GABA, Experimental Myopia, and Ocular Growth in Chick

Abstract

purpose. To learn whether γ-aminobutyric acid (GABA) participates in retinal mechanisms that influence refractive development.

methods. White leghorn chicks, some of which wore a unilateral goggle to induce myopia, received daily intravitreal injections of agonists or antagonists to the major GABA receptor subtypes. Eyes were studied with refractometry, ultrasound, and calipers. Retinas of other chicks wearing unilateral goggles were assayed for GABA content.

results. Antagonists to GABA_A or GABA_A0r (formerly known as GABA_C) receptors inhibited form-deprivation myopia. GABA_A antagonists showed greater inhibition of myopic growth in the equatorial than the axial dimension. A GABA_A0r antagonist displayed parallel inhibition in the axial and equatorial dimensions. A GABA_A0r agonist but not GABA_A agonists altered the myopic refraction of goggled eyes. GABA_B receptor antagonists, more so than an agonist, also slowed development of myopia, inhibiting axial growth more effectively than equatorial expansion of goggled eyes. When administered to nongoggled eyes, GABA_A or GABA_A0r agonists or antagonists also altered eye growth, chiefly stimulating it. Only a GABA_A agonist induced a myopic refraction. Several of these agents stimulated eye growth in the axial, but not the equatorial, dimension. Retinal GABA content was slightly reduced in goggled eyes.

conclusions. GABA_A, GABA_A0r, and GABA_B receptors modulate eye growth and refractive development. The anatomic effects of these drugs reinforce the notion that eye shape and not just eye size is regulated. A retinal site of action is consistent with the known ocular localizations of GABA and its receptors and with the altered retinal biochemistry in form-deprived eyes.

Convincing evidence has identified a central role for the retina in the mechanism linking visual input with refractive development. ^{1 2} Based on biochemical and histochemical analyses and on the antimyopia effects of specific drugs, several retinal neurotransmitters have been implicated in this process. ^{2 3 4 5 6 7 8} For some, the evidence is strong; for others, it is more fragmentary. Many of these neurotransmitters localize to one or another subtype of retinal amacrine cell. Poorly understood characteristics of the visual image modulate eye growth, ⁹ but putative involvement of amacrine cells is consistent with their processing of complex image features. ^{10 11}  

γ-Aminobutyric acid (GABA), a widely distributed inhibitory amino acid neurotransmitter in the central nervous system and retina, ¹⁰ localizes to a large and diverse retinal cell population ¹² and participates in the signaling of both amacrine and horizontal cells. ^{10 13 14} In the retina, GABA colocalizes and/or interacts in functional pathways with dopamine ^{12 15} and acetylcholine, ^{16 17 18 19 20} neurotransmitters implicated in refractive development. ^{2 7 8 21 22} Because many putative GABAergic retinal neurons are relatively resistant to quisqualic acid toxicity and experimental myopia still develops after ocular administration of this neurotoxin, it has been proposed that retinal GABA may be required for myopic eye growth. ³ Given these indications that retinal GABA may participate in refractive development, we sought added evidence for a potential role of retinal GABA in the control of refractive development primarily by investigating the effects of ocular administration in chicks of GABA drugs (i.e., drugs with characteristic specificity to one or more of the major GABA receptor subtypes). 

GABA receptors traditionally have been classified into three major subtypes: GABA_A, GABA_B, and GABA_C receptors. ²³ GABA_A and GABA_C receptors each consist of ligand-gated chloride channels. Most GABA_A receptors are believed to contain five subunits from multiple subunit classes (α1-6, β1-4, γ1-3, δ, ε, θ, and/or π). ^{24 25} GABA_C receptors contain one or more of three different ρ subunits, which can complex with proteins of the other subunit classes. ^{24 26} Despite distinct pharmacology, structure, genetics, and function, ²⁶ GABA_C receptors have been reclassified as the GABA_A0r subtype of the GABA_A receptor family. ²⁴ We here use the general term GABA_A receptors for the large family of bicuculline-sensitive GABA receptors and GABA_A0r receptors for the bicuculline-insensitive, ρ-containing GABA_A receptor subset formerly termed GABA_C receptors. GABA_B receptors are metabotropic, G-protein–linked receptors coupled to adenylate cyclase or to Ca²⁺ and K⁺ channels. ^{27 28}  

We studied each of these major GABA receptor subtypes, because GABA_A, GABA_A0r, and GABA_B receptors are expressed in vertebrate retina. ²⁹ In fact, the predominant location of GABA_A0r receptors in central nervous system tissues is in the retina. GABA_A receptors occur on both pre- and postsynaptic sites of many types of retinal neurons. GABA_A0r receptors are found mainly but not exclusively on bipolar cells. GABA_B receptors tend to localize postsynaptically on amacrine and ganglion cells. Conforming to these generalities, the chick retina contains many putative GABAergic amacrine and horizontal cells and many putative GABAergic nerve fibers in both the inner and outer plexiform layers. ^{3 16 17 30 31} By immunohistochemistry, GABA_A receptors occur in both outer and inner plexiform layers and distinct types of amacrine cell somata. ³² GABA_A0r receptors also localize to both plexiform layers, evidently on the processes of bipolar cells. ³³ In situ hybridization in chick retina identifies GABA_A0r mRNA at retinal levels corresponding to the somata of horizontal, bipolar, amacrine, and perhaps ganglion cells. ³⁴ GABA_B receptors have not yet been identified biochemically or localized at a cellular level in chick retina, to our knowledge. 

Methods

White leghorn chicks (Truslow Farms, Chestertown, MD) were reared under a 12-hour light–dark cycle as described. ⁸ Chicks were anesthetized with inhalation ether for all goggle applications and drug injections. Form-deprivation myopia was induced by securing a unilateral translucent white plastic goggle to the periorbital feathers with cyanoacrylate glue. The experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Intraocular Drug Administrations

All goggle applications and/or drug administrations began when the chicks reached 1 week of age. At about 4 hours into the light phase, a 10-μL intravitreal injection of either drug or vehicle was administered under aseptic conditions to the goggled or experimental nongoggled eye, with all contralateral eyes receiving vehicle injections at the same time. Within each series, the experimental eye was alternated between left and right. 

Table 1 lists the studied drugs, their affinities to GABA receptor subtypes, the suppliers, and the ranges of daily doses. The daily doses for specific experiments are provided in the figures or in the Results section. Table 1 also provides an estimate of the maximum drug concentration (in micromolar) potentially achievable in the vitreous humor, based on the assumptions of rapid and uniform drug distribution into a liquid vitreous volume of 150 μL. ⁴⁰ These calculated estimates of maximum drug concentrations at the inner limiting membrane provide only rough approximations of potential drug levels at retinal receptors because these calculations do not account for factors that may facilitate or impede drug access to intraretinal receptors, such as lipid permeability, molecular weight/charge, diffusional loss, degradation, cellular uptake, or leakage from the injection site. These estimates also cannot be related to the time dependence of drug binding at retinal receptors for modulating eye growth, which is unknown for all drugs. The number of chicks studied at each drug dose is shown in Figures 1 and 4 or in the Results section. 

Eye Measurements

After 4 days of treatment, the chicks were anesthetized on the fifth day with an intramuscular mixture of ketamine (20 mg/kg) and xylazine (5 mg/kg) for ocular examinations; on that day, they received no intraocular injections. Ocular refractions with a Hartinger-type refractometer and A-scan ultrasonography were performed as described. ⁴¹ While still under general anesthesia, the chicks were decapitated, and the axial and equatorial dimensions of enucleated eyes were measured with digital calipers. Because the equatorial profile of the chick eye is elliptical, the equatorial dimension is reported as the mean of the shortest and longest equatorial diameters. 

Regarding axial length differences specifically, ultrasound determines the distance from cornea to inner retinal surface and thus excludes any choroidal or scleral contribution, but calipers measure from cornea to outer sclera and include the thickness of both the choroid and the sclera. The comparative differences in axial length by these two methods can be differentially affected by growth-related changes in choroidal thickness, and the range of the differences between the two measurement techniques fell within the approximate 500-μm reactivity of the chick choroidal thickness previously determined over 4 days in separate studies. ^{42 43} Data on anterior chamber depth and lens thickness are not reported for most experiments, because these parameters were unaffected in almost all cohorts. The only exceptions, lens and anterior chamber dimensions for chicks that received CACA and lens thickness for nongoggled chicks that received CGP46381, are described in a separate section. 

Biochemical Assays

To assay retinal GABA, ⁴⁴ the same goggle type was placed over one eye of 1-day-old chicks. At 2 weeks of age, the chicks were decapitated 4 hours into the light cycle. The eyes were enucleated immediately, chilled in iced saline, measured in axial and equatorial dimensions with calipers to confirm ocular expansion, and dissected on ice as quickly as possible. The retinas were immediately frozen and stored individually in liquid nitrogen. At the time of assay, each frozen retina was placed in 0.5 mL of 0.1 M HClO₄ with 0.3 mM 5-aminovaleric acid HCl as an internal standard at 4°C and homogenized. The homogenate was centrifuged at 4°C for 15 minutes at 14,000 rpm, and the supernatant was filtered with a 13-mm syringe filter with a 0.2 μm nylon membrane (Acrodisc; Gelman Sciences, Ann Arbor, MI). For derivatization, 0.02 mL of the filtered supernatant was incubated for 6 minutes at room temperature with 0.18 mL of 15% carbonate buffer (pH 9.6) containing 5 mM o-phthalaldehyde (OPA; Sigma-Aldrich, St. Louis, MO), 50% methanol and 5 mM 2-methyl-propanethiol (Sigma-Aldrich). A portion (25 μL) of the derivatized sample was separated on a reversed-phase column (ODS, 5 μm, 4.6 mm × 25 cm, Ultrasphere C₁₈; Beckman Coulter, Fullerton, CA) on a high-pressure liquid chromatography system with an electrochemical detector (HPLC-ED; LC-4C; BioAnalytical Systems, West Lafayette, IN). The column was eluted with a mobile phase of 58% 0.1 M Na acetate (pH 5.0) and 42% acetonitrile, at a flow rate of 1.0 mL/min, and read by the detector with a glassy carbon working electrode at +0.7 V versus an Ag/AgCl reference electrode. To assay protein, the centrifugation pellet was dissolved in 1.0 mL of 1.0 M NaOH. Ten microliters was measured with a protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard, according to the manufacturer’s instructions. GABA levels are reported in micrograms per milligram protein. 

Data Analysis

For goggled chicks, the primary outcome measure was the drug effect on the ocular response to visual deprivation, comparing individual doses to each other and to the contemporaneous vehicle-treated control for each drug. The differences in refraction and each size measurement between goggled and contralateral eyes were assessed by one-way analysis of variance (ANOVA). Potential drug effects on vehicle-treated contralateral eyes were evaluated by one-way ANOVA of the same parameters on the nongoggled eyes of the goggled cohorts. For nongoggled chicks, the primary outcome measure was comparison of drug-treated to vehicle-treated contralateral eyes, and a two-way repeated measures ANOVA (one-factor replication, using the eye as the replicated factor) was applied separately to the refraction and size parameters. This latter test also enabled assessing vehicle-treated eyes for possible contralateral drug effects. The outcomes of the two-way ANOVA are described mainly in terms of the primary drug-treated to contralateral eye comparison. In a few instances in which this primary outcome measure did not reach statistical significance but the overall dose or eye-dose interaction terms yielded P < 0.05, these secondary outcomes are described. When the ANOVA assumptions of normality or equal variance were not met, the corresponding ANOVA was performed on rank transformed data. If the ANOVA identified a treatment effect, the Tukey test for multiple pair-wise post hoc comparisons was used to identify differences between specific groups, assuming P < 0.05 for statistical significance. The figures show the probabilities when the overall ANOVA reached a significance level of at least P < 0.05, and the post hoc tests for within-group comparisons appear in Tables 2 and 3 . Retinal levels of GABA in goggled and contralateral nongoggled eyes were compared by Student’s paired t-test. Results from different cohorts of chicks tested with the same drug, along with the corresponding vehicle-treated control, were pooled for analysis. Data are shown as mean ± SEM and were analyzed on computer (SigmaStat; SPSS, Inc., Chicago, IL). 

Results

The multiple effects of GABA drugs on refraction and ocular components are summarized in Table 4 , which shows the statistically significant drug effects in Figures 1 2 3 4 5 . 

Goggled Eyes, GABA_A Agents

GABA_A agonists had no effect on form-deprivation myopia. Neither the mixed GABA_A/partial GABA_A0r agonist muscimol (daily doses of 10, 50, 100, or 200 μg; n = 9 to 10/group; data not shown) nor the mixed GABA_A/GABA_A0r agonist TACA (daily doses of 10 or 100 μg; n = 10 to 13/group; data not shown) had statistically significant effects on refraction or size measurements when given to goggled eyes. 

GABA_A antagonists chiefly limited equatorial expansion of goggled eyes. The classic GABA_A antagonist bicuculline in daily doses up to 50 μg did not affect myopic refraction or axial measures in goggled eyes, but it reduced equatorial diameter (Figs. 1A 2 ; Table 2 ). Higher daily doses of bicuculline (100 or 200 μg) doses caused retinal whitening, interpreted as gross retinal edema or other toxicity and accordingly were not analyzed. 

The GABA_A antagonist SR95531 caused pronounced and dose-dependent inhibition of equatorial expansion in goggled eyes (Fig. 2 ; Table 2 ). SR95531 also reduced the myopic refraction in goggled eyes, with the 50-μg daily dose being the most effective (Fig. 1A ; Table 2 ). Although the direction of the trend for SR95531 to reduce axial length and vitreous chamber depth of goggled eyes corresponded to the reduced myopic refraction (Fig. 2) , none of these length effects reached statistical significance (P = 0.1 or greater). Unlike bicuculline, SR95531 at the doses used did not cause ocular toxicity detectable during in vivo ocular examinations or by inspection of bisected enucleated eyes. 

Goggled Eyes, GABA_A0r Agents

The GABA_A0r agonist CACA had a modest influence on the refractive response to a goggle (Fig. 1B ; Table 2 ). The greatest myopia occurred at the 200-μg dose, which was statistically greater than that at either the 10- or 50-μg doses. However, none of the ultrasound or caliper measurements were altered statistically, when compared with the goggled vehicle-treated controls (Fig. 2) . 

The GABA_A0r antagonist TPMPA inhibited the response to goggle wear (Figs. 1 2 ; Table 2 ). It reduced myopic refraction, and it blocked axial and vitreous chamber elongation measured by ultrasound and the equatorial expansion measured by calipers. Any effect on axial growth measured by calipers did not reach statistical significance. 

Goggled Eyes, GABA_B Agents

The GABA_B agonist baclofen had a weak, antimyopia effect. It partially reduced the myopic refractive error beneath a goggle (Fig. 1C , Table 2 ), but neither the ultrasound nor caliper measurements revealed statistically significant inhibition of myopic growth at the two doses tested (Fig. 3) . 

Among GABA_B antagonists, CGP46381 inhibited the myopic refractive shift, the axial and vitreous chamber elongation and the equatorial expansion of eyes beneath a goggle (Figs. 1C 3 ; Table 2 ). SCH50911 reduced the myopia in goggled eyes (Figs. 1C 3 ; Table 2 ). Whereas SCH50911 showed a trend toward reduced equatorial diameter (P = 0.06), none of the length measures in goggled eyes differed statistically from the contralateral control eyes (Fig. 3) . 2OH-saclofen similarly reduced the myopic refraction but had no statistically significant effect on ocular dimensions, as measured by ultrasound or calipers (Figs. 1C 3 ; Table 2 ). CGP35348 had no statistical effect on the myopic shift in refraction or excessive growth of goggled eyes, measured by either ultrasound or calipers (n = 9 to 18/group; data not shown). 

Nongoggled Eyes, GABA_A Agents

In contrast to the absence of effect on form-deprivation myopia, the GABA_A agonist muscimol induced myopia in nongoggled chicks (Fig. 4 ; Table 3 ), with a maximum refractive effect at the 50-μg dose. Consistent with this refractive shift, muscimol stimulated axial growth measured by either ultrasound or calipers and deepened the vitreous chamber; it also increased equatorial diameter (Fig. 5 ; Table 3 ). The mixed GABA_A/GABA_A0r agonist TACA, however, influenced neither refraction nor ocular size measurements of drug-treated eyes compared with the contralateral vehicle-treated control (daily doses of 10 or 100 μg; n = 10/group; data not shown). 

The GABA_A antagonist SR95531 also stimulated growth of nongoggled eyes (Figs. 4 5 ; Table 3 ), but with fewer statistically significant effects than muscimol. Compared with vehicle-treated contralateral eyes, SR95531 enhanced axial growth measured by calipers and lengthened the vitreous chamber by a comparable amount. Although not significant for the primary drug-treated versus contralateral eye comparison (Fig. 5) , the ultrasound measures revealed an axial length effect in the interaction of eye and dose (Table 3) . For refraction, the myopic shift of up to 1 to 2 D did not reach statistical significance (P = 0.07), when comparing drug-treated with contralateral eyes, but there was an overall dose effect (P = 0.046). SR95531 had no significant effect on equatorial diameter. Given its potential for retinal toxicity (see above), bicuculline was not tested in nongoggled chicks. 

Nongoggled Eyes, GABA_A0r Agents

Although it did not affect refraction (Fig. 4) , the selective GABA_A0r agonist CACA slightly stimulated axial length detected by ultrasound (Fig. 5 ; Table 3 ), with a trend for increased vitreous chamber length (Fig. 5 ; P = 0.056). 

When injected into nongoggled eyes, the GABA_A0r antagonist TPMPA stimulated both axial growth and vitreous chamber length by a modest degree, but reduced equatorial diameter (Figs. 4 5 ; Table 3 ). The slight myopic refractive shift reached statistical significance in the dose (P = 0.02), but not in the comparison of drug-treated versus contralateral eyes (P = 0.14; Fig. 4 ). 

Nongoggled Eyes, GABA_B Agents

When administered to nongoggled eyes, the GABA_B agonist baclofen caused moderate vitreous chamber deepening. A comparable amount of axial elongation reached statistical significance when measured by calipers but not by ultrasound (Fig. 5 ; Table 3 ). None of the other effects of baclofen on nongoggled eyes, including refractions (Fig. 4) , reached statistical significance. 

The most effective GABA_B antagonist against myopia, CGP46381, lengthened the vitreous chamber of nongoggled eyes (Fig. 5 ; Table 3 ). Comparable increases in axial length by ultrasound or caliper measurements did not reach statistical significance. CGP46381did not exert statistically identifiable changes in refraction (Fig. 4) or equatorial diameter (Fig. 5) . 

Anterior Chamber and Lens Effects

Only two agents affected anterior segment parameters. Neither drug altered measurements in the primary statistical comparisons of drug-treated to vehicle-treated contralateral eyes in either goggled or nongoggled cohorts, but instead only in secondary dose comparisons (described earlier). The most consistent anterior segment effects were exerted by the GABA_A0r agonist CACA. In goggled chicks, the anterior chamber depths within drug-treated goggled eyes (P = 0.044) and vehicle-treated contralateral eyes (P = 0.046) varied overall between CACA doses. In goggled eyes, the shallowest anterior chambers occurred in chicks that received bilateral vehicle (1.14 ± 0.03 mm) and the deepest anterior chambers in those that received the 10-μg dose (1.30 ± 0.03 mm; P = 0.023, Tukey test), but no specific post hoc comparisons reached statistical significance for the contralateral eyes. In goggled chicks, CACA exerted no statistically significant effect on lens thickness. 

In nongoggled cohorts, CACA exerted a statistically significant effect on both anterior chamber depth and lens thickness in the overall dose comparisons (P ≤ 0.001 for anterior chamber; P = 0.001 for lens). As with the goggled cohorts, the anterior chambers of drug- and vehicle-treated eyes of nongoggled chicks that received the 10-μg dose were deeper by some 0.08 to 0.14 mm than those of eyes that received the three higher doses (P ≤ 0.02 for each comparison, Tukey test). The lens was thinnest in both eyes of nongoggled chicks with the deepest anterior chambers (i.e., those that received the 10-μg dose). The lens was thinner overall in chicks that received the 10-μg dose (2.22 ± 0.01 mm) than in those that received either the 100-μg (2.34 ± 0.03 mm; P = 0.009, Tukey test) or 200-μg dose (2.35 ± 0.03 mm; P = 0.002, Tukey test). 

The GABA_B antagonist CGP46381 was the only other drug with an anterior segment effect, specifically lens thickness in nongoggled eyes. Although it did not influence lens thickness in the primary outcome measure of drug- to vehicle-treated eyes, CGP46381 exerted a statistically significant lens effect in the dose comparison (P = 0.03). The lenses of drug-treated eyes of chicks that received the 100-μg dose were some 0.07 mm thinner than those of chicks that received the 10-μg dose (P = 0.03, Tukey test), with no differences identified in vehicle-treated eyes. CGP46381 had no effect on anterior chamber depth in nongoggled eyes and no effect on either lens thickness or anterior chamber depth in goggled eyes. 

Contralateral Eyes

With some drugs, refractions or size measurements suggested potential effects in the contralateral vehicle-treated eyes, but only a limited number of parameters were affected in these instances. Those effects with a significance level of P < 0.05 are described. 

After administration of the GABA_A agonist muscimol to nongoggled chicks, the refractions of vehicle-treated eyes varied with the muscimol dose (P < 0.05): 5 μg (−0.86 ± 0.58 D), 10 μg (+0.11 ± 0.82 D), 50 μg (−1.78 ± 1.21 D), and 200 μg (−3.82 ± 0.83 D), with the 10- and 200-μg doses differing from each other significantly (P = 0.002, Tukey test). No ultrasound or caliper measurements were affected in the vehicle-treated eyes of nongoggled chicks, and no parameter of contralateral vehicle-treated eyes was altered when muscimol was given to goggled chicks. 

For the GABA_A antagonist SR95531, contralateral effects occurred only in the vitreous chamber; but the dimension and direction of effect differed between goggled and nongoggled groups. For goggled chicks, contralateral effects were measured for the equatorial diameter by calipers (P = 0.02). The contralateral eyes of chicks that received the highest 100-μg dose were some 0.4 mm wider (11.96 ± 0.13 mm) than those that received the 1-μg dose (11.53 ± 0.10 mm; P = 0.046, Tukey test) and 10-μg dose (11.50 ± 0.10 mm; P = 0.03, Tukey test). In contrast, the contralateral effect in nongoggled chicks occurred in vitreous chamber length, measured by ultrasound. The vitreous chamber contralateral to eyes that received the intermediate 50-μg SR95531 dose was some 0.14 mm shorter (5.16 ± 0.04 mm) than in chicks that received the lowest 5-μg dose (5.30 ± 0.05 mm; P = 0.048, Tukey test). 

For the GABA_A0r antagonist TPMPA, only ultrasound identified a contralateral effect on axial length in goggled chicks (P = 0.046). The axial lengths of eyes contralateral to those that received the 10-μg and higher doses were some 0.1 to 0.2 mm shorter (e.g., 8.49 ± 0.08 mm for 10 μg) than those that received lower doses and bilateral vehicle (e.g., 8.63 ± 0.05 mm for vehicle), but the Tukey test identified no specific comparisons as statistically significant. In nongoggled chicks, a contralateral axial length effect was identified only by calipers. In chicks that received the highest dose 200-μg dose the axial length was shorter by some 0.4 mm (8.45 ± 0.11 mm) than in chicks that received the lowest 10-μg dose (8.90 ± 0.06 mm; P = 0.027, Tukey test). 

With the GABA_B drugs, only two agents exerted contralateral effects, only in goggle experiments, and only by caliper measurements. Eyes contralateral to goggled eyes that received either the highest 200-μg dose of CGP46381 or the vehicle were smaller in caliper-measured axial (P = 0.004) and equatorial (P = 0.014) dimensions by some 0.3 mm than those treated with the other doses. The Tukey test identified only the contralateral equatorial diameter of the vehicle controls as statistically smaller than the 100-μg dose (P = 0.036), with no specific pair-wise comparisons identified for axial length. CGP35348, an ineffective drug reducing the primary response of goggled to contralateral eyes, induced a contralateral effect in caliper axial measurements only (P < 0.001). Eyes contralateral to those that received the 100-μg CGP35348 dose were larger (by some 0.26–0.36 mm) than those contralateral to vehicle treated eyes or to those that received the 1- and 500-μg doses (P < 0.05, Tukey test for most comparisons). 

Retinal Biochemistry

By HPLC-ED assay, GABA levels in nongoggled eyes measured 10.8 ± 0.2 μg/mg protein, consistent with published levels in the chick retina. ⁴⁵ GABA levels in contralateral goggled eyes measured 10.3 ± 0.2 μg/mg protein. Although the magnitude of this difference is small, the reduction in retinal GABA of myopic eyes reached statistical significance (n = 23 pairs of eyes; P < 0.02, two-tailed Student’s paired t-test). 

Discussion

GABA drugs altered eye growth in chicks and influenced both form-deprivation myopia and the development of eyes with normal visual input. As summarized in Table 4 , active drugs in goggled eyes generally reduced myopic refraction and excessive growth. In nongoggled chicks, active drugs stimulated eye growth and, in one instance, induced frank myopia. 

GABA Drug Effects on Form-Deprivation Myopia

Agents from each class of GABA drugs altered the progression of experimental myopia (Table 4) . Evidence for involvement of other retinal neurotransmitters in regulating refractive development has included the antimyopia activity of drugs with affinities to the corresponding receptors. ^{2 3 4 5 6 7 8} By analogy, the antimyopia activity of GABA drugs points to involvement of retinal GABA in myopic eye growth, as previously suggested from studies of experimentally induced lesions. ³  

Antagonists but not agonists to GABA_A receptors showed an unusual inhibitory activity against form-deprivation myopia, markedly reducing equatorial expansion of the vitreous chamber without significantly altering the axial dimensions of goggled eyes. Because equatorial eye dimensions have seldom been reported in comparable studies, other means of exerting selective equatorial effects may have been overlooked. Nonetheless, GABA_A antagonists represent the first drug class reported to inhibit the growth of goggled eyes chiefly in the equatorial dimension. 

The GABA_A0r receptor antagonist TPMPA largely eliminated the myopic refractive shift and significantly reduced both the axial measures by ultrasound and the equatorial expansion of goggled eyes. The anatomic pattern of TPMPA’s activity against myopic growth, reducing overall vitreous cavity expansion, differs from the equatorial inhibition of GABA_A antagonists. The modest, perhaps biphasic, effect on the refraction of goggled eyes from the GABA_A0r receptor agonist CACA was not accompanied by significantly altered ocular dimensions. 

For GABA_B selective drugs, both agonists and antagonists showed variable antimyopia activities. The antagonist CGP46381 in particular inhibited both the myopic refractive response and the excessive growth in both axial and equatorial dimensions. This anatomic pattern differs from two other classes of antimyopia drugs interacting with G-protein–linked receptors, dopamine agonists, and muscarinic antagonists, for which available data suggest a drug action primarily in the axial dimension. ^{7 21}  

GABA Drug Effects on Nongoggled Chicks

As with goggled eyes, agents from each class of GABA drugs exerted at least some effect on the refractive development of nongoggled eyes (Table 4) . The active drugs generally stimulated axial eye growth, with effects on refraction and equatorial diameter varying between drugs. Drugs interacting with GABA_A and GABA_A0r receptor subtypes were effective, but agents selective for GABA_B receptors showed less-pronounced effects. The GABA_A agonist muscimol in particular increased eye size in axial and equatorial dimensions and was the only drug to induce a statistically significant myopic shift in refraction. In terms of ocular geometry, the GABA_A0r receptor agonist CACA and antagonist TPMPA each stimulated eye growth selectively in the axial dimension of the eye, without altering refraction. The geometry of the TPMPA effect was unusual, as the equatorial diameter actually narrowed despite the elongated axial dimensions. 

Perhaps, the responsiveness of both goggled and nongoggled eyes to GABA drugs relates to the broad retinal distribution of GABA receptors or to the specific receptor subtypes, all areas for future study. Because the enlarged eyes that received muscimol became myopic, but those that received drugs with a different specificity remained emmetropic, GABA_A drugs may be useful in dissecting emmetropization mechanisms. 

Another drug that influences the refractive development of both goggled and nongoggled chick eyes, the muscarinic antagonist pirenzepine, inhibits form-deprivation myopia ²¹ and also slows eye growth without visual deprivation, inducing a corresponding hyperopic shift in refraction. ⁴⁶ The parallel inhibitory actions of pirenzepine on the growth of goggled and nongoggled chicks contrasts with the behavior of GABA drugs (e.g., muscimol and TPMPA), with which the growth effects differ between goggled and nongoggled chicks (Table 4) . 

Other nontoxic neuropharmacologic drugs that influence experimental myopia generally have not altered the growth and refractive development of eyes with intact visual input, perhaps because vision-dependent mechanisms governing eye growth dominate the effects of these drugs. Specifically, dopamine agonists, opiates, and basic fibroblast growth factor each inhibit form-deprivation myopia, but have not been found to alter growth or refraction of nonoccluded eyes. ^{7 47 48}  

Most other drugs that have influenced the growth and refraction of nongoggled eyes of chicks are frank neurotoxins, such as kainic acid, N-methyl-d-aspartate, tetrodotoxin, and others. ^{8 48 49 50 51} A frequently studied nicotinic antagonist in brain research, chlorisondamine, inhibits the growth of nonoccluded eyes, but it exerts an unusual toxic effect on the retinal pigment epithelium. ⁸ A nontoxic nicotinic antagonist that inhibits myopia in chicks, mecamylamine, did not alter the growth of nongoggled eyes. ⁸  

Anterior Segment Effects

The lens and/or anterior chamber effects of the GABA_A0r receptor agonist CACA and the GABA_B receptor antagonist CGP46381 were evident only in dose comparisons and not in the primary comparison of drug- and vehicle-treated eyes. Nonetheless, they raise the question of whether retinal GABA mechanisms influence anterior segment development. Optically, increasing anterior chamber depth shifts the image plane posteriorly. Increasing lens thickness, if associated with increased lens surface curvature and power, displaces the image plane anteriorly. ⁵² No shifts in refraction were detected, however, for either of these drugs under the conditions affecting the anterior segment: both goggled and nongoggled cohorts for CACA and nongoggled cohorts for CGP46381. In nongoggled chicks that received CACA, the anterior chamber and lens changes may have balanced each other optically to nullify any measurable influence on refraction, but it is unclear whether such compensation accounts for the absence of refractive effects in the other conditions in which only one anterior segment parameter was affected. Other reports have suggested a potential role for the retina in anterior segment development, including altered anterior chamber depth caused by toxins that cause lesions in specific retinal neurons ^{49 53 54} and induction of corneal astigmatism by the wearing of cylindrical lenses. ^{55 56} A cellular or signaling mechanism by which the retina might modulate anterior segment growth remains obscure, and a direct drug effect on the anterior segment cannot be excluded. Determination of the extent and mechanism of GABA influences on the anterior segment requires further study. 

Contralateral Effects

A few of the responses to some GABA drugs suggested effects in contralateral vehicle-treated eyes. In most instances, the statistically significant contralateral effects occurred in only a single parameter rather than in multiple parameters and were different and/or absent between goggled and nongoggled cohorts, even though contralateral eyes always experienced unimpaired visual input. Because contralateral eyes were used as the control in the primary analysis, learning the biological implications of any of these contralateral effects, including the extent to which they may comprise drug-effects per se or drug–vision interactions, requires direct study. It is notable that no contralateral effects indicated a need to alter the general conclusions of the primary drug-treated to contralateral vehicle-treated eye comparisons. 

The most provocative of the contralateral effects, a myopic refraction developed in vehicle-treated eyes of chicks treated with the GABA_A agonist muscimol, notably at the higher doses. Because the results were analyzed as the difference between drug-treated and contralateral vehicle-treated eyes (Fig. 4) , the 3 to 4 D of myopia in contralateral eyes could account for the apparent loss of the myopic refractive shift in the drug-treated eyes at the 200-μg dose. Because no evidence emerged in measurements by ultrasound or caliper of a growth effect in contralateral eyes in the muscimol-treated cohort, it is possible that myopia in contralateral eyes resulted from a change in corneal curvature, a parameter not measured in the current study. In chicks wearing defocusing spectacle lenses, the growth of the defocused eye adjusts to compensate for the spectacle lens. ⁵⁷ The contralateral eyes of chicks wearing a unilateral defocusing lens also experience refractive shifts in the same direction as the lens-wearing eyes, ⁵⁸ suggesting the possibility of “yoking” of the two eyes in responding to unilateral optical defocus. In the present study, muscimol was the only drug given to nongoggled chicks that changed the refraction in drug-treated eyes—in this instance, inducing myopia. Further work is needed to determine whether the induction of myopia in contralateral eyes at the higher muscimol doses involves a direct drug effect, perhaps from systemic absorption, or instead an analogous refractive “yoking” of the two eyes in response to optical defocus, with the optical power of the anterior segment of vehicle-treated eyes adjusting to conform with the myopia of the drug-treated opposite eyes. 

Refractive Versus Axial Length Effects

Lack of correspondence occurred between refraction and axial length changes in some experiments, but the pattern differed between goggled and nongoggled series. Because we did not perform keratometry or phakometry, we cannot provide full explanation for whether a biological response or measurement variability accounted for specific discrepancies between refractive and axial length measurements. Among goggled cohorts, the most prominent pattern was a drug-induced shift in refraction, with shifts in axial measurements not consistently reaching statistical significance (Table 4 ; Figs. 1 2 3 ). Except for muscimol, the drugs affecting the growth of nongoggled eyes altered eye dimensions without affecting refraction (Table 4 ; Figs. 4 5 ). As a potential biological explanation for the nongoggled cohorts, the anterior segments of drug-treated but enlarged eyes that remained emmetropic may have adjusted optically to compensate for the elongated axial components. Because of the small magnitude of the growth effects in nongoggled eyes (Fig. 5) , any corresponding changes in cornea or lens curvatures may not have induced sufficient anterior segment modification to be detectable by ultrasound. 

GABA Drugs and Eye Shape

Effects of neurotoxins on vitreous chamber form, ⁴⁹ bulging of the vitreous chamber in eyes wearing a partial goggle, ^{1 59} inhibition of axial elongation but not equatorial expansion by dopaminergic and muscarinic drugs in form-deprivation myopia, ^{2 7 21} and selective axial elongation as the initial response to constant-light rearing ^{41 60} all have suggested that the shape of the vitreous chamber is controlled, presumably through retinal activity. The ocular responses to GABA drugs similarly implicate the retina in the control of eye form. Depending on the particular drug and presumably the corresponding receptor subtype and depending on whether visual input was impaired or intact, GABAergic drugs exerted generalized, selective axial or selective equatorial effects on eye shape. The response to the GABA_A0r antagonist TPMPA in nongoggled chicks provides a particularly convincing illustration of the modulation of eye shape per se, because this drug concurrently lengthened the eye and narrowed its equatorial diameter. 

GABA_A and GABA_A0r receptors may have distinct roles in modulating eye shape (Table 4) . In goggled eyes, GABA_A antagonists acted chiefly to inhibit equatorial expansion, but the GABA_A0r antagonist TPMPA inhibited growth in both axial and equatorial dimensions. In nongoggled eyes, the GABA_A agonist muscimol expanded the vitreous chamber in both axial and equatorial dimensions, but the GABA_A0r agonist CACA caused only modest axial lengthening. Also in nongoggled eyes, the GABA_A antagonist SR95531 stimulated axial growth, but the GABA_A0r antagonist TPMPA both stimulated axial growth and inhibited equatorial expansion. 

For a clinical perspective on eye shape, selective axial elongation of the vitreous chamber is believed to characterize the ocular morphology of many but not all human myopic eyes. ^{61 62} So far, the initial growth response to disrupting the dark phase of a 12–12-hour light–dark cycle by constant lighting ⁴¹ and the GABA drug responses in the current study are the only known or published experimental conditions that induce selective axial elongation of the vitreous chamber. 

Underlying Mechanisms

The complexities of GABA drug effects (Table 4) do not permit unambiguous interpretation of underlying pharmacologic mechanisms. For each of the major GABA receptor subtype classes, agonists and antagonists at a particular subtype did not provide reciprocal effects in either goggled or in nongoggled chicks. Agonists and antagonists to some receptor subtypes actually exerted effects in the same direction, such as some of the growth responses of nongoggled cohorts to agonists and antagonists to either GABA_A or GABA_A0r receptors. The dose–response curves tended to be complicated, because some of the optimally effective drug doses occurred in the middle of the tested range. U-shaped or inverted U-shaped dose–response curves showing such behavior, termed hormesis, are increasingly being recognized in biological responses to drugs. ⁶³ The GABA_B antagonists in particular showed marked differences in their effectiveness against form-deprivation myopia. The retina expresses a multiplicity of retinal GABA receptor subtypes, but their molecular subunit compositions are not extensively characterized. ^{24 25 26} Besides bioavailability and other important pharmacokinetic considerations, the complexity of the ocular responses may reflect the specific types of GABA receptor subunits in the retina, the intraretinal distribution of GABA receptors, the interactions of putative GABAergic neurons with other retinal cells that modulate eye growth, and/or differential drug affinities to specific or multiple GABA receptor subtypes. 

Despite the complexities, the growth responses substantiate a role for retinal GABA in modulating refractive development, especially the inhibition of form-deprivation myopia by the GABA_A0r antagonist TPMPA and the GABA_B antagonist CGP46381 and the stimulatory effects in nongoggled eyes of TPMPA and the GABA_A agonist muscimol. GABA and its receptors are expressed by diverse retinal neurons, but neither GABAergic nerves nor GABA receptors have yet been described in nonretinal tissues of the eye, supporting the hypothesis that GABA drugs act locally at the retina. The activity of the GABA_A0r receptor antagonist TPMPA particularly suggests retinal participation in eye development because of the receptor’s predominant localization to this tissue. ^{26 35} With other neurotransmitters and modulators, altered retinal levels have supported potential retinal involvement in regulation of eye growth, ^{4 7 64 65} and we assayed retinal GABA, seeking parallel information in the current study. The reduced retinal concentration of GABA in form-deprived myopic eyes was small in magnitude, and the large number of putative retinal GABAergic neurons complicates interpreting these biochemical results. Retinal levels of a neurotransmitter can reflect the sum of many biochemical and physiological processes, not addressed in the present study. Only a subset of GABAergic neurons may be affected or several subsets may be differentially affected after form deprivation, either of which may account for the low magnitude of the effect. Still, in the context of the refractive effects of GABA drugs, the biochemistry is consistent with the general notion that the retina modulates eye growth. ^{1 2}  

Summary

GABA drugs both inhibit form-deprivation myopia and influence the growth of eyes with normal visual input, thus implicating GABA receptors in the mechanism that modulates eye growth and refractive development. Both ionotropic (GABA_A and GABA_A0r) and metabotropic (GABA_B) receptors are implicated by the drug responses. The complex anatomic effects of these drugs reinforce the notion that retinal mechanisms modulate the shape and not just the overall size of the developing chick eye. A retinal mechanism is consistent with the known ocular localizations of GABA and its receptors, with the developmental responses of the eye to these drugs and with the small reduction in retinal GABA in form-deprived eyes. Although GABA probably has many roles in the retina, the nature of the GABA drug effects on the growth of both goggled and nongoggled eyes and the predominant localization of GABA_A0r receptors in retina compared with other regions of the central nervous system ²⁶ suggest that GABA pharmacology may be useful in studying retinal mechanisms that modulate the growth or geometric form of the eye and may provide leads to novel approaches to the inhibition of myopia. 

²  Present affiliations: Department of Ophthalmology, Nissay Hospital, Osaka, Japan; and the

³  Division of Ophthalmology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania.

 Supported by National Eye Institute Grant EY-07354, the Paul and Evanina Bell Mackall Foundation Trust, and a grant from Valley Forge Pharmaceuticals, Inc., Irvine, California. RAS is the recipient of a Research to Prevent Blindness Physician-Scientist Award, New York, New York.

 Submitted for publication July 31, 2002; revised December 3, 2002 and March 19, 2003; accepted April 17, 2003.

 Disclosure: R.A. Stone, Valley Forge Pharmaceuticals, Inc. (F, I, C, P); J. Liu, None; R. Sugimoto, None; C. Capehart, None; X. Zhu, None; K. Pendrak, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Drugs, Activity, and Dose Ranges

Drug	Pharmacologic Activity*	Chemical Name and Drug Supplier, †	Dose Ranges (per 10-μL injection)	Calculated Vitreous Level, ‡ (μM)
GABAA drugs
Muscimol	Mixed GABAA and GABAA0r agonist	Muscimol hydrobromide (R)	5–200 μg; 25.6–1020 nmol	160–6410
TACA	Mixed GABAA and GABAA0r agonist	trans-4-Aminocrotonic acid (T)	10–100 μg; 98.9–989 nmol	618–6180
Bicuculline	Antagonist	(−)-Bicuculline methobromide (R)	0.01–50 μg; 0.0216–108 nmol	0.135–676
SR95531	Antagonist	6-Imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (T)	1–100 μg; 2.72-272 nmol	17.0–1700
GABAA0r drugs
CACA	Agonist	cis-4-Aminocrotonic acid (R)	10–200 μg; 98.9–1980 nmol	618–12360
TPMPA	Antagonist	(1,2,5,6-Tetrahydropyridine-4-yl)methylphosphinic acid (R)	0.1–200 μg; 0.621–1240 nmol	3.89–7760
GABAB drugs
Baclofen	Agonist	R(+)-Baclofen (R)	10–100 μg; 40–400 nmol	250–2500
CGP46381	Antagonist	(3-Aminopropyl)(cyclohexylmethyl)phosphinic acid (T)	1–200 μg; 4.56–912 nmol	28.5–5701
SCH50911	Antagonist	(+)-(2S)-5,5-Dimethyl-2-morpholineacetic acid (T)	10–200 μg; 57.7–1150 nmol	361–7217
2OH-saclofen	Antagonist	2-Hydroxysaclofen (R)	10–200 μg; 37.6–753 nmol	235–4700
CGP35348	Antagonist	(3-Aminopropyl)(diethoxymethyl)phosphinic acid (T)	1-500 μg; 4.44–2220 nmol	27.8–13880

Figure 1.

 

Drug effects on refractions of goggled eyes are shown for drugs selective for (A) GABA_A, (B) GABA_A0r, and (C) GABA_B receptors. n = number of chicks in each cohort. Probabilities apply to the ANOVA comparison of goggled with contralateral nongoggled eyes. NS, not significant. For pair-wise comparisons by the Tukey test, see Table 2 .

Figure 4.

 

Drug effects on refractions of nongoggled eyes. The refractive effects are shown for drugs that influenced at least one parameter, refraction or size measurements (see Fig. 5 ), when administered unilaterally to nongoggled chicks. The probabilities shown apply to the ANOVA comparison of drug-treated with contralateral vehicle-treated eyes. NS, not significant in the drug-treated to contralateral vehicle-treated eye comparison. †Effects reached statistical significance in the dose comparison, but not in the drug-treated to vehicle-treated eye comparison. For pair-wise comparisons, see Table 3 . n = number of chicks in each cohort.

Table 2.

 

Post Hoc Pair-wise Comparisons of Drug Effects on Goggled Eyes

Drug	Specificity	Refraction	Axial Length			Vitreous Chamber Depth (Ultrasound)	Equatorial Diameter (Calipers)
								Ultrasound	Calipers
Bicuculline	GABAA antagonist	NS	NS	NS	NS	10 μg vs. control;
							10 μg vs. 0.01 μg
SR95531	GABAA antagonist	50 μg vs. control	NS	NS	NS	100 μg vs. control, 1 μg;
							50 μg vs. control, 1 μg;
						10 μg vs. control, 1 μg
CACA	GABAA0r agonist	200 μg vs. 50 μg, 10 μg	NS	NS	NS	NS
TPMPA	GABAA0r antagonist	200 μg vs. control;	100 μg vs. control;	NS	200 μg vs. control;	200 μg vs. control, 1 μg, 0.1 μg;
			100 μg vs. control;	10 μg vs. control			100 μg vs. control;
		50 μg vs. control;			10 μg vs. control;	100 μg vs. control, 10 μg, 1 μg, 0.1 μg;
		10 μg vs. control			1 μg vs. control
						50 μg vs. control
Baclofen	GABAB agonist	10 μg vs. control	NS	NS	NS	NS
CGP46381	GABAB antagonist	200 μg vs. control;	100 μg vs. control	100 μg vs. control;	200 μg vs. control;	200 μg vs. control;
			100 μg vs. control, 1 μg;		10 μg vs. control	100 μg vs. control, 1 μg	100 μg vs. control
		50 μg vs. control;
SCH50911	GABAB antagonist	50 μg vs. control	NS	NS	NS	NS
2OH-Saclofen	GABAB antagonist	100 μg vs. control, 10 μg	NS	NS	NS	NS

Table 3.

 

Post Hoc Pair-wise Comparisons of Drug Effects on Nongoggled Eyes

Drug	Specificity	Refraction	Axial Length			Vitreous Chamber Depth (Ultrasound)	Equatorial Diameter (Calipers)
								Ultrasound	Calipers
Muscimol	GABAA, GABAA0r agonist	50, 10 μg	200, 50, 10, 5 μg	200, 50, 10 μg	200, 50, 10 μg	200, 50, 10 μg
SR95531	GABAA antagonist	*	, ‡	100 μg	100, 50 μg	NS
CACA	GABAA0r agonist	NS	50 μg	NS	NS	NS
TPMPA	GABAA0r antagonist	, †	10 μg	200, 100 μg	, ∥	200, 100 μg
Baclofen	GABAB agonist	NS	NS	, §	100 μg	NS
CGP46381	GABAB antagonist	NS	NS	NS	10 μg	NS

Table 4.

 

Overview of Significant GABA Drug Effects on Both Goggled and Nongoggled Eyes

Drug	Drug Effects on Form-Deprived Eyes											Drug Effects on Eyes with Unimpaired Visual Input
		Drug Activity	Refraction	Axial Length (Ultrasound)	Axial Length (Caliper)	Vitreous Chamber	Equatorial Diameter	Refraction	Axial Length (Ultrasound)	Axial Length (Caliper)	Vitreous Chamber	Equatorial Diameter
GABAA drugs
Muscimol	Agonist	−	−	−	−	−	↓↓	↑↑↑	↑↑↑	↑↑↑	↑↑↑
TACA	Agonist	−	−	−	−	−	−	−	−	−	−
Bicuculline	Antagonist	−	−	−	−	↓↓	Not tested	Not tested	Not tested	Not tested	Not tested
SR95531	Antagonist	↑	−	−	−	↓↓↓	−	−	↑	↑↑	−
GABAA0r drugs
CACA	Agonist	↓↑	−	−	−	−	−	↑↑	−	−	−
TPMPA	Antagonist	↑↑↑	↓↓	−	↓↓↓	↓↓↓	−	↑↑	↑↑	↑↑	↓↓↓
GABAB drugs
Baclofen	Agonist	↑	−	−	−	−	−	−	↑	↑	−
CGP46381	Antagonist	↑↑↑	↓↓	↓↓↓	↓↓↓	↓↓	−	−	−	↑	−
SCH50911	Antagonist	↑	−	−	−	−	Not tested	Not tested	Not tested	Not tested	Not tested
2OH-saclofen	Antagonist	↑↑↑	−	−	−	−	Not tested	Not tested	Not tested	Not tested	Not tested
CGP35348	Antagonist	−	−	−	−	−	Not tested	Not tested	Not tested	Not tested	Not tested

Figure 2.

 

Effects of GABA_A- and GABA_A0r-selective drugs on dimensions of goggled eyes. The number of chicks in each experimental group appears in Figure 1 . Probabilities apply to the ANOVA comparison of goggled with contralateral nongoggled eyes. NS, not significant. For pair-wise comparisons, see Table 2 .

Figure 3.

 

Effects of GABA_B drugs on dimensions of goggled eyes. The influences of drugs selective of the GABA_B receptor subtype are shown. The number of chicks in each experimental group appears in Figure 1 . Probabilities apply to the ANOVA comparison of drug-treated goggled to contralateral nongoggled eyes. NS, not significant. For pair-wise comparisons, see Table 2 .

Figure 5.

 

Drug effects on dimensions of nongoggled eyes. Effects are shown for drugs that influenced at least one parameter when administered to one eye of nongoggled chicks. The number of chicks in each cohort appears in Figure 4 . Probabilities apply to the ANOVA comparison of drug-treated to contralateral vehicle-treated eyes. NS, not significant in the drug-treated to contralateral vehicle-treated eye comparison. ‡Effects reached statistical significance in the dose–eye interaction only, not in the drug-treated to contralateral vehicle-treated eye comparison. For pair-wise comparisons, see Table 3 .

The authors thank Maureen G. Maguire for valuable statistical advice.