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Melatonin is an output signal of an endogenous circadian clock of retinal photoreceptors, with highest levels occurring at night. Melatonin synthesized in the retina appears to act as a paracrine signal by binding to specific receptors in the eye. We have previously demonstrated that RNA encoding the Mel(1b) and Mel(1c) melatonin receptor subtypes is expressed in the Xenopus laevis retina. The goal of this study was to determine the distribution of the Mel(1b) and Mel(1c) receptor subtype RNA expression in the retina, and to determine if the level of expression of these receptors exhibits a diurnal rhythm. Sections of frog neural retina were analyzed by in situ hybridization with 35S-labeled Xenopus Mel(1c) and Mel(1b) riboprobes. Hybridization was present in cells of the inner nuclear layer and the ganglion cell layer. Moreover, there was hybridization in the photoreceptors, which has not been previously reported. To test the hypothesis that retinal melatonin receptor mRNA undergoes a diurnal rhythm of expression, total RNA was isolated from frog neural retinas obtained at 3-h intervals during a 24-h period. The total RNA was used in real-time PCR assays to quantify the differences in Mel(1b) and Mel(1c) receptor mRNA expression at various circadian times. Both the Mel(1b) and Mel(1c) receptor RNA demonstrated a diurnal rhythm of expression, with peak levels occurring late in the light period, and lowest levels late in the dark period. These results support the hypothesis that RNA encoding melatonin receptors undergo a diurnal rhythm of expression. To further investigate the possible expression of the Mel(1a) receptor subtype in Xenopus retina, we generated Mel(1a) PCR products in genomic DNA, and in reverse-transcribed neural retina and retinal pigment epithelium (RPE) RNA. The identity of the PCR product was confirmed by sequencing. Therefore, all three known Xenopus melatonin receptor subtypes appear to be expressed in the neural retina and RPE.
Fig. 1. Expression of melatonin receptor subtypes in Xenopus neural retina, RPE, and genomic DNA. Agarose gel electrophoresis shows PCR products from Xenopus genomic DNA that was amplified with primers complementary to the Mel1a [A], Mel1b [B], and Mel1c [C] DNA sequences. All three primer sets amplified the PCR product of the expected size (arrows; Mel1a: 459 bp, Mel1b: 388 bp, and Mel1c: 251 bp). Reverse-transcribed neural retina (NR) and RPE RNA also resulted in PCR amplification of all three melatonin receptor subtypes. No specific PCR product was detected with Mel1a primers following a reverse transcription reaction in the absence of reverse transcriptase (A). STD=molecular weight standards.
Fig. 2. Melatonin 1c and 1b receptor in situ hybridization in sections of Xenopus retina. (A and B) One-micron DGD-embedded Xenopus retina section hybridized with Xenopus Mel1c melatonin receptor antisense riboprobe. A diffuse band of hybridization (represented by black silver grains) is located over cells of the inner nuclear layer (INL) and over the photoreceptor inner segments (IS) (cones can be identified by characteristic lipid droplet). Some positive cells in the INL and GCL are indicated by arrows. (C) Retina section treated with the Mel1b antisense riboprobe, demonstrates a lower level of specific hybridization to photoreceptors and inner retinal neurons. (D) Retina section treated with a sense (control) riboprobe, demonstrates the level of nonspecific background hybridization throughout the entire thickness of the retina. ONL, outer nuclear layer; INL, inner nuclear layer; OS, outer segments of photoreceptors; IS, inner segments of photoreceptors (×300 magnification).
Fig. 3. Diurnal rhythm of Mel1b and Mel1c melatonin receptor subtype RNA expression. Quantitative real-time RTâPCR analysis was performed on RNA samples obtained from pooled neural retinas of four frogs at 3-h intervals during a 24-h period. Tissues obtained in the first four time points (Circadian Time [CT] 3, 6, 9 and 12) were retrieved in the light, whereas tissues obtained in the second four time points (CT 15, 18, 21 and 24) were retrieved under dim red light. (a) Xenopus Mel1b melatonin receptor RNA levels normalized to GAPDH RNA levels. The late afternoon (CT 9) shows a peak level of RNA expression, and lowest levels occur during the dark period. (B) Xenopus Mel1c melatonin receptor RNA levels normalized to GAPDH RNA levels. The late afternoon (CT 9) shows a peak level of RNA expression, and lowest levels occur during the dark period, similar to that seen for the Mel1b receptor. Note that the RNA levels at CT 12, when the lights are still on, decline from the peak levels of CT 9, suggesting the presence of a true circadian rhythm, rather than merely a light-driven response. (C) GAPDH and β-actin RNA levels demonstrate fairly constant levels of expression throughout a 24-h period. Each data point in the two receptor RNA graphs is the mean of triplicate samples±the standard deviation. Each data point in the actin/GAPDH graph is the mean of duplicate samples. The difference between CT 9 and CT 21 of the Mel1c RNA expression is statistically significant (P<0.05), as measured by a Studentâs t-test. The difference between CT 9 and CT 21 of the Mel1b RNA expression is not statistically significant. However, if the transition time points (CT 3 and 24) are not included, the difference between the CT 6, 9 and 12 (light) group vs. the CT 15, 18, and 21 group (dark) of both the Mel1b and Mel1c receptor expression is statistically significant (P<0.01 for Mel1c, and P<0.0002 for Mel1b).
