February 2017
Volume 58, Issue 2
Open Access
Retina  |   February 2017
Alterations of Glutamate and γ-Aminobutyric Acid Expressions in Normal and Myopic Eye Development in Guinea Pigs
Author Affiliations & Notes
  • Li Guoping
    Shandong University of Traditional Chinese Medicine, Jinan, China
  • Ye Xiang
    Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases; Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases in Universities of Shandong; Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan, China
  • Wu Jianfeng
    Shandong University of Traditional Chinese Medicine, Jinan, China
  • Guo Dadong
    Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases; Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases in Universities of Shandong; Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan, China
  • Huang Jie
    Shandong University of Traditional Chinese Medicine, Jinan, China
  • Jiang Wenjun
    Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases; Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases in Universities of Shandong; Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan, China
  • Guo Junguo
    Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases; Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases in Universities of Shandong; Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan, China
  • Bi Hongsheng
    Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases; Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases in Universities of Shandong; Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan, China
    Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China
  • Correspondence: Bi Hongsheng, Eye Institute of Shandong University of Traditional Chinese Medicine, No. 48#, Yingxiongshan Road, Jinan 250002, China; hongshengbibi1@163.com
Investigative Ophthalmology & Visual Science February 2017, Vol.58, 1256-1265. doi:10.1167/iovs.16-21130
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Li Guoping, Ye Xiang, Wu Jianfeng, Guo Dadong, Huang Jie, Jiang Wenjun, Guo Junguo, Bi Hongsheng; Alterations of Glutamate and γ-Aminobutyric Acid Expressions in Normal and Myopic Eye Development in Guinea Pigs. Invest. Ophthalmol. Vis. Sci. 2017;58(2):1256-1265. doi: 10.1167/iovs.16-21130.

      Download citation file:


      © 2017 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

Purpose: The retina has an important role in the signal transmission related to eye development and myopia. Glutamate (Glu) and γ-aminobutyric acid (GABA) are the major excitatory and inhibitory neurotransmitters in the retina, which can affect the development of both the eye and myopia. Nevertheless, change in the balance between the excitatory and inhibitory neurotransmitters still is unclear during development of eyes and myopia. The purpose of this study was to explore the alterations of the ratio of Glu to GABA (RGG), which mediates the balance between the excitatory and inhibitory neurotransmitters in retina in the development of the eyes and lens-induced myopia (LIM) in a guinea pig model.

Method: An LIM guinea pig model was established using a −10 diopter (D) negative lens. The levels of Glu, GABA, and the dynamic change of RGG were measured in the retina in normal and myopia guinea pigs at four time points (i.e., 0, 2, 4, and 6 weeks after onset of LIM). Considering that Glu and GABA are related closely to the occurrence of myopia, we further studied the changes of RGG in the retina in LIM guinea pigs.

Result: Our results showed that the RGG was upregulated and was well correlated with diopter and axial length than either Glu or GABA during the development of normal eyes. Besides, we observed that the content of the RGG in the retina in myopia eyes was higher than that of Glu and GABA in normal subjects and was an obviously positive correlation.

Conclusions: Taken together, our findings suggest that the RGG has a pivotal role in eye development and myopia. The abnormal retina signal induced by the unbalanced ratio between Glu and GABA is related closely to the occurrence of myopia.

Glutamate (Glu), a major excitatory neurotransmitter, not only affects the transmission of visual information in the brain and retina, but also impacts the development of the retina and the whole eyes.14 Meanwhile, it has been demonstrated that a synthetic Glu receptor also could induce myopia.5 Similarly, γ-aminobutyric acid (GABA), a traditional inhibitory transmitter, has a crucial role in the nerve signaling transmission and development of retinal and entire eyes,68 and is correlated with the occurrence and development of myopia.9,10 Studies have confirmed that in retinal signal circuits, photoreceptors and bipolar cells release Glu, which is responsible for the radial flow of the visual signal in the retina and mediates feed forward, whereas horizontal cells receive direct input from photoreceptors and, in turn, provide negative feedback to cone photoreceptors.11,12 Meanwhile, horizontal cells and amacrine cells participate in the retinal circuits through the lateral pathway, which is mediated by GABA.1214 By means of these kinds of neural circuits, the processed signals are transmitted to the lateral geniculate nucleus (LGN) and to the visual cortex finally. Besides processing neural signals, excitatory and inhibitory signals affect synapse maturation and neuronal plasticity, and further impact the development of the eyes.15 In addition, cooperation between early GABAergic inputs and Glu receptor could mediate the development of retinal signal circuits.16 If the balance between them was disturbed, it would cause a series of pathologic changes including retinal dysplasia.17 Therefore, the balance between excitatory and inhibitory signals from the retina is very important for the development of the eyes. 
Both Glu and GABA have an important role in the development of eyes. Nevertheless, it still is unclear how Glu, GABA, as well as the balance between excitatory and inhibitory transmitters vary in normal eye development, and the role of the balance between Glu and GABA in the myopia development still is unknown. To study the alteration of excitatory and inhibitory molecules in normal and myopic eye development, we selected the representative molecules of excitatory and inhibitory neurotransmitters, that is Glu and GABA, to investigate the levels of Glu and GABA in normal and lens-induced myopic (LIM) guinea pigs at several developmental time points. Our results facilitated the understanding of the role of the balance between excitatory and inhibitory neurotransmitters in the development of eyes and myopia. 
Methods
Animals
Three-week-old, pigmented guinea pigs (Cavia procellus) were obtained from the Laboratory Animal Co., LTD of Henan Kangda (Henan, China). All animals were raised with food and water ad libitum and the indoor temperature was maintained at 25°C. An average illumination in the cage was approximately 275 Lux under a 12-hour light/dark cycle with lights on at 8 AM and off at 8 PM. Four or five guinea pigs were raised together in a transparent plastic cage (20 × 30 × 35 cm). Animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee of the Shandong University of Traditional Chinese Medicine and adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmologic and Vision Research. 
The changes in the ratio of Glu to GABA are most important during emmetropization and myopic development. The present study included the investigations of the changes of retinal GABA and Glu. Guinea pigs were divided randomly into seven groups: 0-week normal group, 2-week normal group and LIM group, 4-week normal group and LIM group, and 6-week normal group and LIM group. Each group included 8 guinea pigs. 
The concave lenses were purchased from Shanghai Million New Optical Glasses Co., LTD (Shanghai, China), and were fabricated by polymethyl methacrylate (PMMA) with 11 mm diameters and large optic zones (10–11 mm). To induce myopia in guinea pigs, −10 diopter (D) lenses were mounted onto a self-made frame using surgical tapes and were glued onto the right eyes of the guinea pigs.10 To ensure the effectiveness of LIM in guinea pigs, lenses were cleaned in the morning and in the evening twice daily. 
Retinoscopy and Ultrasonography
First, 1% cyclopentolate hydrochloride (Alcon, Ft. Worth, TX, USA) was applied to reach a completely dilated pupil and cycloplegia. The retinoscopy for all animals was performed with a streak retinoscope (YZ24; 66 Vision Tech. Co., Ltd, China) and trial lenses in a dark room. The operation was made by the same optometrist at a distance of 20 cm. The mean value of the refractive errors was defined as the refraction of the eyes, which was along the vertical and horizontal meridians of three repeated measurements.18,19 
A-scan ultrasonography (Cinescan; Quantel Medical, Coumon-d'Auvergne, France) was used to measure the total axial length (defined as the distance from the corneal surface to the RPE).20 The ultrasonic probe emission frequency was 11 MHz.18,21 The conducting velocities of 1557.5, 1723.3, and 1540 m/s were used in the anterior chamber, lens, and the vitreous chamber, respectively.2224 Oxybuprocaine hydrochloride (Santen Pharmaceutical, Osaka, Japan), a topical anesthesia, was administered before the measurement of the eye axial length. During the measurement of axial length, the probe was focused as near as possible on the center of the cornea, and perpendicular to the plane of the cornea. The axial length was obtained from the mean value of 10 repeated measurements to minimize the effect of an obvious outlier value. 
High Performance Liquid Chromatography (HPLC)
An HPLC system with Chromeleon software (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the content of retinal Glu and GABA. Before the experiment, all animals were anesthetized with intraperitoneal injections of 5% choral hydrate (6 mL/kg), and then the eyes were enucleated immediately and were put on a Petri dish filled with iced saline. The anterior segment of the eyeball then was removed from the corneoscleral limbus. After isolation and weighing of the retina, samples were stored in Eppendorf tubes and then put in liquid nitrogen immediately. For HPLC determination, every frozen retina was ground with pure water according to the proportion of 1:10 (mg: μL), and then was centrifuged at 4°C at 12,000g for 30 minutes, followed by filtration of the supernatant through a Millipore filter with 0.22 μm pore size. To efficiently determine the components, the samples were subjected to derivatization, according to the following procedures: 50 μL phenylisothiocyanate (PITC; Sigma-Aldrich Corp., St. Louis, MO, USA) and 50 μL triethylamine (Sigma-Aldrich Corp.) were incubated with the filtered supernatant (100 μL) at room temperature for 1 hour, then 300 μL n-hexane (Sigma-Aldrich Corp.) was added in the above mixture and blended for 15 minutes at room temperature, followed by centrifugation at 12,000g for 15 minutes at 4°C. All derivative samples were analyzed using an HPLC–UV spectrophotometer at 254 nm (Ultimate 3000 UPLC; Dionex Softron, Germering, Germany) with a reversed-phase column (ODS, 3 μm, 4.6 × 150 mm, Atlantis dC18; Waters Corporation, Milford, MA, USA). The mobile phase A (pH = 6.4) was composed of a mixture of 93% sodium acetate (0.1 M, pH = 6.4) and 7% acetonitrile. The mobile phase B was composed of methanol, acetonitrile, and ultrapure water (1:3:1, vol/vol/vol). The mobile phases were filtered with a 0.22 μm nylon filter and degassed under ultrasound for 30 minutes. The column was eluted with a mobile phase as follows: 0 minutes, 100% A; 6 minutes, 94% A; 15 minutes, 91% A; 19 minutes, 55% A; 26 minutes, 0% A, and 30 minutes, 100%A. The column temperature was set to 35°C, and the flow rate was 0.8 ml/minutes. In the meantime, standard solutions (composition of either GABA or Glu) were calculated wisth five different concentrations (39.9, 79.8, 159.5, 319, and 638 μg/ml for GABA, and 38.3, 76.6, 153.25, 306.5, and 613 μg/ml for Glu, respectively) using the same procedure. The contents of GABA and Glu in the retina were calculated by the formula: content of GABA or Glu (μmol/g) = content of sample (μg/ml) × volume of sample (ml)/weight of the retina detected (g)/molecular mass of GABA (Mr = 103.1206) or Glu (Mr = 147.13076). 
Statistical Analysis
Statistical analysis was performed using SPSS software (Version 17.0; SPSS, Inc., Chicago, IL, USA). The results were expressed with mean ± SEM. All values for the lens-induced eyes were statistically compared to those of the fellow eyes within the same group using a paired sample t-test. The mean interocular difference used an independent sample t-test between the LIM and normal groups. Statistical analysis among groups was performed by 1-way ANOVA, and statistical significance was considered when P < 0.05. 
Results
Changes in Refractive Error and Axial Length
Consistent with previous studies (see Table),10,11 the axial length was elongated (P > 0.05, 1-way ANOVA) and the diopter was decreased (P > 0.05, 1-way ANOVA) during normal eye development. By contrast, the axial length and the diopter showed a more obvious change during the myopic development (both P < 0.001, 1-way ANOVA). 
Table
 
Alterations of the Axial Length and Refraction in Lens-Induced Myopia Guinea Pigs
Table
 
Alterations of the Axial Length and Refraction in Lens-Induced Myopia Guinea Pigs
Variations of Glutamate in the Retina During Eye Development
Using the HPLC technique, we determined the Glu content in the retina during normal and myopic eye development. We observed that the content of Glu in the retina was increased by 160.8% at the end of the 4-week treatment period in normal eye development (P > 0.05, post hoc test) compared to that at week 0, whereas it decreased by 59.4% at the end of the 6-week treatment period compared to that at 4 weeks, and the content at 6 weeks showed no apparent difference compared to that at week 0 (P > 0.05, ANOVA, post hoc test). Meanwhile, the change of Glu level showed a consistency in binocular retina (P > 0.05, 1-way ANOVA; Fig. 1A). During myopic eye development, the Glu level in the retina showed a similar tendency, but showed a greater increase (P < 0.01, 1-way ANOVA) at the end of the 4-week treatment period. Compared to that at week 0, the Glu content in LIM eyes was increased by 481.7% at the end of the 4-week treatment period (P < 0.001, post hoc test). By contrast, the Glu level of the myopic eye was decreased by 64.9% at the end of 6 weeks of treatment (P < 0.01, post hoc test) compared to that at 4 weeks, whereas the decreased level of Glu was not different from that in normal eyes (P > 0.05, post hoc test). Though there was a similar tendency, the level of Glu in LIM eyes was higher than that in the fellow eyes at every time point (1.352 ± 0.062 vs. 0.628 ± 0.06 μmol/g at 2 weeks, P = 0.016; 2.036 ± 0.45 vs. 0.887 ± 0.45 μmol/g at 4 weeks, P = 0.0015; 0.714 ± 0.055 vs. 0.360 ± 0.051 μmol/g at 6 weeks, P = 0.023. paired sample t-test, Fig. 1C). 
Figure 1
 
Expressions of Glu and GABA in retina in normal and LIM guinea pigs. High performance liquid chromatography analysis was performed to measure the levels of Glu and GABA in retina in normal (A, B) and LIM (C, D) guinea pigs (n = 8 per group). *P < 0.05, **P < 0.01 compared to fellow eyes. LIM eye, lens-induced myopia eye; LIM Fellow eye, lens-induced myopia fellow eye.
Figure 1
 
Expressions of Glu and GABA in retina in normal and LIM guinea pigs. High performance liquid chromatography analysis was performed to measure the levels of Glu and GABA in retina in normal (A, B) and LIM (C, D) guinea pigs (n = 8 per group). *P < 0.05, **P < 0.01 compared to fellow eyes. LIM eye, lens-induced myopia eye; LIM Fellow eye, lens-induced myopia fellow eye.
To investigate the correlation between the level of Glu and the diopter and axial length, we further performed a correlation analysis to clarify this issue. From the 0- to 4-week time points, we found that the level of Glu was correlated negatively with the diopter and was correlated positively with the axial length in LIM eyes (R = −0.884, P < 0.001, Fig. 2E; R = 0.940, P < 0.001, Fig. 2G, respectively; 1-way ANOVA) and in normal eyes (R = −0.695, P = 0.002, Fig. 2A; R = 0.857, P < 0.001, Fig. 2C; respectively; 1-way ANOVA). Nevertheless, the Glu levels from the 4- to 6-week time points were correlated positively with the diopter and was correlated negatively with the axial length (R = 0.717, P = 0.006, Fig. 2F; R = −0.868, P < 0.001, Fig. 2H; respectively; 1-way ANOVA) in LIM eyes and in normal eyes (R = 0.646, P = 0.043, Fig. 2B; R = −0.802, P = 0.005, Fig. 2D, respectively; 1-way ANOVA). 
Figure 2
 
Regression analyses between glutamate content and diopter/axial length in LIM guinea pigs. The correlation between Glu content and diopter in LIM guinea pigs was evaluated (n = 8 per group). The result indicated that there was a correlation between the levels of Glu content and axial length.
Figure 2
 
Regression analyses between glutamate content and diopter/axial length in LIM guinea pigs. The correlation between Glu content and diopter in LIM guinea pigs was evaluated (n = 8 per group). The result indicated that there was a correlation between the levels of Glu content and axial length.
Alteration of GABA in Retina During Eye Development
Our results showed that Glu, a main excitatory neurotransmitter, varied in eye development. Due to the importance of the balance between excitation and inhibition to normal retinal function, its change will affect normal and myopic eye development. Thus, we explored the alteration of GABA in the retina by HPLC. During normal eye development, the level of GABA in the retina was increased by 21.8% at the end of the first 4 weeks in normal eye development compared to that at week 0 (P > 0.05, post hoc test), and was decreased by 17.1% at the end of 6 weeks than that at the end of 4 weeks (P > 0.05, post hoc test). Similarly, the change of GABA level showed consistency in binocular retina (P > 0.05, 1-way ANOVA, Fig. 1B). Meanwhile, the GABA level in the retina showed a similar consistency during myopic eye development, and exhibited more extended tendency (P < 0.001, 1-way ANOVA) in the first 4-week period. Compared to the level at week 0, the GABA content in LIM eyes was increased by 76.8% at the end of 4 weeks of treatment (P < 0.001, post hoc test). By contrast, the GABA content in the myopic eye was decreased by 25.4% at the 6-week time point (P < 0.01, post hoc test) compared to that at 4 weeks, and the decreased tendency differed in normal eyes (P > 0.05, post hoc test). Despite the same change tendency, the level of GABA in LIM eyes was higher than that in fellow eyes at every time point (21.45 ± 0.56 vs. 16.80 ± 0.43 μmol/g, P = 0.014 at 2 weeks; 26.34 ± 0.52 vs. 18.63 ± 0.45 μmol/g, P = 0.0021 at 4 weeks; 19.650 ± 0.47 vs. 15.450 ± 0.39 μmol/g, P = 0.019 at 6 weeks, paired sample t-test, Fig. 1D). 
Given that the expression of the Glu was correlated with diopter and axial length, is there also a certain correlation between the changes of GABA and either diopter or axial length? Based on this consideration, we made a relevant correlation analysis. From 0 to 4 weeks, the content of GABA was weakly negatively correlated with the diopter and was moderately positively correlated with the axial length (R = −0.5, P = 0.044, Fig. 3A; R = 0.785, P < 0.001, Fig. 3C; respectively, 1-way ANOVA) in normal eyes; These correlations were obvious in LIM eyes (R = −0.876, P < 0.001, Fig. 3E; R = 0.940, P < 0.001, Fig. 3G; respectively, 1-way ANOVA). Compared to the level at week 0, the contents of the GABA were correlated positively with the diopter and correlated negatively with the axial length from 4 to 6 weeks (R = 0.715, P = 0.006, Fig. 3F; R = −0.881, P < 0.001, Fig. 3H; respectively, 1-way ANOVA) in LIM eyes; these correlations also were weak in normal eyes (R = 0.474, P = 0.041, Fig. 3B; R = −0.779, P = 0.008, Fig. 3D; respectively, 1-way ANOVA). 
Figure 3
 
Regression analyses between GABA content and diopter/axial length in LIM guinea pigs. The correlation between GABA content and diopter in LIM guinea pigs was evaluated (n = 8 per group). The result indicated that there was a correlation between the levels of Glu content and axial length.
Figure 3
 
Regression analyses between GABA content and diopter/axial length in LIM guinea pigs. The correlation between GABA content and diopter in LIM guinea pigs was evaluated (n = 8 per group). The result indicated that there was a correlation between the levels of Glu content and axial length.
Correlation Among the Ratio of Glu to GABA (RGG) and Diopter and Axial Length
Previous studies have suggested that the neural network information transfer process is dependent on the balance between excitation and inhibition.15,16,25 Correct transfer of the neural signal is vital to retinal and visual development. Therefore, taking the RGG as a simplistic mode of balance between excitation and inhibition. We further explored whether this balance varies during normal and myopic eye development. We found that during normal eye development, the RGG between two eyes had no apparent difference (all P > 0.05, paired sample t-test, Fig. 4A), and RGG showed a tendency similar to that of either Glu or GABA. The RGG was increased by 231.8% from 0 to 4 weeks (P > 0.05, post hoc test) and was decreased by 76.6% from 4 to 6 weeks (P > 0.05, post hoc test), and these results were similar to that at week 0 for RGG (P > 0.05, post hoc test). Similar to the tendency of Glu and GABA in LIM eye development, the RGG of LIM eye also showed an extended variance. From 0 to 4 weeks, the RGG in LIM eye was increased by 243.7% (P < 0.005, post hoc test), and was decreased by 62.7% from 4 to 6 weeks (P < 0.01, post hoc test), and there was no difference compared to that at week 0 (P > 0.05, post hoc test). In comparison with the fellow eyes, the change of RGG in LIM eyes was extended (P = 0.014 at 2 weeks; P = 0.008 at 4 weeks, paired sample t-test, Fig. 4B). 
Figure 4
 
Ratio of Glu to GABA content in retina in LIM guinea pigs at different time-points. The results indicated that the RGG was elevated in normal (control) and LIM guinea pigs (n = 8 per group), and LIM enhances the RGG. *P < 0.05, **P < 0.01.
Figure 4
 
Ratio of Glu to GABA content in retina in LIM guinea pigs at different time-points. The results indicated that the RGG was elevated in normal (control) and LIM guinea pigs (n = 8 per group), and LIM enhances the RGG. *P < 0.05, **P < 0.01.
Moreover, we also examined the correlation between the RGG and either the diopter or the axial length. Figure 5 shows the scatter plots between the RGG and either diopter or axial length; this result indicated that there was a correlation between the RGG and diopter and axial length. The RGG at 0 to 4 or 4 to 6 weeks in LIM guinea pigs also was highly correlated to the diopter and axial length (R = −0.912, P < 0.001 Fig. 5E; R = 0.923, P < 0.001, Fig. 5G; R = 0.719, P = 0.006, Fig. 5F; R = −0.790, P = 0.001, Fig. 5H; respectively, 1-way ANOVA), but poorly correlated with those in normal groups (R = −0.715, P = 0.001, Fig. 5A; R = 0.835, P < 0.001, Fig. 5C; R = 0.663, P = 0.037, Fig. 5B; R = −0.787, P = 0.007, Fig. 5D, respectively, 1-way ANOVA). 
Figure 5
 
Correlation between the RGG content and diopter/axial length in LIM guinea pigs. The result indicated that there was a correlation between the RGG and either diopter or axial length.
Figure 5
 
Correlation between the RGG content and diopter/axial length in LIM guinea pigs. The result indicated that there was a correlation between the RGG and either diopter or axial length.
Discussion
In the present study, we investigated the alterations of the level of retinal Glu and GABA during normal and myopic eye development, and further explored the underlying correlations between Glu and GABA levels, the RGG and either diopter or axial length. We noted that during normal eye development, both of the Glu and GABA levels showed an inconsistent style; they were increased from 0 to 4 weeks, and further decreased to the normal level at the 6-week time point. Meanwhile, the levels of Glu, GABA, and the RGG showed a significant correlation with the diopter and with the axial length during normal eye development. However, in comparison with normal eye development, both of the levels of Glu and GABA in LIM eyes exhibited various changes, and the levels of Glu, GABA, and the RGG showed a more significant correlation with the diopter and with the axial length during LIM eye development. 
Neurotransmitter and Visual Development
Currently, a large number of studies have revealed the role of various neurotransmitters in normal refractive development of the eye, such as dopamine (DA),26 acetylcholine,27 nitric oxide,28,29 and retinoic acid.18,30 Glu and GABA, the major excitatory and inhibitory neurotransmitters in retina and other parts of the central nervous system,31,32 also were closely related to the neural development, which is associated with the visual system in the rabbit, chick, and rat eye.3335 On one hand, studies found that the Glu and its receptor were associated with the excitatory synaptic development and the circulation of the retinotectal system,1,3 and the excitotoxicity induced by Glu could lead to the abnormal development of the retina and the occurrence of diseases, such as retinal ischemia, neuronal necrosis, and glaucoma.3638 On the other hand, studies also found that GABA would affect the growth and development of retinal neurons in culture, and its agonist baclofen may stimulate the visual system developing of Xenopus laevis and the retinal ganglion cell axons extent.39,40 These studies suggest that Glu and GABA are associated with eye development. Our study showed that retinal Glu and GABA had significant correlations with the diopter and axial length. Therefore, it seems reasonable to conclude that the expressions of retinal Glu and GABA have an important role in eye development. 
Compared to other organs, growth of the eye is regulated by homeostatic control mechanisms, including the balance of the excitation and inhibition.41,42 Moreover, it is well known that the balance between the excitatory and inhibitory neurotransmitters is mainly mediated by Glu and GABA and the dynamic interplay could enrich visual processing by enhancing retinal ability to respond to a rapidly changing visual environment.43 Therefore, the RGG usually is chosen to represent the dynamic interplay between the excitatory and inhibitory neurotransmitters under different experimental conditions.14,44 Following consideration of the important role of the retina in the regulation of information transmission related to eye development, we selected RGG of the retina as a target to study its possible underlying role in normal and myopic eye development. 
Previous studies found that during early neural development the regulation of the glutamatergic and GABAergic activity was relatively weak, and both increased as development progressed.42,45 In addition, the balance of excitatory and inhibitory synaptic inputs can be homeostatically regulated in an activity-dependent manner.42,46 In the present study, the RGG was increased during normal and myopic eye development, and showed a better correlation with diopter and axial length. One possible explanation for this is that the GABA content is regulated to match the level of Glu or in contrast. No matter what kind of adjusting method, the ultimate goal is to maintain the balance of the RGG and to adjust the normal visual development. Although previous studies indicate that early depolarizing GABAergic transmission might be crucial for the coordination of subsequent levels of excitatory and inhibitory inputs,32 the specific regulation mechanism of the RGG on visual development still needs to be explored further when taking into account the selection of different parts of the study. 
Relationship Between the RGG and Myopia
The occurrence and the potential regulation mechanism in myopia has been a subject of interest for scientists in the present.47 Previous studies have suggested that neurotransmitters, such as dopamine,48 GABA,10 and acetylcholine,49 may have a pivotal role in myopic development. Specially, the expression of GABA in retina was upregulated in myopia.10,50 In addition, it has been shown that GABAergic antagonists enhance the protective effect of brief periods of normal vision on the development of form deprivation myopia (FDM), whereas agonists inhibit the protective effects.9 Similarly, our study demonstrated a significant correlation between a rising of GABA content and either diopter or axial length. Besides, we also detected that there was a higher relevance between Glu content and diopter and axial length in LIM eyes than in normal. 
Previous studies revealed that when injection of a single and large dose of N-methyl-D-days (NMDA) into the chick eye would induce ocular growth.51 Moreover, studies have found that MK801, a Glu receptor antagonist, can effectively inhibit the FDM in chickens.5,52 All of these findings suggest that GABA and Glu are closely related to the occurrence of myopia. 
One hypothesis that emerges from these ideas is that the RGG is closely related to the occurrence and development of myopia. Previous study indicated that the contents of GABA and Glu were upregulated in myopia eyes in chick53,54 and guinea pigs,55 and our experimental results obtained the same result. Although in the normal development process the contents of Glu and GABA also were increased, the difference is that the RGG represented the balance between the excitatory and inhibitory neurotransmitters and always keeps an equilibrium state.41 A possible explanation for the occurrence of myopia is that the balance between the excitatory and inhibitory neurotransmitters is disturbed. In other words, the increased GABA content could not respond to the changes of the Glu level, and the relative expression is beyond the needed range of normal development. The processes whether the abnormal visual signal directly leads to changes in the expression of GABA in the retina, or by acting on the molecules related to the GABA expression, such as dopamine9 or Cl,32 remains to be determined. 
More interestingly, we also observed that all of the contents of Glu and GABA were decreased at 6 weeks, yet the value still was higher than that at week 0. In contrast, the RGG change was similar to the value at week 0. This difference may be due to the following reason. On one hand, we infer that the GABA and Glu could affect each other's expression. This is consistent with the general idea that depolarizing GABAergic transmission is required for the formation of glutamatergic synapses, which in turn would regulate the development of inhibitory GABAergic inputs.56 Alternatively, the changes of the related parameters associated with emmetropization in guinea pigs may be near to the end at 6 weeks after LIM, which is developed to 9 weeks of age.57 In this process, the RGG may be affected by other molecules. This is consistent with a recent series of studies that some candidate molecules appear to regulate the balance of glutamatergic and GABAergic synapses, such as neurexin and the scaffolding protein PSD-95.5860 
Relationship Between Retina and Either Normal or Myopia Development
It is well known that the information transmission in the retina is closely related to the occurrence and development of myopia. When the optic nerve is transected or action potential was inhibited, the degree of myopia will be influenced, whereas the occurrence of FDM would not be affected.6163 Previous investigations also have shown that if half covered for the retina with diffusers or negative lenses, only that half of the eye became enlarged and myopic, and if positive lenses cover half of the retina, only the half showed inhibited eye growth.41,64 Furthermore, Park et al.65 found that photoreceptor degeneration may alter dopamine metabolism and increase the sensitivity of the myopia occurrence. All of these studies have shown that the structure and information transmission in the retina has essential roles in the occurrence and development of myopia. 
Taking into account the role of the retina in the transmission of visual information, we hypothesized that the myopia-triggered signals are abnormal signals derived from the retina. In addition, retinal signal transduction depends on the feedback and the negative feedback mechanism.25 Moreover, this mechanism is mediated by Glu and GABA which is expressed by photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, and ganglion cells.43 However, due to the complex transmission of the visual information network, the specific regulatory mechanism still must further investigated. 
Finally, there are deficiencies in the present study. Although we selected the RGG to study the role of the balance between the excitatory and inhibitory molecules in the development in normal and myopia guinea pigs, it did not fully and accurately reflect the changes of the excitatory and inhibitory molecules in retina. The reason is that in the information transmission processing system, neural network has a variety of inhibitory and excitatory neurotransmitters, such as glycine, which also could adjust the balance between the excitation and inhibition molecules.25 Therefore, considering the interactive effect of the neurotransmitters, the underlying mechanism of the balance between the excitation and inhibition molecules should be further explored. 
Conclusions
In summary, we assume that the trigger signal of myopia is caused by the abnormal expressions of the retina neurotransmitters. The balance of the excitatory and inhibitory neurotransmitters of the retina is essential to eye development and myopia. It still is a long way to explore the underlying mechanism. 
Acknowledgments
Supported by the National Natural Science Foundation of China (81303081), the National Ministry of Science & Technology (2015BAI04B04), and the Development program of Science & Technology of Shandong province (2014GGH219004), the Natural Science Foundation of Shandong province (ZR2014HP059). 
Disclosure: L. Guoping, None; Y. Xiang, None; W. Jianfeng, None; G. Dadong, None; H. Jie, None; J. Wenjun, None; G. Junguo, None; B. Hongsheng, None 
References
Aamodt SM, Shi J, Colonnese MT, Veras W, Constantine-Paton M. Chronic NMDA exposure accelerates development of GABAergic inhibition in the superior colliculus. J Neurophysiol. 2000; 83: 1580–1591.
Zhang LI, Tao HW, Holt CE, Harris WA, Poo M. A critical window for cooperation and competition among developing retinotectal synapses. Nature. 1998; 395: 37–44.
Rajan I, Witte S, Cline HT. NMDA receptor activity stabilizes presynaptic retinotectal axons and postsynaptic optic tectal cell dendrites in vivo. J Neurobiol. 1999; 38: 357–368.
Ruthazer ES, Akerman CJ, Cline HT. Control of axon branch dynamics by correlated activity in vivo. Science. 2003; 301: 66–70.
Fischer AJ, Seltner RL, Stell WK. N-methyl-D-aspartate-induced excitotoxicity causes myopia in hatched chicks. Canad J Ophthalmol. 1997; 32: 373–377.
Ben-Ari Y. Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci. 2002; 3: 728–739.
Owens DF, Kriegstein AR. Is there more to GABA than synaptic inhibition? Nat Rev Neurosci. 2002; 3: 715–727.
Harauzov A, Spolidoro M, DiCristo G, et al. Reducing intracortical inhibition in the adult visual cortex promotes ocular dominance plasticity. J Neurosci. 2010; 30: 361–371.
Schmid KL, Strasberg G, Rayner CL, Hartfield PJ. The effects and interactions of GABAergic and dopaminergic agents in the prevention of form deprivation myopia by brief periods of normal vision. Exp Eye Res. 2013; 110: 88–95.
Sha F, Ye X, Zhao W, et al. Effects of electroacupuncture on the levels of retinal γ-aminobutyric acid and its receptors in a guinea pig model of lens-induced myopia. Neuroscience. 2015; 287: 164–174.
Yang XL. Characterization of receptors for glutamate and GABA in retinal neurons. Prog. Neurobiol. 2004; 73: 127–150.
Gabernet L, Jadhav SP, Feldman DE, Carandini M, Scanziani M. Somatosensory integration controlled by dynamic thalamocortical feed-forward inhibition. Neuron. 2005; 48: 315–327.
Wehr M, Zador AM. Balanced inhibition underlies tuning and sharpens spike timing in auditory cortex. Nature. 2003; 426: 442–446.
Akerman CJ, Cline HT. Depolarizing GABAergic conductances regulate the balance of excitation to inhibition in the developing retinotectal circuit in vivo. J Neurosci. 2006; 26: 5117–5130.
Zhu JJ, Malinow R. Acute versus chronic NMDA receptor blockade and synaptic AMPA receptor delivery. Nature Neurosci. 2002; 5: 513–514.
Leinekugel X, Khalilov I, McLean H, et al. GABA is the principal fast-acting excitatory transmitter in the neonatal brain. Adv Neurol. 1999; 79: 189–201.
Turrigiano GG. The self-tuning neuron: synaptic scaling of excitatory synapses. Cell. 2008; 135: 422–435.
McFadden SA, Howlett MH, Mertz JR. Retinoic acid signals the direction of ocular elongation in the guinea pig eye. Vision Res. 2004; 44: 643–653.
Lu F, Zhou X, Zhao H, et al. Axial myopia induced by a monocularly-deprived facemask in guinea pigs: a non-invasive and effective model. Exp Eye Res. 2006; 82: 628–636.
Iyamu E, Iyamu J, Obiakor CI. The role of axial length-corneal radius of curvature ratio in refractive state categorization in a nigerian population. ISRN Ophthalmol. 2011; 2011: 138941.
Zhong X, Ge J, Nie H, Chen X, Huang J, Liu N. Effects of photorefractive keratectomy-induced defocus on emmetropization of infant rhesus monkeys. Invest Ophthalmol Vis Sci. 2004; 45: 3806–3811.
Zhou X, Lu F, Xie R, et al. Recovery from axial myopia induced by a monocularly deprived facemask in adolescent (7-week-old) guinea pigs. Vision Res. 2007; 47: 1103–1111.
Lu F, Zhou X, Jiang L, et al. Axial myopia induced by hyperopic defocus in guinea pigs: a detailed assessment on susceptibility and recovery. Exp Eye Res. 2009; 89: 101–108.
Jiang L, Schaeffel F, Zhou X, et al. Spontaneous axial myopia and emmetropization in a strain of wild-type guinea pig (Cavia porcellus). Invest Ophthalmol Vis Sci. 2009; 50: 1013–1019.
Soto F, Bleckert A, Lewis R, et al. Coordinated increase in inhibitory and excitatory synapses onto retinal ganglion cells during development. Neural Devel. 2011; 6: 31.
Chen JC, Brown B, Schmid KL. Retinal adaptation responses revealed by global flash multifocal electroretinogram are dependent on the degree of myopic refractive error. Vision Res. 2006; 46: 3413–3421.
Stone RA, Lin T, Laties AM. Muscarinic antagonist effects on experimental chick myopia. Exp Eye Res. 1991; 52: 755–758.
Nickla DL, Wilken E, Lytle G, Yom S, Mertz J. Inhibiting the transient choroidal thickening response using the nitric oxide synthase inhibitor l-NAME prevents the ameliorative effects of visual experience on ocular growth in two different visual paradigms. Exp Eye Res. 2006; 83: 456–464.
Nickla DL, Wildsoet CF. The effect of the nonspecific nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on the choroidal compensatory response to myopic defocus in chickens. Optom Vis Sci. 2004; 81: 111–118.
McFadden SA, Howlett MH, Mertz JR, Wallman J. Acute effects of dietary retinoic acid on ocular components in the growing chick. Exp Eye Res. 2006; 83: 949–961.
Fagiolini M, Fritschy JM, Low K, Mohler H, Rudolph U, Hensch TK. Specific GABAA circuits for visual cortical plasticity. Science. 2004; 303: 1681–1683.
Akerman CJ, Cline HT. Refining the roles of GABAergic signaling during neural circuit formation. Trends Neurosci. 2007; 30: 382–389.
Stafford BK, Park SJ, Wong KY, Demb JB. Developmental changes in NMDA receptor subunit composition at ON and OFF bipolar cell synapses onto direction-selective retinal ganglion cells. J Neurosci. 2014; 34: 1942–1948.
Bedore J, Martyn AC, Li AK, et al. Whole-retina reduced electrophysiological activity in mice bearing retina-specific deletion of vesicular acetylcholine transporter. PLoS One. 2015; 10: e0133989.
Zhou EK, Xu HP. GABAergic regulation of spontaneous spike patterns in the developing rabbit retina. Neurosci Lett. 2015; 600: 137–142.
Vorwerk CK, Zurakowski D, McDermott LM, Mawrin C, Dreyer EB. Effects of axonal injury on ganglion cell survival and glutamate homeostasis. Brain Res Bull. 2004; 62: 485–490.
Ishikawa M. Abnormalities in glutamate metabolism and excitotoxicity in the retinal diseases. Scientifica. 2013; 2013: 528940.
Zucker RS, Regehr WG. Short-term synaptic plasticity. Ann Rev Physiol. 2002; 64: 355–405.
Spoerri PE. Neurotrophic effects of GABA in cultures of embryonic chick brain and retina. Synapse. 1988; 2: 11–22.
Ferguson SC, McFarlane S. GABA and development of the Xenopus optic projection. J Neurobiol. 2002; 51: 272–284.
Wallman J, Winawer J. Homeostasis of eye growth and the question of myopia. Neuron. 2004; 43: 447–468.
Liu G. Local structural balance and functional interaction of excitatory and inhibitory synapses in hippocampal dendrites. Nature Neurosci. 2004; 7: 373–379.
Sagdullaev BT, Eggers ED, Purgert R, Lukasiewicz PD. Nonlinear interactions between excitatory and inhibitory retinal synapses control visual output. J Neurosci. 2011; 31: 15102–15112.
Ge S, Goh EL, Sailor KA, Kitabatake Y, Ming GL, Song H. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature. 2006; 439: 589–593.
Hollrigel GS, Soltesz I. Slow kinetics of miniature IPSCs during early postnatal development in granule cells of the dentate gyrus. J Neurosci. 1997; 17: 5119–5128.
Kilman V, van Rossum MC, Turrigiano GG. Activity deprivation reduces miniature IPSC amplitude by decreasing the number of postsynaptic GABA(A) receptors clustered at neocortical synapses. J Neurosci. 2002; 22: 1328–1337.
Chia A, Li W, Tan D, Luu CD. Full-field electroretinogram findings in children in the atropine treatment for myopia (ATOM2) study. Doc Ophthalmol Adv Ophthalmol. 2013; 126: 177–186.
Mitchelson F. Muscarinic receptor agonists and antagonists: effects on ocular function. Hand Exp Pharmacol. 2012; 263–298.
Stone RA, Pardue MT, Iuvone PM, Khurana TS. Pharmacology of myopia and potential role for intrinsic retinal circadian rhythms. Exp Eye Res. 2013; 114: 35–47.
Frederikse PH, Kasinathan C. Lens GABA receptors are a target of GABA-related agonists that mitigate experimental myopia. Med Hypoth. 2015; 84: 589–592.
Quinlan EM, Olstein DH, Bear MF. Bidirectional, experience-dependent regulation of N-methyl-D-aspartate receptor subunit composition in the rat visual cortex during postnatal development. Proc Natl Acad Sci U S A. 1999; 96: 12876–12880.
Fischer AJ, Seltner RL, Poon J, Stell WK. Immunocytochemical characterization of quisqualic acid- and N-methyl-D-aspartate-induced excitotoxicity in the retina of chicks. J Comp Neurol. 1998; 393: 1–15.
Stone RA, Liu J, Sugimoto R, Capehart C, Zhu X, Pendrak K. GABA, experimental myopia, and ocular growth in chick. Invest Ophthalmol Vis Sci. 2003; 44: 3933–3946.
Cheng ZY, Wang XP, Schmid KL, Liu L. Identification of GABA receptors in chick retinal pigment epithelium. Neurosci Lett. 2013; 539: 43–47.
Cheng ZY, Wang XP, Schmid KL, et al. GABAB receptor antagonist CGP46381 inhibits form-deprivation myopia development in guinea pigs. BioMed Res Int. 2015; 2015: 207312.
Chudotvorova I, Ivanov A, Rama S, et al. Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses. J Physiol. 2005; 566: 671–679.
Zhou X, Qu J, Xie R, et al. Normal development of refractive state and ocular dimensions in guinea pigs. Vision Res. 2006; 46: 2815–2823.
Graf ER, Zhang X, Jin SX, Linhoff MW, Craig AM. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell. 2004; 119: 1013–1026.
Prange O, Wong TP, Gerrow K, Wang YT, El-Husseini A. A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin. Proc Natl Acad Sci U S A. 2004; 101: 13915–13920.
Levinson JN, Chery N, Huang K, et al. Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1beta in neuroligin-induced synaptic specificity. J Biol Chem. 2005; 280: 17312–17319.
Norton TT, Essinger JA, McBrien NA. Lid-suture myopia in tree shrews with retinal ganglion cell blockade. Vis Neurosci. 1994; 11: 143–153.
Troilo D, Gottlieb MD, Wallman J. Visual deprivation causes myopia in chicks with optic nerve section. Curr Eye Res. 1987; 6: 993–999.
Wildsoet C. Neural pathways subserving negative lens-induced emmetropization in chicks--insights from selective lesions of the optic nerve and ciliary nerve. Curr Eye Res. 2003; 27: 371–385.
Wallman J, Gottlieb MD, Rajaram V, Fugate-Wentzek LA. Local retinal regions control local eye growth and myopia. Science. 1987; 237: 73–77.
Park H, Tan CC, Faulkner A, et al. Retinal degeneration increases susceptibility to myopia in mice. Mol Vis. 2013; 19: 2068–2079.
Figure 1
 
Expressions of Glu and GABA in retina in normal and LIM guinea pigs. High performance liquid chromatography analysis was performed to measure the levels of Glu and GABA in retina in normal (A, B) and LIM (C, D) guinea pigs (n = 8 per group). *P < 0.05, **P < 0.01 compared to fellow eyes. LIM eye, lens-induced myopia eye; LIM Fellow eye, lens-induced myopia fellow eye.
Figure 1
 
Expressions of Glu and GABA in retina in normal and LIM guinea pigs. High performance liquid chromatography analysis was performed to measure the levels of Glu and GABA in retina in normal (A, B) and LIM (C, D) guinea pigs (n = 8 per group). *P < 0.05, **P < 0.01 compared to fellow eyes. LIM eye, lens-induced myopia eye; LIM Fellow eye, lens-induced myopia fellow eye.
Figure 2
 
Regression analyses between glutamate content and diopter/axial length in LIM guinea pigs. The correlation between Glu content and diopter in LIM guinea pigs was evaluated (n = 8 per group). The result indicated that there was a correlation between the levels of Glu content and axial length.
Figure 2
 
Regression analyses between glutamate content and diopter/axial length in LIM guinea pigs. The correlation between Glu content and diopter in LIM guinea pigs was evaluated (n = 8 per group). The result indicated that there was a correlation between the levels of Glu content and axial length.
Figure 3
 
Regression analyses between GABA content and diopter/axial length in LIM guinea pigs. The correlation between GABA content and diopter in LIM guinea pigs was evaluated (n = 8 per group). The result indicated that there was a correlation between the levels of Glu content and axial length.
Figure 3
 
Regression analyses between GABA content and diopter/axial length in LIM guinea pigs. The correlation between GABA content and diopter in LIM guinea pigs was evaluated (n = 8 per group). The result indicated that there was a correlation between the levels of Glu content and axial length.
Figure 4
 
Ratio of Glu to GABA content in retina in LIM guinea pigs at different time-points. The results indicated that the RGG was elevated in normal (control) and LIM guinea pigs (n = 8 per group), and LIM enhances the RGG. *P < 0.05, **P < 0.01.
Figure 4
 
Ratio of Glu to GABA content in retina in LIM guinea pigs at different time-points. The results indicated that the RGG was elevated in normal (control) and LIM guinea pigs (n = 8 per group), and LIM enhances the RGG. *P < 0.05, **P < 0.01.
Figure 5
 
Correlation between the RGG content and diopter/axial length in LIM guinea pigs. The result indicated that there was a correlation between the RGG and either diopter or axial length.
Figure 5
 
Correlation between the RGG content and diopter/axial length in LIM guinea pigs. The result indicated that there was a correlation between the RGG and either diopter or axial length.
Table
 
Alterations of the Axial Length and Refraction in Lens-Induced Myopia Guinea Pigs
Table
 
Alterations of the Axial Length and Refraction in Lens-Induced Myopia Guinea Pigs
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×