The avian retina and pineal gland contain autonomous circadian oscillators and photo-entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehydebased photopigments than the retinaldehyde-based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0-4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.

The avian pineal gland, like that of mammals, displays a striking circadian rhythm in the synthesis and release of the hormone melatonin. However, the pineal gland plays a more prominent role in avian circadian organization and differs from that in mammals in several ways. One important difference is that the pineal gland in birds is relatively autonomous. In addition to making melatonin, the avian pineal contains photoreceptors and a circadian clock (thus, an entire circadian system) within itself. Furthermore, avian pineals retain their circadian properties in organ or dispersed cell culture, making biochemical components of regulatory pathways accessible. Avian pinealocytes are directly photosensitive, and novel candidates for the unidentified photopigments involved in the regulation of clock function and melatonin production, including melanopsin, pinopsin, iodopsin, and the cryptochromes, are being evaluated. Transduction pathways and second messengers that may be involved in acute and entraining effects, including cyclic nucleotides, calcium fluxes, and protein kinases, have been, and continue to be, examined. Moreover, several clock genes similar to those found in Drosophila and mouse are expressed, and their dynamics and interactions are being studied. Finally, the bases for acute and clock regulation of the key enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT), are described. The ability to study entrainment, the oscillator itself, and a physiological output in the same tissue at the same time makes the avian pineal gland an excellent model to study the bases and regulation of circadian rhythms.

Melatonin is an important hormone regulating circadian clocks in birds, but the specific cellular sites of action are not completely known. The present study was designed to determine whether astrocytes derived from chick brain contained functional melatonin receptors. Primary cell cultures of diencephalon astrocytes that express glial fibrillary acidic protein (GFAP), but not neuron-specific enolase (NSE) immunoreactivity, were employed to determine the cellular distribution and physiological role for the three known receptor subtypes. Saturation and Scatchard analysis of 2-[(125)I]iodomelatonin binding demonstrated melatonin receptor binding with a high affinity and a pharmacological profile similar to that obtained from brain. In situ hybridization for receptor subtypes revealed Mel(1A) and Mel(1C) receptor mRNA, but not Mel(1B). Administration of pharmacological levels of melatonin acutely inhibited forskolin-stimulated 2-deoxyglucose (2DG) uptake, while rhythmic administration of physiological levels of melatonin gradually imposed a rhythm in 2DG uptake and of the release of both lactate and pyruvate into the medium. These results indicate that (1) there are functional Mel(1A) and Mel(1C) melatonin receptors in astrocyte-rich cultures, and (2) rhythmic administration of melatonin plays an important role in the regulation of astrocytic metabolic activity. Together, the data suggest that the circadian secretion of melatonin probably plays a role in the global metabolic economy of the avian brain through rhythmic regulation of metabolism in astrocytes.

The circadian secretion of melatonin is a critical component in circadian and seasonal rhythms in many vertebrate species. This hormone is produced by photoreceptors and cell types derived from photoreceptors in vertebrate retinas and pineal complexes via circadian regulation of the biosynthetic enzymes arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase at both transcriptional and posttranscriptional levels. The question of whether other multicellular animals and organisms from other taxa produce melatonin in a homologously regulated pathway is at this point unclear, but preliminary evidence suggests that vertebrate and insect melatonin are produced by convergent or parallel phylogenies. The existence and function of algal and plant melatonin is worthy of further study but is unresolved at this point. In vertebrates, the role of melatonin in behavioral and systems physiology follows two phylogenetic patterns. First, the circadian regulation of visual system structures, including the hypothalamic suprachiasmatic area, the inner retina, and retinorecipient and integrative visual structures, is a primitive characteristic among vertebrate species. Second, the relative loss of visual regulation and the presence of melatonin binding in the pars tuberalis of the adenohypophysis among mammals is a derived characteristic because these characteristics are present in this group only.

The indoleamine hormone melatonin is synthesized and released by photoreceptors during the night within the chick retina, and confers timing information to modulate retinal physiology. Three subtypes of melatonin receptor with nearly identical pharmacological profiles have been described in chickens and are present in the retina. In this study, the spatial localization and temporal pattern of the mRNA for each of these receptors within the retina are described. The localization and rhythmicity of receptor mRNA were analyzed using in situ hybridization and RNase protection assay, respectively, with probes against specific nucleotide sequences encoding these receptors. Mel1A and Mel1C receptor mRNA have similar patterns of expression, primarily in the inner segments of photoreceptors, vitread portion of the inner nuclear layer, and in the retinal ganglion cell layer. Mel1B receptor mRNA is expressed at higher levels in the retina, with expression in photoreceptors, throughout the inner nuclear layer, and in the ganglion cell layer. Mel1A receptor mRNA is rhythmic in both light:dark (LD) cycles and in constant darkness (DD); Mel1A peaks during midday and mid-subjective day, respectively. Mel1C receptor mRNA is also rhythmically expressed in LD, but with a lower amplitude, such that transcript is high during the day and low during the night. In DD, Mel1C rhythms become 180 deg out of phase with a slight increase at night. Mel1B mRNA expression was highly variable and arrhythmic.