CRISPR/Cas9 mediated mutation of the mtnr1a melatonin receptor gene causes rod photoreceptor degeneration in developing Xenopus tropicalis.

Nighttime surges in melatonin levels activate melatonin receptors, which synchronize cellular activities with the natural light/dark cycle. Melatonin receptors are expressed in several cell types in the retina, including the photon-sensitive rods and cones. Previous studies suggest that long-term photoreceptor survival and retinal health is in part reliant on melatonin orchestration of circadian homeostatic activities. This scenario would accordingly envisage that disruption of melatonin receptor signaling is detrimental to photoreceptor health. Using in vivo CRISPR/Cas9 genomic editing, we discovered that a small deletion mutation of the Mel1a melatonin receptor (mtnr1a) gene causes a loss of rod photoreceptors in retinas of developing Xenopus tropicalis heterozygous, but not homozygous mutant tadpoles. Cones were relatively spared from degeneration, and the rod loss phenotype was not obvious after metamorphosis. Localization of Mel1a receptor protein appeared to be about the same in wild type and mutant retinas, suggesting that the mutant protein is expressed at some level in mutant retinal cells. The severe impact on early rod photoreceptor viability may signify a previously underestimated critical role in circadian influences on long-term retinal health and preservation of sight. These data offer evidence that disturbance of homeostatic, circadian signaling, conveyed through a mutated melatonin receptor, is incompatible with rod photoreceptor survival.

Identification of active retinaldehyde dehydrogenase isoforms in the postnatal human eye.

BACKGROUND/OBJECTIVES: Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined. METHODOLOGY: Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye. PRINCIPAL FINDINGS: In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels. CONCLUSIONS/SIGNIFICANCE: Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA.

Diurnal variation of tight junction integrity associates inversely with matrix metalloproteinase expression in Xenopus laevis corneal epithelium: implications for circadian regulation of homeostatic surface cell desquamation.

BACKGROUND AND OBJECTIVES: The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium. METHODOLOGY/PRINCIPAL FINDINGS: Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime. CONCLUSIONS/SIGNIFICANCE: MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals.

Role of melatonin and its receptors in the vertebrate retina.

Melatonin is a chemical signal of darkness that is produced by retinal photoreceptors and pinealocytes. In the retina, melatonin diffuses from the photoreceptors to bind to specific receptors on a variety of inner retinal neurons to modify their activity. Potential target cells for melatonin in the inner retina are amacrine cells, bipolar cells, horizontal cells, and ganglion cells. Melatonin inhibits the release of dopamine from amacrine cells and increases the light sensitivity of horizontal cells. Melatonin receptor subtypes show differential, cell-specific patterns of expression that are likely to underlie differential functional modulation of specific retinal pathways. Melatonin potentiates rod signals to ON-type bipolar cells, via activation of the melatonin MT2 (Mel1b) receptor, suggesting that melatonin modulates the function of specific retinal circuits based on the differential distribution of its receptors. The selective and differential expression of melatonin receptor subtypes in cone circuits suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons and thus promote dark adaptation.

Whole slide images and digital media in pathology education, testing, and practice: the Oklahoma experience.

Examination of glass slides is of paramount importance in pathology training. Until the introduction of digitized whole slide images that could be accessed through computer networks, the sharing of pathology slides was a major logistic issue in pathology education and practice. With the help of whole slide images, our department has developed several online pathology education websites. Based on a modular architecture, this program provides online access to whole slide images, still images, case studies, quizzes and didactic text at different levels. Together with traditional lectures and hands-on experiences, it forms the back bone of our histology and pathology education system for residents and medical students. The use of digitized whole slide images has a.lso greatly improved the communication between clinicians and pathologist in our institute.

Melatonin receptors are anatomically organized to modulate transmission specifically to cone pathways in the retina of Xenopus laevis.

Melatonin receptors have been identified in several retinal cell types, including photoreceptors, horizontal cells, amacrine cells, and ganglion cells. Recent reports suggest that melatonin potentiates signaling from rods to inner retinal neurons. However, the organization of the melatonin receptors mediating this action in the outer plexiform layer (OPL) is not clear. To assess melatonin receptor localization in the OPL, double-label confocal immunohistochemistry for Mel1a or Mel1b melatonin receptors was performed in combination with markers for cone photoreceptors (calbindin, XAP-1) and ON bipolar cells (guanine nucleotide binding protein alpha, Goα) on the retina of Xenopus laevis. Both Mel1a and Mel1b receptors were specifically associated with processes contacting the pedicles of cones, but localized to processes from different sets of second-order neurons. Mel1a receptors localized to the large axonal processes of horizontal cells, while Mel1b receptors localized to the dendrites of OFF bipolar cells. Both receptors also localized to third-order amacrine and ganglion cells and their processes in the inner plexiform layer. This study indicates that Mel1a and Mel1b melatonin receptors are expressed specifically in the Xenopus OPL to modulate transmission from cones to horizontal cells and OFF bipolar cells, respectively; they are second-order neurons that predominantly contact ribbon synapses and display OFF responses to light. When combined with results from recent physiological studies, the current results suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons across species, although the precise mechanisms by which melatonin enhances this transmission are likely to vary in a species-dependent manner.

Light-induced tyrosine phosphorylation of rod outer segment membrane proteins regulate the translocation, membrane binding and activation of type II α phosphatidylinositol-5-phosphate 4-kinase.

Type II phosphatidylinositol 5-phosphate 4-kinase (PIPKIIα) catalyzes the synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)), an essential lipid second messenger that may be involved in the regulation of phototransduction, neuroprotection, and morphogenesis in the vertebrate retina. Here we report that in rodent and transgenic frogs, the light-mediated activity and membrane binding of PIPKIIα in rod outer segments (ROS) is dependent on tyrosine phosphorylation of ROS proteins. The greater type II α PIP kinase activity in the light-adapted ROS membrane results from light-driven translocation of PIPKIIα from the rod inner segment to ROS, and subsequent binding to the ROS membrane, thus improving access of the kinase to its lipid substrates. These results indicate a novel mechanism of light regulation of the PIPKIIα activity in photoreceptors, and suggest that the greater PIPKIIα activity in light-adapted animals and the resultant accumulation of PI-4,5-P(2) within the ROS membrane may be important for the function of photoreceptor cells.

Increased hyaluronan synthase-2 mRNA expression and hyaluronan accumulation with choroidal thickening: response during recovery from induced myopia.

PURPOSE: Several studies have convincingly shown that in chicks, compensation for imposed focus involves immediate changes in choroid thickness. The molecular events associated with choroidal thickening and the regulation of the choroidal response are largely unknown. METHODS: Form-deprivation myopia was induced in the right eyes of 2-day-old chicks by the application of translucent occluders for 10 days and was followed by unrestricted vision for an additional 1 to 20 days (recovery). Individual choroids were isolated from treated and control eyes and used for reverse transcription-quantitative PCR, hyaluronan (HA) localization with biotinylated hyaluronic acid binding protein (b-HABP), and analyses of HA size and concentration by size exclusion chromatography-multiangle laser light scattering (SEC-MALLS). RESULTS: HAS2 gene expression increased significantly after 6 hours of unrestricted vision (>7-fold) and peaked at 24 hours (>9-fold). In untreated eyes, HA was localized to perivascular sheaths of larger choroidal blood vessels; however, after 4 to 15 days of recovery, intense labeling for HA was detected throughout the thickened choroidal stroma. Analyses of choroidal HA by SEC-MALLS indicated that HA concentration was significantly increased in recovering choroids compared with controls after 4 to 8 days of recovery (≈3.5-fold). CONCLUSIONS: Newly synthesized HA accumulates in the choroidal stroma of recovering eyes and is most likely responsible for the stromal swelling observed during recovery from myopia. This HA accumulation is initiated by a rapid increase in choroidal expression of the HAS2 gene in response to myopic defocus.

Melatonin receptor expression in Xenopus laevis surface corneal epithelium: diurnal rhythm of lateral membrane localization.

PURPOSE: Melatonin receptors are seven-pass G protein-coupled receptors located in many tissues throughout the body, including the corneal epithelium (CE), and relay circadian signals to the target cells. The purpose of this study was to determine more precisely the cellular distribution of the melatonin receptors in the surface cells of the CE of Xenopus laevis, and to examine the relative distribution of melatonin receptor subtype expression at different times during the circadian cycle. METHODS: Cryostat sections and whole corneas of adult Xenopus laevis were processed for immunocytochemistry using antibodies specific for each of the three melatonin receptor subtypes (Mel1a, Mel1b, and Mel1c). For the circadian studies, corneas were obtained from euthanized frogs at 4-h intervals during a 24-h period under a 12 h:12 h light-dark cycle. Double-label immunocytochemistry was performed using a Mel1a antibody in combination with antibodies against Mel1b, Mel1c, or the zonula occludens protein ZO-1. Corneal whole-mount specimens and corneal sections were analyzed by laser-scanning confocal microscopy. RESULTS: All three melatonin receptor subtypes were expressed on the surface and sub-superficial layer of CE cells, but with different sub-cellular distributions. The Mel1a receptor was highly localized to the lateral plasma membrane of the surface CE, but also displayed cytoplasmic localization at some times of day, especially at night. Mel1c showed a similar pattern of labeling to Mel1a, but there were some distinctive differences, insofar as the Mel1c receptors were usually located immediately basal to the Mel1a receptors. The relative degree of membrane and cytoplasmic labeling of the Mel1c receptor also oscillated during the 24-h period, but was out of phase with the changes that occurred in the Mel1a receptor localization. Furthermore, in the late afternoon time point, the Mel1a and Mel1c receptors were highly co-localized, suggestive of heterodimerization, whereas at other time points, the two receptors were distinctly not co-localized. Double-label immunocytochemistry of Mel1a and ZO-1 demonstrated that the Mel1a receptor was located basal to the tight junctions, on the lateral membrane in very close proximity to the ZO-1 protein. CONCLUSIONS: Mel1a, Mel1b, and Mel1c receptor subtypes are expressed in the lateral plasma membrane of the Xenopus surface CE, at a position in close proximity to the tight junctions that form the corneal diffusion barrier. The very close association of the Mel1a receptors to the ZO-1 peripheral membrane tight junction proteins is suggestive of a potential role for melatonin in influencing the rate of tight junction formation or breakdown. The transient co-localization of Mel1a and Mel1c late in the light period is suggestive of formation of heterodimers that may influence receptor responsiveness and/or activity during specific periods of the day. The dynamic daily changes in melatonin receptor subtype expression and localization in the surface CE supports the concept that melatonin signaling may affect circadian activities of the surface epithelium of the cornea.

Ocular expression of avian thymic hormone: changes during the recovery from induced myopia.

PURPOSE: Several studies suggest that postnatal ocular growth is under the control of factors within the eye that regulate the rate of scleral extracellular matrix remodeling and the rate of ocular elongation. A microarray analysis was employed to identify some of the factors involved in the regulation of visually guided ocular growth. Gene expression was compared in the retina-retinal pigmented epithelium (RPE)-choroid of chick eyes that were decelerating in the rate of ocular growth ("recovering" from myopia) as compared with contralateral control eyes. METHODS: Form-deprivation myopia was induced in the right eyes of two-day-old chicks by the application of translucent occluders. Following 10 days of deprivation, occluders were removed and chicks were provided unrestricted vision for an additional 1-7 days (recovery). After one and four days of recovery, chicks were sacrificed, retina, RPE, and choroid were isolated, and mRNA was subjected to microarray analysis using a chicken immune system 4000 gene microarray. In addition, whole eyes and isolated ocular tissues (retina and RPE, choroid, sclera, and extraocular muscle) of treated and control eyes were subjected to real-time PCR, immunohistochemistry, and western blot analyses to verify gene expression results. RESULTS: Following one day of recovery, only one gene, avian thymic hormone (ATH) was highly upregulated (+12.3 fold). ATH gene and protein expression were confirmed in the retina and choroid as well as in the sclera and extraocular muscle. A significant increase in ATH protein was detected in choroids from treated eyes following four days of recovery as compared to contralateral controls (p<0.05; Wilcoxon signed-rank test). CONCLUSIONS: ATH is expressed in several ocular tissues and is specifically and rapidly (within one day) upregulated in the choroids of chick eyes recovering from induced myopia. This upregulation corresponds to the onset of choroidal thickening and increased choroidal vascular permeability. The identification of ATH in ocular tissues and its increased protein accumulation in the choroid during recovery from induced myopia suggest a novel role for this protein in the choroidal response to myopic defocus.

Activation and membrane binding of retinal protein kinase Balpha/Akt1 is regulated through light-dependent generation of phosphoinositides.

Akt is a phospholipid-binding protein and the downstream effector of the phosphoinositide 3-kinase (PI3K) pathway. Akt has three isoforms: Akt1, Akt2, and Akt3. All of these isoforms are expressed in rod photoreceptor cells, but the individual functions of each isoform are not known. In this study, we found that light induces the activation of Akt1. The membrane binding of Akt1 to rod outer segments (ROS) is insulin receptor (IR)/PI3K-dependent as demonstrated by reduced binding of Akt1 to ROS membranes of photoreceptor-specific IR knockout mice. Membrane binding of Akt1 is mediated through its Pleckstrin homology (PH) domain. To determine whether binding of the PH domain of Akt1 to photoreceptor membranes is regulated by light, various green fluorescent protein (GFP)/Akt1-PH domain fusion proteins were expressed in rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The R25C mutant PH domain of Akt1, which does not bind phosphoinositides, failed to associate with plasma membranes in a light-dependent manner. This study suggests that light-dependent generation of phosphoinositides regulates the activation and membrane binding of Akt1 in vivo. Our results also suggest that actin cytoskeletal organization may be regulated through light-dependent generation of phosphoinositides.

The human ubiquitin conjugating enzyme, UBE2E3, is required for proliferation of retinal pigment epithelial cells.

PURPOSE: Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation. METHODS: UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27(Kip1) were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for beta-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells. RESULTS: UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27(Kip1), and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse. CONCLUSIONS: UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation.

Circadian rhythms in the eye: the physiological significance of melatonin receptors in ocular tissues.

Many biological processes display circadian rhythms in activity, which presumably operate to coordinate cellular functions with daily environmental oscillations. The diurnal changes in environmental illumination are conveyed by the retina to the brain to entrain circadian rhythms throughout the body. Many ocular tissues themselves exhibit circadian rhythms of activity to optimize specific processes which require coordination with the light-dark cycle. The circadian signaling molecule, melatonin, is secreted into the circulation from the pineal gland, and is also produced within specific ocular cells such as retinal photoreceptors, ciliary epithelial cells, and perhaps cells of the lens. Melatonin appears to entrain many aspects of the biological clock via activation of specific G-protein-coupled integral membrane melatonin receptors. Melatonin receptors have been identified in many ocular tissues, including the neural retina, retinal pigment epithelium, ciliary body, cornea, sclera, and lens. This review will describe the circadian rhythmicity of some of the functions of these various ocular tissues, and will attempt to correlate these circadian activities with the expression of specific G-protein-coupled melatonin receptors, the role of melatonin in the regulation of circadian activity in ocular tissues, and its potential role in ocular diseases.

Influence of dietary melatonin on photoreceptor survival in the rat retina: an ocular toxicity study.

Previous studies have shown that melatonin treatment increases the susceptibility of retinal photoreceptors to light-induced cell death. The purpose of this study was to evaluate under various conditions the potential toxicity of dietary melatonin on retinal photoreceptors. Male and female Fischer 344 (non-pigmented) and Long-Evans (pigmented) rats were treated with daily single doses of melatonin by gavage for a period of 14 days early in the light period or early in the dark period. In another group, rats were treated 3 times per week with melatonin early in the light period, and then exposed to high intensity illumination (1000-1500 lx; HII) for 2h, and then returned to the normal cyclic lighting regime. At the end of the treatment periods, morphometric measurements of outer nuclear layer thickness (ONL; the layer containing the photoreceptor cell nuclei) were made at specific loci throughout the retinas. In male and female non-pigmented Fischer rats, melatonin administration increased the degree of photoreceptor cell death when administered during the nighttime and during the day when followed by exposure to HII. There were some modest effects of melatonin on photoreceptor cell death when administered to Fischer rats during the day or night without exposure to HII. Melatonin treatment caused increases in the degree of photoreceptor cell death when administered in the night to male pigmented Long-Evans rats, but melatonin administration during the day, either with or without exposure to HII, had little if any effect on photoreceptor cell survival. In pigmented female Long-Evans rats, melatonin administration did not appear to have significant effects on photoreceptor cell death in any treatment group. The results of this study confirm and extend previous reports that melatonin increases the susceptibility of photoreceptors to light-induced cell death in non-pigmented rats. It further suggests that during the dark period, melatonin administration alone (i.e., no HII exposure) to pigmented male rats may have a toxic effect on retinal cells. These results suggest that dietary melatonin, in combination with a brief exposure to high intensity illumination, induces cellular disruption in a small number of photoreceptors. Chronic exposure to natural or artificial light and simultaneous intake of melatonin may potentially contribute to a significant loss of photoreceptor cells in the aging retina.

Melatonin receptors in chick ocular tissues: implications for a role of melatonin in ocular growth regulation.

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 Mel(1a), Mel(1b), and Mel(1c) 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 Mel(1a) 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 Mel(1a) and Mel(1b) occurring during the night and peak levels of Mel(1c) 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.

Stimulation of melatonin receptors decreases calcium levels in xenopus tectal cells by activating GABA(C) receptors.

To investigate the physiological effects of melatonin receptors in the Xenopus tectum, we have used the fluorescent indicator Fluo-4 AM to monitor calcium dynamics of cells in tectal slices. Bath application of KCl elicited fluorescence increases that were reduced by melatonin. This effect was stronger at the end of the light period than at the end of the dark period. Melatonin increased gamma-aminobutyric acid-C (GABA(C))-receptor activity, as demonstrated by the ability of the GABA(C)-receptor antagonists, picrotoxin and TPMPA, to abolish the effects of melatonin. In contrast, neither the GABA(A)-receptor antagonist bicuculline nor the GABA(B)-receptor antagonist CGP 35348 diminished the effects of melatonin. RT-PCR analyses revealed expression of the 3 known melatonin receptors, MT1 (Mel1(a)), MT2 (Mel1(b)), and Mel1(c). Because the effect of melatonin on tectal calcium increases was antagonized by an MT2-selective antagonist, 4-P-PDOT, we performed Western blot analyses with an antibody to the MT2 receptor; the data indicate that the MT2 receptor is expressed primarily as a dimeric complex and is glycosylated. The receptor is present in higher amounts at the end of the light period than at the end of the dark period, in a pattern complementary to the changes in melatonin levels, which are higher during the night than during the day. These results imply that melatonin, acting by MT2 receptors, modulates GABA(C) receptor activity in the optic tectum and that this effect is influenced by the light-dark cycle.

Localization of Mel1b melatonin receptor-like immunoreactivity in ocular tissues of Xenopus laevis.

The circadian signaling molecule, melatonin, is produced by pinealocytes and retinal photoreceptors. In the retina, melatonin is thought to diffuse into the inner retina to act as a paracrine signal of darkness by binding to specific receptors in retinal neurons. The retinal cell locations of the Mel1a and Mel1c melatonin receptor types have been reported, but the localization of the Mel1b receptor, which is the most highly expressed melatonin receptor type in the retina, is unknown. To determine the cellular distribution of Mel1b melatonin receptor protein in the Xenopus laevis retina and other ocular tissues, polyclonal antibodies were raised against a peptide fragment of the X. laevis Mel1b receptor. Western blot analysis of several ocular tissues revealed the presence of one or more immunoreactive bands in the sclera, cornea, lens, retinal pigment epithelium (RPE)/choroid, and neural retina. In the neural retina, the major immunoreactive bands displayed electrophoretic mobilities corresponding to approximately 35, 42, 45, and 80 Kd. Sections of X. laevis eyes were analyzed by immunocytochemistry and confocal microscopy, in combination with antibodies against the Mel1a melatonin receptor, a rod photoreceptor-specific protein, opsin, and two amacrine cell-specific markers, tyrosine hydroxylase (TOH; dopaminergic cells) and glutamic acid decarboxylase (GAD; GABA-ergic cells). Mel1b immunoreactivity was localized to the apical membranes of RPE cells, and punctate Mel1b immunoreactivity was observed in both rod and cone photoreceptor inner segments. Presumptive horizontal cells that ramify in the outer plexiform layer (OPL) were immunoreactive for Mel1b, and were exclusive of the Mel1a immunoreactivity present in the OPL. Neither TOH nor GAD co-localized with the Mel1b immunoreactivity that was present in the inner plexiform layer (IPL), suggesting that Mel1b is not expressed in dopaminergic or GABA-ergic amacrine cells. Mel1b immunoreactivity was observed in ganglion cells of the retina, a population of cells covering the outer surface of the outer fibrous layer of the sclera, and in lens fibers located in the outer regions of the lens. These results suggest that melatonin may influence retinal function by binding to receptors on RPE and photoreceptor cells, and by acting on neurons of the inner retina that do not use dopamine or GABA as a neurotransmitter. Furthermore, melatonin may bind to receptors on cells located in the sclera and lens, perhaps to modify the growth or function of these ocular tissues.

Direct modulation of rod photoreceptor responsiveness through a Mel(1c) melatonin receptor in transgenic Xenopus laevis retina.

PURPOSE: Retinal circadian signals may have a role in maintaining the normal function and health of photoreceptors. Melatonin is an output of the retinal circadian oscillator and provides nocturnal signaling that is mediated through specific G-protein-coupled receptors. Melatonin receptors are expressed in retinal photoreceptor cells, and this study was undertaken to test the hypothesis that melatonin directly increases photoreceptor responses through melatonin receptors. METHODS: Transgenic Xenopus laevis frogs were generated using a DNA construct containing a Xenopus opsin promoter driving expression of a melatonin Mel(1c) receptor-green fluorescent protein (GFP) fusion protein (XOP-MEL(1c)-GFP). Electroretinogram (ERG) analysis on transgenic and normal tadpole eyes was performed in response to melatonin treatment, and the eyes were subsequently examined by confocal microscopy and GFP immunocytochemistry. RESULTS: XOP-MEL(1c)-GFP transgenic frogs demonstrated GFP immunoreactivity in rod photoreceptor inner segments throughout the retina, indicating the rod-specific expression of the Mel(1c)-GFP fusion protein. ERG analysis of transgenic tadpole eyes showed that 1 to 100 nM melatonin increased the a- and b-wave amplitudes. Control transgenic (XOP-GFP) and normal frogs exhibited only modest ERG responses to 100-nM melatonin treatment. The effect of melatonin on a- and b-wave amplitudes in XOP-MEL(1c)-GFP transgenic frogs was dose dependent, with ERG responses occurring at physiological concentrations. CONCLUSIONS: The results suggest that melatonin, acting through Mel(1c) receptors on rod photoreceptor membranes, directly stimulates the responsiveness of rod photoreceptors to light. This supports the hypothesis that melatonin acts both as an intracrine and paracrine circadian signal of darkness, and binds to specific receptors in photoreceptors and other retinal cells to increase visual sensitivity.

Melatonin receptor expression in the cornea and sclera.

The scornea and sclera have been shown to exhibit circadian rhythms in cellular proliferation, wound healing and extracellular matrix synthesis. The distribution of melatonin Mel1a and Mel1c receptors was examined in the cornea and sclera of the Xenopus laevis eye in order to determine whether melatonin may potentially influence the growth and/or development of these ocular tissues. Sections of adult X. laevis eyes were analyzed by immunocytochemistry and confocal microscopy, using antibodies prepared against specific peptide sequences of the Xenopus Mel1a and Mel1c receptor proteins. Antibodies were pre- incubated with their appropriate antigenic peptides to control for non-specific labelling. Analysis of the distribution of Mel1a and Mel1c receptor immunoreactivity in the Xenopus eye revealed that both the Mel1a and Mel1c receptors were located in the outer fibrous layer (OFL) of the sclera, with Mel1c labelling being the most prominent. Similarly, Mel1a and Mel1c (Mel1c mostly) were also located in cells of the inner fibrous layer (IFL) with Mel1c being most abundant. The chondrocytes of the cartilaginous layer also appeared to express Mel1a, Mel1c, or both receptors. Both Mel1a and Mel1c receptor immunoreactivity were observed in the corneal epithelium and endothelium. Whereas the Mel1a antibody labelled the entire corneal epithelial layer, the Mel1c antibody labelled only the most superficial layer of epithelial cells. Cell processes of fibroblasts of the corneal stroma were immunoreactive for either Mel1a or Mel1c receptors. The identification of Mel1a and Mel1c receptors in restricted distributions in the cornea and sclera suggests that melatonin may play a role in the cellular physiology of these ocular tissues.

Differential distribution of melatonin receptors in the pituitary gland of Xenopus laevis.

A major target site for melatonin action is thought to be the pituitary gland. We have detected differential expression and co-localization of the Mel(1a) and Mel(1c) receptors in cells of the Xenopus laevis pituitary gland. Sections of Xenopus pituitary glands were labeled with Mel(1a) and/or Mel(1c) antibodies, in combination with antibodies to arginine vasotocin (AVT), alpha-melanocyte stimulating hormone (alpha-MSH), prolactin (PRL), and luteinizing hormone (LH). Mel(1a) immunoreactivity was localized to cells of the pars intermedia and to elements within the pars nervosa. Mel(1c) immunoreactivity was also localized to the pars nervosa, and significant labeling was also observed in discrete clusters of cells in the pars distalis. Mel(1a) was absent from the pars distalis, while Mel(1c) was absent from the pars intermedia. Mel(1a) and Mel(1c) were co-localized in the pars nervosa. AVT was present in the pars nervosa, and appeared to be localized to the cell clusters of the pars distalis in which the Mel(1c) receptor was localized. alpha-MSH co-localized with the Mel(1a) receptor in the pars intermedia. LH appeared to localize to many of the cells in the pars distalis, with the notable exception of the Mel(1c) receptor-positive clusters of cells. PRL did not appear to co-localize with either melatonin receptor. The pattern of differential expression of the Mel(1a) and Mel(1c) receptors suggests that the receptors specifically mediate the cellular response to melatonin binding in the specific cell populations.