (red); the empty arrow indicates a cell colabeled with PAX6 and c-fos; the oblique arrow indicates a cell colabeled with PAX6 and D1R. Labeling patterns in panels (B2B6) are identical to those in panel (B1). (B2) GAD67 staining. (B3) PV staining. The blue arrow indicates a HC, and the white arrows indicate PV+ ACs. (B4) CHX10 staining. (B5) GO αstaining. (B6) Recoverin staining. (C) The number (C1) and percentage (C2) of D1R+ cells in different types of retinal neurons in the INL. (D) The number (D1) and percentage (D2) of c-fos+ cells in different types of D1R+ retinal neurons in the INL. Bright light increased the c-fos expression in BCs, especially in the D1R+ ON-BCs. *P < 0.05, **P < 0.01, independent t-test.
Figure 4
 
c-Fos and D1R expression in different types of retinal neurons. (A) A schematic illustration for costaining of specific neuron type markers and c-fos in Drd1a-tdTomato mice. (B) The distribution of c-fos and D1R in different retinal neurons in NL and BL. (B1) The filled arrow indicates a cell colabeled with PAX6 (white), c-fos (green), and D1R (red); the empty arrow indicates a cell colabeled with PAX6 and c-fos; the oblique arrow indicates a cell colabeled with PAX6 and D1R. Labeling patterns in panels (B2B6) are identical to those in panel (B1). (B2) GAD67 staining. (B3) PV staining. The blue arrow indicates a HC, and the white arrows indicate PV+ ACs. (B4) CHX10 staining. (B5) GO αstaining. (B6) Recoverin staining. (C) The number (C1) and percentage (C2) of D1R+ cells in different types of retinal neurons in the INL. (D) The number (D1) and percentage (D2) of c-fos+ cells in different types of D1R+ retinal neurons in the INL. Bright light increased the c-fos expression in BCs, especially in the D1R+ ON-BCs. *P < 0.05, **P < 0.01, independent t-test.
Table 1
 
Primary Antibody Details
Table 1
 
Primary Antibody Details
Table 2
 
Biometric Results of Each Treatment Group
Table 2
 
Biometric Results of Each Treatment Group
Table 3
 
D1R and c-Fos Expression Patterns in Different Types of Retinal Neurons in NL and BL Environments
Table 3
 
D1R and c-Fos Expression Patterns in Different Types of Retinal Neurons in NL and BL Environments
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