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(Investigative Ophthalmology and Visual Science. 2006;47:25-33.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0195
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Melatonin Receptors in Chick Ocular Tissues: Implications for a Role of Melatonin in Ocular Growth Regulation

Jody A. Summers Rada1 and Allan F. Wiechmann1,2

1From the Departments of Cell Biology and 2Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The influences of diurnal rhythms involving a variety of ocular parameters are implicated in the development of myopia. The purpose of this study was to determine the expression of the melatonin receptor subtype proteins in chick ocular tissues and to examine the role of the circadian signaling molecule melatonin in normal ocular growth and the exaggerated ocular growth associated with the development of myopia.

METHODS. Expression of the Mel1a, Mel1b, and Mel1c melatonin receptor proteins in ocular tissues was examined by Western blot analyses, slot blot analyses, and immunocytochemistry. For examining the effect of melatonin on ocular growth, chicks were maintained on a 12-hour light–dark cycle and were monocularly form-vision deprived in one eye with a translucent occluder for 5 days. During the 5-day treatment period, chicks were injected systemically during the early dark period with melatonin (0.6 mg) or 2% ethanol vehicle control. Ocular dimensions of normal and deprived eyes were examined by high frequency A-scan ultrasound.

RESULTS. Immunocytochemical analysis of chick ocular tissues revealed the cellular distribution of the Mel1a receptor subtype in the cornea, choroid, sclera, and retina. Western blot and slot blot analyses demonstrated that all three receptors were present in these tissues and they demonstrated distinct diurnal rhythms of protein expression in the retina-RPE-choroid, with peak levels of Mel1a and Mel1b occurring during the night and peak levels of Mel1c during the day. Systemic administration of melatonin resulted in significant changes in anterior chamber depth, vitreous chamber depth, and choroidal thickness of form-deprived and/or control eyes.

CONCLUSIONS. Results of this study show that all three melatonin receptor subtypes are expressed in retinal and extraretinal ocular tissues of the chick eye. The finding that administration of melatonin alters the growth of several ocular tissues in both control and form-deprived eyes suggests that melatonin, acting through specific melatonin receptors in ocular tissues, plays a role in ocular growth and development. This conclusion suggests that the action of melatonin, combined with expression of melatonin receptors, is involved in the regulation of the diurnal rhythm of ocular growth.

In the vertebrate eye, various physiological parameters have been reported to operate under the influence of daily rhythms. A regular diurnal rhythm of light and dark periods has been shown to be essential for normal ocular elongation and emmetropization (accurate refractive development). In chicks, the rate of ocular elongation exhibits a pronounced growth rhythm, with peak growth occurring during the day and slowed growth occurring at night under normal visual conditions.1 2 3 In eyes with induced myopia, increases in the amount of nighttime ocular growth result in overall increased axial length.1 2 3 Rearing chickens in constant light or constant darkness also produces excessive ocular elongation in otherwise normal eyes.4 5 6 Together, these results suggest that normal ocular growth requires a diurnal rhythm of pattern (or form) vision.

The indoleamine hormone melatonin transmits daily and seasonal timing information to a variety of tissues in essentially all vertebrate species. Melatonin is rhythmically synthesized by pinealocytes, retinal photoreceptors, and ciliary epithelial cells7 8 on a diurnal rhythm with peak levels occurring in the dark period.7 9 The circadian rhythm of melatonin synthesis and release by retinal cells has been shown to modulate a variety of biological rhythms in the eye, including circadian photoreceptor outer segment disc shedding,10 11 regulation of horizontal cell sensitivity to light,12 modulation of dopamine release,13 photomechanical movements,14 and circadian changes in intraocular pressure.15 The specific functions of melatonin are mediated by G-protein-coupled receptors. Three melatonin receptors (Mel1a, Mel1b, and Mel1c) have been cloned.16 17 18 The Mel1a receptor sequence is homologous to the mammalian MT1 melatonin receptor, and the Mel1b receptor is homologous to the mammalian MT2 receptor. The Mel1c receptor has been cloned from Xenopus laevis, chickens, and zebrafish,18 but an equivalent receptor has not been found to be expressed in mammals. In mammals, MT1 receptors are expressed primarily in the suprachiasmatic nucleus and the pars tuberalis and appear to mediate the circadian and reproductive effects of melatonin.19 20 Mammalian MT2 receptors are expressed in the retina and various brain areas and may mediate the effects of melatonin on retinal physiology, such as the modulation of dopamine release.19 20 All the mammalian and nonmammalian melatonin receptor subtypes studied so far appear to be negatively coupled to cyclic adenosine monophosphate (AMP) synthesis, although alternative signaling pathways have also been reported in a variety of tissues and species.19 20 Immunocytochemical labeling of Mel1a, Mel1b, and Mel1c melatonin receptors in Xenopus retinas has demonstrated intense labeling for melatonin receptors on amacrine cells in the inner plexiform layer (Mel1a, Mel1c), horizontal cells (Mel1a, Mel1b, and Mel1c), rod and cone photoreceptors (Mel1b and Mel1c), ganglion cells (Mel1b and Mel1c), and RPE (Mel1b) (Weichmann AF, et al. IOVS 2004;45:ARVO E-Abstract 4294).20 21 22

Results presented in the present study indicate that Mel1a, Mel1b, and/or Mel1c melatonin receptor subtypes are expressed in the chick retina as well as in the cornea, ciliary body, choroid, retina, and sclera of chick eyes in distinct tissue specific patterns. In the chick retina, the expression of each melatonin receptor subtype had distinct rhythms of protein expression over a 24-hour period. Moreover, administration of melatonin resulted in alterations in several parameters of ocular growth in normal and form-deprived (myopic) eyes. Taken together, these data indicate that the differential distributions and rhythms of melatonin receptor subtypes in chick ocular tissues mediate distinct downstream cellular functions of melatonin and suggest possible roles for melatonin in modulating rhythms in ocular growth, anterior chamber depth, aqueous humor production, and intraocular pressure.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
White Leghorn chickens (Gallus gallus) were obtained as 2-day old hatchlings from Ideal Breeding Poultry Farms (Cameron, TX). Birds were housed in temperature-controlled brooders with a 12-hour light–dark cycle (lights on at 6 AM and off at 6 PM) and were fed food and water ad libitum. To induce myopia, form deprivation was introduced with translucent plastic occluders cut from the bottoms of 15-mL round-bottomed test tubes and affixed with cyanoacrylate adhesive to the feathers around the right eye of 2-day-old chicks. Occluders remained in place for 5 days. Melatonin (0.6 mg/chick in 2% ethanol) was administered via intraperitoneal injection at the beginning of the dark phase to monocularly deprived chicks (n = 25). This dosage was designed to achieve a final concentration of 10 mg/kg, based on an average chick weight of 60 g and is comparable with several previous studies in which melatonin was applied systemically to rats and mice in doses ranging from 1 to 30 mg/kg.23 24 25 Moreover, previous studies of rats indicate that approximately 60 picograms from a single 100-µg intraperitoneal dose of melatonin reaches the retina, with the peak dose occurring 15 minutes after injection and a subsequent decrease to baseline levels within 2 hours.26 Therefore, based on these measurements, we estimate that approximately 6 µg of melatonin reached the eye shortly after each intraperitoneal injection of melatonin, and baseline levels were established well before the next injection. Melatonin injections were administered daily for the duration of the deprivation period (5 days). A control group (n = 25) of monocularly deprived chicks received injections of vehicle (2% ethanol). Ocular measurements were made with high-frequency ultrasound, before and after the form deprivation and melatonin treatment periods, and the differences in anterior chamber depth between the first and last measurements were compared in melatonin-treated and untreated eyes.

Tissue Preparation
Normal 7-day-old chicks were anesthetized with isoflurane (Iso-thesia; Vetus Animal Health, North Chicago, IL) and euthanatized by an overdose of isoflurane. For immunocytochemistry, eyes were enucleated, a small window was cut in the equatorial region of each eye, and tissues were fixed for 18 hours at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), followed by immersion in 30% sucrose in phosphate buffer for 16 to 20 hours at 4°C. Eyes were then embedded and frozen in optimal cutting temperature (OCT) embedding compound (Tissue-Tek, Sakura FineTek, Torrance, CA). Sagittal 10-µm-thick sections were cut on a cryostat microtome and collected on glass slides. For Western blot analysis, chick ocular tissues were isolated from normal eyes of 7-day-old chicks, placed separately into 1.5-mL microcentrifuge tubes, and immediately snap frozen in liquid nitrogen. The animals were cared for in accordance with the NIH guidelines as described in Principles of Laboratory Animal Care and according to the ARVO Statement for the use of Animals in Ophthalmic and Vision Research.

Antibody Development
Polyclonal antibodies directed against a peptide corresponding to a region of an intracytoplasmic loop of the chicken Mel1a, Mel1b, and Mel1c receptors (Mel1a, KPD NNP RLK PHD FR; Mel1b, SKG GTD GQK SKP SP; and Mel1c, HSL RYD KLF NLK NT), were generated in goats, rabbits, and chickens, respectively (Invitrogen, Carlsbad, CA). The 14-amino acid peptides were conjugated to a multiple antigenic peptide (MAP) lysine scaffold and used to immunize the animals. Pooled antisera against the receptor peptides were affinity purified against the antigen peptide conjugated to a solid support matrix. The peptide synthesis, conjugation, immunizations, and affinity purification were all performed by Invitrogen.

Western Blot Analyses
For retinal and RPE extractions, each sample was homogenized in 200 µL extraction buffer (19 mM Tris [pH 7.4] containing 9.5 mM EGTA, 4.8 mM iodoacetic acid, 3 mM MgCl2, 1.5 mM phenylmethylsulfonyl fluoride (PMSF), 10% sucrose, 1% NP-40, and 13 mM leupeptin) on ice with a homogenizer (Rotor-Stador; VirTis, Gardiner, NY). For cornea, lens, and scleral extractions, frozen tissues were pulverized in microcentrifuge tubes under liquid nitrogen with a homogenizer mounted on a standard drill (Dounce; Bellco Glass, Vineland, NJ) Pulverized samples were homogenized in 200 µL of extraction buffer with the Rotor-Stador homogenizer. All tissue homogenates were then centrifuged at 100,000g with a micro ultracentrifuge at 4°C for 1 hour. Supernatants were collected, assayed for protein using the Bradford assay (Bio-Rad, Hercules, CA) and used in Western blot experiments. Ocular tissue lysates were electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels under reducing conditions. Five micrograms of total protein from each tissue was mixed with an equal volume of 2% SDS loading buffer (60 mM Tris-HCl [pH 6.8], 2.5% SDS, 0.1% ß-mercaptoethanol, and 0.25% glycerol), and boiled for 5 minutes. Proteins were separated by SDS-PAGE, electrophoretically transferred to a nitrocellulose membrane, and subjected to Western blot analysis with affinity-purified polyclonal anti-Mel1a, -Mel1b, or -Mel1c receptor antibodies, followed by incubation with rabbit anti-goat IgG, goat-anti-rabbit IgG, or rabbit anti-chicken IgG conjugated to alkaline phosphatase. Immunoreactive proteins were detected with a chemiluminescent substrate (Western Star; Tropix, Bedford, MA). Control blot analyses were performed as just described, except that incubation with the anti-Mel1a, -Mel1b, or -Mel1c antibody was omitted.

Diurnal Expression of Melatonin Receptor Protein
Chicks were reared in a 12-hour light–dark cycle for 10 days, at which time birds were euthanatized every 4 hours over a 24-hour period (5 birds/time point). Both eyes were enucleated and immediately snap frozen in liquid nitrogen. Retina-RPE-choroid complexes were later isolated from both eyes of each chick, and tissue homogenates were prepared as described for Western Blot analyses, and 5 µg of total protein was applied in triplicate to wells of a 48-well slot blot apparatus (Bio-Dot SF; Bio-Rad). Briefly, slot blot wells were rinsed with TBS buffer and membranes were removed from the apparatus, blocked with protein solution, and incubated with anti-Mel1a and anti-Mel1b antibodies, followed by incubation with the appropriate secondary antibody and detection with chemiluminescent substrate as described for Western blot analysis. Because of the presence of nonspecific bands on Mel1c Western blot analysis (presumably because the antibody was raised in chickens), quantification of Mel1c receptor protein was determined by Western blot rather than slot blot analysis. Five micrograms of total protein from each tissue homogenate was applied in triplicate to a 10% SDS-PAGE gel and subjected to Western blot. Bands on slot blot and Western blot analyses were digitized and quantified (Chemi Genius2 Bioimaging System; Syngene, Frederick, MD).

Immunohistochemistry
For immunocytochemical localization of the Mel1a receptor in chick ocular tissues, cryostat sections were rinsed in PBS and then incubated in incubation buffer (1% normal goat serum [Sigma-Aldrich, St. Louis, MO], 0.2% Triton X-100, and 0.004% sodium azide in PBS) for 30 minutes at room temperature (RT). Sections were incubated with 2 µg/mL of Mel1a receptor antibody in incubation buffer for 3 days at 4°C. In control experiments, the tissue sections were incubated in 2 µg/mL normal goat immunoglobulin (Sigma-Aldrich) instead of the Mel1a antibody. After the 3-day incubation with the primary antibody, the sections were rinsed in PBS, and incubated in 5 µg/mL Alexa Fluor 488 (green) or 568 (red) conjugated with rabbit anti-goat antibody (Molecular Probes; Eugene, OR) for 30 minutes at RT. Sections were rinsed in PBS, incubated with 0.0005% 4',6'-diamino-2-phenylindole (DAPI) nuclear stain for 10 seconds at RT, followed by a final rinse in PBS. Coverslips were mounted on the slides (Prolong Gold Antifade medium; Molecular Probes), and the immunolabeled sections were viewed by confocal microscopy (510 LSM; Carl Zeiss Meditec GmbH, Jena, Germany).

High-Frequency A-Scan Ultrasound
Chicks were anesthetized with isoflurane (Vedco Inc., St. Joseph, MO) inhalation anesthesia (0.8% in oxygen) and placed on a body and head support device adjacent to the ultrasound probe. A-scan ultrasonography was performed using a 30-mHz polymer transducer (Panametrics, Waltham, MA), linked to a computer interfaced with a an A/D board (model 8100; Sonix, Springfield, VA) similar to that previously described.27 Traces were obtained with a data-acquisition program (designed in LabView Professional, ver. 4.0; National Instruments, Austin, TX) at a sampling frequency of 800 MHz. Peaks corresponding to tissue interfaces were detected with a matched filter algorithm in conjunction with a measurement program (designed in LabView) (Rada K, et al. IOVS 1999;40:ARVO Abstract 4000).

Data Analysis
Measurements of ocular tissue thicknesses and chamber depths were determined from tissue interfaces detected through the use of an automated matched-filter algorithm (Rada K, et al. IOVS 1999;40:ARVO Abstract 4000). The axial length was defined as the distance from the front of the cornea to the front of the sclera, as determined from the sum of the calculated corneal thickness, anterior chamber depth, lens thickness, vitreous chamber depth, retina thickness, and choroid thickness. The growth changes that occurred in each ocular tissue or chamber depth over the treatment period were calculated by subtracting the ocular measurements taken at the beginning of the treatment period from those taken at the end of the experiment. Comparisons between form-deprived and contralateral control eyes were made using two-tailed paired t-tests. Comparisons between groups were made using a one-way analysis of variance (ANOVA). For experiments measuring melatonin receptor protein levels every 4 hours over a 24-hour period, post hoc multiple comparisons were performed using the Scheffé method for pair-wise comparisons to determine whether there were significant differences between groups.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Identification of Melatonin Receptors in Chick Ocular Tissues
Monospecific polyclonal antibodies were developed against intracytoplasmic sequences of the chicken Mel1a, Mel1b, and Mel1c melatonin receptors (Weichmann AF, et al., IOVS 2002;43:ARVO E-Abstract 4294) which were used to determine the distribution of melatonin receptor subtype expression in chick tissues. By using these antibodies in Western blot analysis, we have detected specific Mel1a, Mel1b, and Mel1c receptor protein in the cornea, ciliary body, retina, choroid, and sclera (Fig. 1) .



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  		FIGURE 1. Western blot analyses of chick ocular tissues with Mel1a, Mel1b, and Mel1c receptor antibodies. Whole tissue homogenates were separated by SDS-PAGE, blotted to nitrocellulose, and labeled with affinity-purified antibodies against the Mel1a, Mel1b, and Mel1c melatonin receptors. (A) Multiple Mel1a-immunoreactive bands were present at {approx}42 to 65 kDa in cornea (Co), ciliary body (CB), retina (Ret), choroid (Ch), and sclera (Scl). Preincubation of Mel1a antibodies with (+) the 14 amino acid Mel1a peptide (1 µM final concentration) used to construct the antigen for anti-Mel1a antibody production abolished all major anti-Mel1a immunoreactive bands, with the exception of the bands in the CB lane migrating at ≤35 kDa. (B) Mel1b immunoreactivity was limited to a major band migrating at {approx}48 kDa in the retina, with much lower amounts of this band in the sclera and in the ciliary body at {approx}55 kDa and 25 to 30 kDa. (C) Specific Mel1c immunoreactivity could be detected in the cornea at 45 and 49 kDa; in the ciliary body at 38 and 49 kDa, in the retina at 38 kDa, in the choroid at 30 kDa, and in the sclera at 38 and 42 kDa. The lower-molecular-mass bands (15–20 kDa) in the cornea, retina, and ciliary body homogenates may represent fragments of the Mel1c receptor. No 1°: duplicate blots incubated with anti-goat, rabbit, or chicken IgG-alkaline phosphatase secondary antibody in the absence of anti-Mel1a, anti-Mel1b, or anti-Mel1c, respectively. Molecular mass standards are indicated to the left of the blots.

 
Multiple Mel1a immunoreactive bands were present at {approx}42 to 65 kDa in chick cornea (Co), ciliary body (CB), retina (Ret), choroid (Ch), and sclera (Scl; Fig. 1 ). Because other multiple bands were present on Mel1a Western blots, a peptide-blocking experiment was performed to determine the specificity of the anti-Mel1a antibodies (+Mel1a; 1 µM). Five micrograms of protein extracted from ciliary body, retina, choroid, and sclera were subjected to Western blot analysis with anti-Mel1a antibodies, with and without prior incubation of blots with the 14-amino-acid Mel1a peptide (1 µM final concentration) used to construct the antigen for anti-Mel1a antibody production. Bands migrating at {approx}35 kDa and lower, as well as bands at {approx}62 and {approx}80 kDa present in tissue extracts of ciliary body (CB) remained after peptide blocking, indicating that these bands represent nonspecific immunoreactivity in ciliary body extracts. In contrast, bands in ciliary body extracts migrating at {approx}42 and {approx}60 kDa, as well as all major Mel1a-immunoreactive bands in retina, choroid, and sclera were abolished after the peptide block, suggesting that these bands represent the Mel1a receptor protein in these tissues. The multiple bands at 42 to 65 kDa in ciliary body and retina after incubation with anti-Mel1a may represent homodimerization and/or heterodimerization with other melatonin receptor subtypes as has been described for the MT1 and MT2 melatonin receptor28 as well as other G-protein-coupled receptors, such as rhodopsin,29 the {delta}-opioid receptor,30 the serotonin 5 HT(2C) receptor,31 and the ß2 adrenergic receptor.32 In addition, digestion of retinal and ciliary body extracts with N-glycanase before Western blot analysis with anti-Mel1a had no effect on the migration of immunoreactive bands, suggesting that very little n-glycosylation was present on the melatonin Mel1a receptor protein, and that differences in n-glycosylation are not responsible for the multiple Mel1a-specific bands on the Western blots (data not shown). In contrast to Mel1a immunoreactivity, Mel1b immunoreactivity was limited to a major band migrating at {approx}48 kDa in the retina, with much lower amounts of this band present in the sclera. Immunoreactive bands were also detected in the ciliary body at {approx}55 and 25 to 30 kDa. No bands were present in the cornea or choroid. Specific Mel1c immunoreactivity was detected in the cornea at 45 and 49 kDa, in the ciliary body at 38 and 49 kDa, in the retina at 38 kDa, in the choroid at 30 kDa, and in the sclera at 38 and 42 kDa. The lower-molecular-weight bands (15–20 kDa) in the cornea, retina, and ciliary body homogenates may represent fragments of the Mel1c receptor. The bands at 64 and 28 kDa in the Mel1c-labeled blots were nonspecific, as they were detected with the anti-chicken IgG-alkaline phosphatase secondary antibody in the absence of the primary antibody (Fig. 1 ; no 1°,). Molecular weight standards are indicated to the left of the blots.

Immunohistochemistry
Only anti-Mel1a antibodies were suitable for immunohistochemistry. With confocal microscopy, specific immunolabeling of the Mel1a melatonin receptor was detected in the corneal epithelium, corneal stroma (Fig. 2) , and corneal endothelium (Fig. 3) , as well as in the outer and inner fibrous layers (OFL, IFL) and the cartilaginous layer (CL) of the sclera (Fig. 4) . In the retina, specific Mel1a immunolabeling was detected in the photoreceptor cell layer, in the inner and outer plexiform layers (IPL, OPL), suggestive of horizontal and/or amacrine cell labeling, and in the ganglion cell (GCL) and nerve fiber (NFL) layers (Fig. 5) . In addition, moderate Mel1a immunolabeling was detected in the choroid (Fig. 5) .



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  		FIGURE 2. Confocal image of immunocytochemistry of chick cornea with Mel1a melatonin receptor antibodies. (A) Corneal section incubated with Mel1a receptor antibody labeled with a secondary antibody conjugated to a red fluorescent dye. Mel1a labeling was intense in corneal epithelium (arrowheads), especially in the basal layer of cells and in the keratinocytes of the corneal stroma (arrows). (B) Corneal section incubated with normal goat IgG labeled with a secondary antibody conjugated to red fluorescent dye. Tissues are stained with a blue nuclear dye. EP, corneal epithelium. Scale bar, 100 µm.

 


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  		FIGURE 3. Confocal image of immunocytochemistry of chick corneal endothelium with Mel1a melatonin receptor antibodies. Corneal section incubated with Mel1a receptor antibody labeled with a secondary antibody conjugated to a red fluorescent dye. Mel1a labeling is present in keratinocytes of the corneal stroma (arrowheads) and is intense in the corneal endothelium (arrow). Tissues are stained with a blue nuclear dye. Scale bar, 100 µm.

 


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  		FIGURE 4. Confocal image of immunocytochemistry of chick sclera with Mel1a antibodies. (A) Scleral section incubated with the Mel1a receptor antibody labeled with a secondary antibody conjugated to a red fluorescent dye. Mel1a labeling was present in the OFL (arrowheads), IFL, and on chondrocytes in the CL (arrows). (B) Scleral section incubated with normal goat IgG labeled with a secondary antibody conjugated to red fluorescent dye. Tissues are stained with a blue nuclear dye. Scale bar, 100 µm.

 


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  		FIGURE 5. Confocal image of immunocytochemistry of chick choroid and retina with Mel1a receptor antibodies. (A) Retina-RPE-choroid section incubated with the Mel1a receptor antibody labeled with a secondary antibody conjugated to a red fluorescent dye. Mel1a labeling is present in the photoreceptor (PH) inner segment layer, outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), and nerve fiber layer (NFL). (B) Retina-RPE-choroid section incubated with normal goat IgG labeled with a secondary antibody conjugated to red fluorescent dye. Tissues are stained with a blue nuclear dye. Scale bar, 100 µm.

 
Rhythms in Melatonin Receptor Protein Expression
Melatonin-specific antibodies were used in Western and slot blot analyses to determine the temporal patterns of protein expression for each melatonin receptor subtype in the retina-RPE-choroid complexes isolated from the posterior poles of normal chick eyes over a 24-hour period. A diurnal rhythm in Mel1c receptor protein expression was identified in which levels were high during the early morning and low during the night. In contrast, the rhythms of Mel1a and Mel1b generally appeared to be opposite of Mel1c, with lowest levels occurring in the early morning and highest levels in the evening (Fig. 6) .



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  		FIGURE 6. Diurnal rhythms in Mel1a, Mel1b, and Mel1c receptor protein expression in the retina-RPE-choroid. Retina-RPE-choroid complexes were isolated from the posterior poles of chick eyes over a 24-hour period (12-hour light–dark). Lights went on at 6:00 AM (0 hour of circadian time) and went off at 6:00 PM (12 hours of circadian time). Eyes harvested at the 24-hour circadian time point were harvested in the dark. (A) Mel1a receptor protein was quantified on slot blots with affinity purified anti-Mel1a receptor antibodies. Peak levels of Mel1a receptor protein were detected at 12 hours of circadian time and were significantly increased compared with the circadian time point 0 (P < 0.05, ANOVA; Scheffé post hoc multiple comparisons). (B) Mel1b receptor protein was quantified on slot blots using affinity purified anti-Mel1b receptor antibodies together with slot blot analyses. Levels of Mel1b receptor were low in the morning and rose significantly in the afternoon and evening (*P < 0.01 for circadian time points 8, 12, 16, and 20 hours, compared with circadian time points 0, 4, and 24 hours ANOVA; Scheffé post hoc multiple comparisons). (C) Mel1c receptor protein was quantified on Western blot analysis using affinity purified anti-Mel1c receptor antibodies. Five micrograms of total protein was applied to each well of the Western blot and the 38-kDa band was quantified by densitometry. Mel1c receptor levels were significantly reduced at night, reaching lowest levels at the 16-hour circadian time point (*P < 0.01, compared with circadian time points 0 and 24, ANOVA; Scheffé post hoc multiple comparisons; n = 5 birds in each group).

 
Role of Melatonin Receptors on the Regulation of Ocular Growth
The effect of systemic administration of melatonin was evaluated on normal ocular growth and exaggerated ocular growth associated with form-deprivation myopia. Melatonin (0.6 mg) was administered to chicks undergoing monocular form deprivation induced by intraperitoneal injections at the beginning of the dark cycle (7:00 PM). Ocular measurements were taken before and after 5 days of form deprivation and treatment with melatonin (or ethanol control), with a high-frequency A scan ultrasound used to determine the absolute tissue thicknesses and chamber depths (Table 1) at the end of the treatment period as well as the change in chamber lengths and tissue thicknesses that occurred over the 5-day treatment period (Table 2) . Systemic administration of melatonin at the beginning of the dark cycle resulted in a significant reduction in the depth of the anterior chamber of form-deprived eyes compared with anterior chamber depth of form-deprived eyes from ethanol-deprived chicks (–6.4%, P = 0.030, data not shown). The decrease in anterior chamber depth observed in melatonin-treated, form-deprived eyes reflected a significant reduction in the growth of the anterior chamber in deprived eyes over the 5-day deprivation period (*P = 0.05, Fig. 7A ). Anterior chambers of deprived and control eyes increased in depth by approximately 97 to 106 µm in vehicle-treated (2% ethanol) chicks over the 5-day experimental period. Systemic administration of melatonin resulted in a significant reduction in anterior chamber elongation over the 5-day treatment period ({approx}33µm, P = 0.05) in form-deprived eyes only, whereas contralateral control eyes of melatonin-treated chicks showed a trend toward increased anterior chamber elongation over the 5-day period (133 µm), although this did not reach statistical significance (P = 0.13). Of interest, vitreous chamber depth was affected differentially and in the opposite manner as anterior chamber depth. The vitreous chamber of contralateral control eyes of melatonin-treated chicks was significantly longer than that of contralateral eyes of ethanol control-treated chicks after the 5-day treatment period (P = 0.03, Table 1 ) resulting from significantly more growth over the 5-day treatment period (P = 0.005, Table 2 , Fig. 7B ). Vitreous chamber depth was not affected by melatonin treatment in form-deprived eyes, although a trend toward increased growth over the 5-day treatment period was noted (Table 2 P = 0.06).


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  		TABLE 1. Ocular Measurements after 5 Days of Treatment with Melatonin and Visual Form Deprivation

 

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  		TABLE 2. Change in Ocular Measurements after 5 Days of Treatment with Melatonin and Visual Form Deprivation

 


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  		FIGURE 7. Effect of melatonin on ocular dimensions in control and form-deprived eyes. (A–C) High-frequency A-scan ultrasound measurements taken at the beginning of the treatment period were subtracted from measurements at the end of the treatment period to obtain a measurement of growth changes that occurred in response to systemic administration of melatonin. (A) Melatonin treatment resulted in a significant reduction in the amount of growth in the anterior chamber of deprived eyes over the 5-day deprivation period (*P = 0.05), but had no significant effect on contralateral control eyes (P = 0.13). (B) Melatonin treatment resulted in a significant increase in the amount of growth in the vitreous chamber of contralateral control eyes over the 5-day deprivation period (*P = 0.005), but had no significant effect on form-deprived eyes, although a similar trend was noted (P = 0.06). (C) Melatonin treatment resulted in significant thinning of the retina in form-deprived eyes over the 5-day treatment period compared with form-deprived eyes of ethanol-deprived birds (P = 0.02), but had no significant effect on the change in retinal thickness of contralateral control eyes (P = 0.92). (D) Effect of melatonin on choroidal thickness in control and form-deprived eyes at the end of the treatment period. Ultrasound measurements were taken at the end of the treatment period, and thickness changes were compared between melatonin- and vehicle-treated chicks. Melatonin treatment resulted in a significant decrease in choroidal thickness in form-deprived eyes when measured after the 5-day deprivation period (P = 0.02), but had no significant effect on the choroidal thickness of contralateral control eyes (P = 0.09) In all cases, n = 24 or 25 birds in each group, with significance determined by two-tailed t-test.

 
Retinas of form vision-deprived eyes of melatonin-treated chicks also demonstrated significant thinning over the 5-day deprivation and melatonin treatment period, compared with form-deprived eyes from ethanol-treated chicks (P = 0.02, Fig. 7C ), which resulted in a trend toward thinner retinas in form-deprived eyes of melatonin-treated chicks when measured after the treatment period (P = 0.06, compared with retinal thickness in form-deprived eyes of ethanol-deprived chicks, Table 1 ). Retinas of form-deprived eyes from ethanol-treated chicks were significantly thinner than retinas of contralateral control eyes (P < 0.001, paired t-test). Melatonin treatment also resulted in significant decreases in choroidal thickness in form-deprived eyes after 5 days of drug treatment compared with that ethanol-treated chicks (P = 0.02, Fig. 7D ). As expected, choroids of form-deprived eyes were significantly thinner than choroids of contralateral control eyes in both melatonin and 2% ethanol-treated chicks (P < 0.0001).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Several studies, mostly in chicks, have identified diurnal rhythms in the rate of axial elongation, intraocular pressure, and choroidal thickness, and have shown that alterations in these rhythms, or the phase relationships between them, affect the development of and recovery from myopia (Nickla DL, et al. IOVS 2004;45:ARVO E-Abstract 1158).2 3 33 34 35 36 37

Results in the present study indicate that the Mel1a, Mel1b, and/or Mel1c melatonin receptor subtypes are expressed in retinal and extraretinal locations in chick eyes in distinct tissue specific patterns. The differential distribution of melatonin receptor subtypes in ocular tissues suggests that these receptors mediate distinct downstream cellular functions of melatonin in these tissues. Moreover, the differential effect of systemic treatment of melatonin on form-deprived and contralateral control eyes suggests that form deprivation alters the cellular response to melatonin.

The expression of the membrane melatonin receptors (Mel1a, Mel1b, and Mel1c) on several ocular tissues suggests that they may mediate the melatonin effects on ocular growth. However, other pathways through which melatonin may act should also be considered. For example, there is a class of nuclear melatonin receptors (ROR/RZR) that are associated with melatonin signaling, and they appear to act as transcription factors to directly regulate gene expression.38 These widely distributed nuclear melatonin receptors may be primary targets of the membrane melatonin receptors, or melatonin may perhaps bind to them directly to influence gene expression. In addition, melatonin has well-documented antioxidant effects at pharmacological doses.39 40 Melatonin scavenges free radicals, stimulates activity of antioxidant enzymes, enhances mitochondrial oxidative phosphorylation, and augments the efficacy of other antioxidants.39 Considering the dosages of melatonin administered in this study, and the resultant presumed physiological levels of melatonin reaching the ocular tissues,26 it seems unlikely that the antioxidant properties of melatonin play a prominent role in its effects on ocular growth. Similarly, nuclear melatonin receptors are responsive at much higher dosages than presumed to be delivered to the ocular tissues in this study.38 Furthermore, we have observed that a membrane melatonin receptor antagonist has opposite effects on some aspects of ocular growth from that observed with melatonin administration (unpublished data).

Specific immunolabeling of the Mel1a melatonin receptor was detected in the outer and inner fibrous layers as well as the cartilaginous layer of the sclera, the choroid, retina, and the corneal epithelium, stromal cells, and endothelium. Of note, melatonin has recently been shown to modulate the hydration state of the cornea in vitro (Wahl CM, et al. IOVS 2004;45:ARVO E-Abstract 4293), which in humans is thought to account for daily changes in corneal thickness.41 The intense immunolabeling of Mel1a receptors on chick corneal endothelium supports the hypothesis that melatonin may modulate daily rhythms in corneal hydration/thickness via melatonin receptors on the corneal endothelium.

The results of the present study are the first to characterize the diurnal expression patterns of each melatonin receptor subtype at the protein level and suggest that distinct diurnal rhythms are present in the retina and/or choroid for each melatonin receptor subtype. The results of our quantitative Western blot analyses are in agreement with previous studies of Mel1c mRNA levels, which indicate that Mel1c is rhythmically expressed in Xenopus and chicks, with peak levels for Mel1c RNA occurring in the day.42 43 In contrast to the diurnal rhythm of Mel1c expression, our results indicate that the rhythms of Mel1a and Mel1b receptor protein expression generally appear to be opposite of Mel1c, with lowest levels occurring in the early morning and higher levels in the evening. We hypothesize that rhythms in melatonin receptor expression are superimposed on the circadian rhythm in melatonin synthesis as an additional level of regulation of melatonin’s downstream effects in the various ocular tissues.

Recently, small diurnal fluctuations in axial length have been identified in human eyes, with maximum axial length occurring at midday.44 Diurnal differences in anterior chamber depth have been reported for humans, being 60 µm greater in the morning than later in the day,45 which could contribute to diurnal fluctuations in axial length.44 Our findings that systemic administration of melatonin decreases anterior chamber depth in control eyes, but not in form-deprived eyes, suggests that diurnal fluctuations in anterior chamber depth may be due to a melatonin-mediated mechanism, and this mechanism may be disrupted in eyes undergoing myopia development.

Systemic administration of melatonin in form-deprived chick eyes resulted in significant thinning of the choroids and retinas of form-deprived eyes, and a trend toward choroidal thinning in contralateral eyes. Nitric oxide has been implicated as a factor regulating choroidal thickness in form-deprived eyes and eyes recovering from myopia.46 Intravitreal administration of the nonspecific nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) induced a rapid, transient, and dose-dependent thinning in experimentally thickened choroids (–116 to –219 µm) and in normal choroids (–47 µm). Recently, nitric oxide was also shown to inhibit arylalkylamine-N-acetyltransferase (AA-NAT), the activity of which reflects the changes in retinal melatonin synthesis.47 Taken together, our results and the results of these recent investigations suggest that nitric oxide, through its suppression of AA-NAT activity, may play a role in the modulation of choroidal thickness and permeability through actions on melatonin levels and subsequent effects on the choroidal vasculature, although the melatonin-induced effects observed in the present study were substantially less (–25 to –29 µm) than those induced by NOS inhibition.

Melatonin has been shown to result in photoreceptor cell loss and retinal thinning when administered systemically to albino rats.26 Our finding that melatonin treatment resulted in significant retinal thinning in form-deprived eyes only, suggests that the retinas of myopic eyes (already thinned over the expanding scleral shell) are highly susceptible to the cytotoxic effects of melatonin.

The results of the present study describe the distribution and diurnal expression of Mel1a, Mel1b, and Mel1c melatonin receptor proteins in the chick eye. The finding that melatonin administration alters the growth of several ocular tissues in both control and form-deprived (myopic) eyes suggests that melatonin, acting through specific melatonin receptors in retinal and extraretinal ocular tissues, plays a role in ocular growth and development. Because melatonin was systemically applied, it is possible that some of the ocular effects observed in melatonin-treated chicks may not be due to the direct effect of melatonin on each ocular tissue, but rather may be related to a shift in one or more ocular diurnal rhythms imposed by the melatonin treatment. Indeed, the magnitude of the change in choroidal thickness observed in the present study ({approx}25 µm) is approximately in the range of the diurnal differences in choroidal thickness previously described (16 µm).2 Additional studies are needed to identify the melatonin receptors responsible for mediating the action of melatonin on the growth of each ocular tissue, and how these receptors are altered in ocular growth disorders.


   Acknowledgements
 
The authors thank Kevin G. Rada for assistance with the A-scan ultrasound analyses, Melissa J. Vrieze for technical assistance with the immunocytochemistry, and Ben Fowler and Julie Maier (Oklahoma Medical Research Foundation) for assistance with the confocal microscopy.


   Footnotes
 
Supported by National Eye Institute Grants EY09391, EY13686, EY12191, National Institutes of Health Grant RR017703, and Oklahoma Center for Advancement of Science and Technology (OCAST) Grant HR02-135R.

Submitted for publication February 14, 2005; revised June 2 and August 10, 2005; accepted November 21, 2005.

Disclosure: J.A. Summers Rada, None; A.F. Wiechmann, 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.

Corresponding author: Jody A. Summers Rada, Department of Cell Biology, 940 Stanton L. Young Blvd, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; jody-rada{at}ouhsc.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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