Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types.

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 retinaldehyde-based 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.

Transduction of light in the suprachiasmatic nucleus: evidence for two different neurochemical cascades regulating the levels of Per1 mRNA and pineal melatonin.

The suprachiasmatic nucleus (SCN) contains a circadian clock and regulates melatonin synthesis in the pineal gland. Light exposure during the subjective night acutely increases the mRNA levels of the Period (Per)1 gene in the SCN and acutely suppresses melatonin levels in the pineal gland. Activation of N-methyl-D-aspartate (NMDA) receptors in the SCN has been demonstrated to phase-shift the circadian clock in a manner similar to light. We tested the hypothesis that activation of excitatory amino acid (EAA) receptors in the SCN mediates the acute effects of light on Per1 mRNA levels and pineal melatonin. NMDA, injected into the SCN of Syrian hamsters during the night, acutely suppressed melatonin levels in the pineal gland. Both the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) and the alpha-amino-3-hydroxy-5-methylisoxazoleproprionic acid (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) inhibited the light-induced increase of Per1 mRNA levels in the SCN. In the same animals, however, these antagonists had no effect on the ability of light to suppress pineal melatonin. These results support the hypothesis that EAA receptor activation in the SCN is necessary for the acute effects of light on Per1 mRNA levels. They also indicate that NMDA receptor activation in the SCN is sufficient but may not be necessary for the acute effects of light on pineal melatonin. These data suggest that there may be at least two different neurochemical cascades that transduce the effects of light in the SCN

Photic and circadian regulation of retinal melatonin in mammals.

Several studies have established that melatonin synthesis occurs in the retina of vertebrates, including mammals. In mammals, a subpopulation of photoreceptors (probably the cones) synthesize melatonin. Melatonin synthesis in the retina is elevated at night and reduced during the day in a fashion similar to events in the pineal gland. Both the MT1 and MT2 melatonin receptors are present in the retina and retinal melatonin does not contribute to circulating levels, suggesting that retinal melatonin acts locally as a neurohormone and/or neuromodulator. Melatonin synthesis in the retina of mammals is under the control of a circadian oscillator, and circadian rhythms in melatonin synthesis and/or release have been described for several species of mammals. These rhythms are present in vivo, persist in vitro, are entrained by light and are temperature compensated. The cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the key enzyme in the melatonin biosynthetic pathway) has allowed studies of the molecular mechanisms responsible for the generation of retinal melatonin rhythmicity. The present review focuses on the cellular and molecular mechanisms that regulate melatonin synthesis. In particular, we discuss how the photic environment and the circadian clock interact in determining melatonin levels, in addition to the role that melatonin plays in retinal physiology.

Photic regulation of melatonin in rat retina and the role of proteasomal proteolysis.

Several investigations have shown that illumination at night reduces melatonin level in the mammalian pineal, but the effect of night illumination on the retina is not known. In this study retinas were cultured in a flow-through apparatus and then were exposed to light at ZT 18. Light exposure reduced melatonin levels to the daytime level within 30 min. The reduction of melatonin levels was due to a rapid decrease in the activity of the enzyme AA-NAT; AA-NAT mRNA levels were not affected by illumination. Pre-incubation with lactacystin (25 microM) prevented light-induced reduction of AA-NAT activity and melatonin levels. These results demonstrate that melatonin levels in the mammalian retina are affected by light exposure at night, via proteosomal proteolysis of AA-NAT.

Neuropeptide Y rapidly reduces Period 1 and Period 2 mRNA levels in the hamster suprachiasmatic nucleus.

The mammalian suprachiasmatic nucleus (SCN) contains the main circadian clock. Neuropeptide Y (NPY) that is released from the intergeniculate leaflet of the lateral geniculate body to the SCN, acts in the SCN to advance circadian phase in the subjective day via the NPY Y2 receptor. We used semi-quantitative in situ hybridization to determine the effect of NPY on circadian clock genes, Period 1 (Per1) and Period 2 (Per2), expression in SCN slices. Addition of NPY to the brain slices in the subjective day resulted in reduction of Per1 and Per2 mRNA levels 0.5 and 2 h after treatment. NPY Y1/Y5 and Y2 agonists decreased Per1 within 0.5 h. These results suggest that NPY may induce phase shifts by mechanisms involving or resulting in reduction of Per1 and Per2 mRNA levels.

The mammalian circadian clock shop.

In mammals, a master circadian pacemaker driving daily rhythms in behavior and physiology resides in the suprachiasmatic nucleus (SCN). The SCN contains multiple circadian oscillators that synchronize to environmental cycles and to each other in vivo. Rhythm production, an intracellular event, depends on more than eight identified genes. The period of the rhythms within the SCN also depends upon intercellular communication. Many other tissues also retain the ability to generate near 24 -h periodicities although their place in the organization of circadian timing is still unclear. This paper focuses on the tissue-, cellular- and molecular-level events that generate and entrain circadian rhythms in behavior in mammals and emphasizes the apparent differences between the SCN and peripheral oscillators.

Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse.

BACKGROUND: Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. RESULTS: cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. CONCLUSION: The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

The circadian system of reptiles: a multioscillatory and multiphotoreceptive system.

Many parameters exhibited by organisms show daily fluctuations that may persist when the organisms are held in constant environmental conditions. Rhythms that persist in constant conditions with a period close to 24 h are called circadian. Although nowadays most research in this field is focused on the molecular and genetic aspects--and therefore mostly on two animal models (Drosophila and mouse)--the study of alternative animal models still represent a useful approach to understanding how the vertebrate circadian system is organized, and how this fascinating time-keeping system has changed throughout the evolution of vertebrates. The present paper summarizes the current knowledge of the circadian organization of Reptiles. The circadian organization of reptiles is multioscillatory in nature. The retinas, the pineal, and the parietal eye (and, possibly, the suprachiasmatic nuclei of the hypothalamus, SCN) contain circadian clocks. Of particular interest is the observation that the role these structures play in the circadian organization varies considerably among species and within the same species in different seasons. Another remarkable feature of this class is the redundancy of circadian photoreceptors: retinas of the lateral eyes, pineal, parietal eye, and the brain all contain photoreceptors.

Melatonin circadian rhythm in the retina of mammals.

Melatonin has been traditionally considered to be derived principally from the pineal gland. However, several investigations have now demonstrated that melatonin synthesis occurs also in the retina (and in other organs as well) of several vertebrate classes, including mammals. As in the pineal, melatonin synthesis in the retina is elevated at night and reduced during the day. Since melatonin receptors are present in the retina and retinal melatonin does not contribute to the circulating levels, retinal melatonin probably acts locally as a neuromodulator. Melatonin synthesis in the retinas of mammals is under control of a circadian oscillator located within the retina itself, and circadian rhythms in melatonin synthesis and/or release have been described for several species of rodents. These rhythms are present in vivo, persist in vitro, are entrained by light, and are temperature compensated. The recent cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the only enzyme unique to the melatonin synthetic pathway) will facilitate localizing the cellular site of melatonin synthesis in the retina and investigating the molecular mechanism responsible for the generation of retinal melatonin rhythmicity. Melatonin has been implicated in many retinal functions, and the levels of melatonin and dopamine appear to regulate several aspects of retinal physiology that relate to light and dark adaptation. In conclusion, it seems that retinal melatonin is involved in several functions, but its precise role is yet to be understood.

Induction of photosensitivity in neonatal rat pineal gland.

Pineal glands removed from neonatal rats at 5, 7, and 9 days of age and explanted into short-term culture, synthesized melatonin when stimulated with norepinephrine (NE); their melatonin synthesis could not be suppressed with bright white light. Dispersed pineal cell cultures or pineal explants prepared from 1-day-old neonates and held in culture for 7 or 9 days also synthesized melatonin when stimulated with NE, but in these cases melatonin synthesis was significantly suppressed by light, demonstrating that the pineals had become photosensitive while in culture. The development of photosensitivity in culture could be partially or completely abolished by the continuous presence of 1 or 10 microm of NE in the culture medium. The pineals of all nonmammalian vertebrates are photoreceptive, whereas those of mammals do not normally respond to light. We hypothesize that a mechanism to suppress pineal photosensitivity by using NE released from sympathetic nerve endings evolved early in the history of mammals.

Circadian clocks: what makes them tick?

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals.

Circadian expression of period 1, period 2, and arylalkylamine N-acetyltransferase mRNA in the rat pineal gland under different light conditions.

Period 1 (Per1), 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) mRNA levels were determined by semi-quantitative in situ hybridization in rat pineal glands. In agreement with previous reports, AA-NAT mRNA levels were rhythmic in light:dark (LD) cycles and the rhythm persisted in constant dim light (DLL). Per1 and Per2 mRNA also showed significant variations both in LD and DLL. AA-NAT and Per1 mRNA levels showed very similar patterns of variations in LD and DLL to one another, whereas Per2 showed a different pattern of expression from AA-NAT and Per1. Exposure to 30 min of light did not affect the expression of the three genes, while exposure to a longer light pulse (1 or 2 h) decreased AA-NAT and Per1 mRNA levels; Per2 mRNA levels were also decreased but only temporarily. Our results demonstrate that Per1 and Per2 expression in the rat pineal is under circadian control, and suggest Per1 may be regulated by the same mechanism which controls the expression of AA-NAT gene. Per2 seem to be controlled by a different mechanism.

Dopamine inhibits melatonin release in the mammalian retina: in vitro evidence.

A circadian oscillator located within the retina controls melatonin synthesis in the retina of mammals. In non-mammalian vertebrates retinal melatonin and dopamine appear to act as mutually inhibitory paracrine signals for night and day, respectively; while in mammals this mutually inhibitory capability has now been fully demonstrated. In this study using a flow-through culture apparatus we investigated melatonin release from cultured retinas of golden hamster (Mesocricetus auratus) in the presence of dopamine or dopaminergic agonists and antagonists. Neural retinas were cultured with medium 199 for 24 h in a flow-through apparatus at the temperature 33 degrees C. During the subjective night the culturing medium was supplemented with dopamine, dopamine receptor antagonists or agonists. At the concentration of 0.1 microM dopamine did not inhibit melatonin release, while at higher dopamine concentration (1 to 1000 microM) melatonin release was inhibited in a dose-dependent manner. These effects appeared to be mediated by a D2/D4 receptor, because D2 and D4 receptor agonists (100 microM), but not D1/D5 receptor agonists (100 microM), inhibited melatonin release. Furthermore, D2/D4 selective receptor antagonists (100 microM) in conjunction with 100 microM dopamine blocked melatonin suppression, whereas a D1/D5 selective receptors antagonist was completely ineffective. Taken together, these results directly demonstrate for the first time that in the retina of mammals dopamine may suppress melatonin, and that suppression is mediated by D2/D4 dopaminergic receptors.

The tau mutation affects temperature compensation of hamster retinal circadian oscillators.

Neural retinas of the golden hamster (Mesocricetus auratus) express circadian rhythms of melatonin synthesis when cultured in constant darkness. Retinas from wild-type hamsters synthesize melatonin with a period close to 24 h, while retinas obtained from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 h, synthesize melatonin with a period close to 20 h. The retinal circadian oscillators of both wild-type and tau mutant hamsters are temperature compensated; however, temperature compensation is adversely affected by the mutation.

The clock in the mouse retina: melatonin synthesis and photoreceptor degeneration.

Melatonin is synthesized rhythmically under control of circadian oscillators by the retinas of non-mammalian vertebrates. Here we report that the retinas of some strains of laboratory mice exhibit robust circadian rhythms of melatonin synthesis which can be entrained by light in vitro. The rd mutation results in progressive loss of the rod and later cone photoreceptors. In mice homozygous for rd retinal melatonin synthesis is rhythmic at postnatal day 28 but not in older animals. Apparently rod photoreceptors are necessary for the expression of the circadian rhythm of melatonin synthesis but not for the synthesis itself. The many genetic and molecular tools available in the mouse can now be applied to analysis of the retinal circadian oscillator.

Multioscillatory circadian organization in a vertebrate, iguana iguana.

The lizard Iguana iguana when kept in constant ambient temperature displays endogenously generated circadian rhythms of body temperature and locomotor activity. Although surgical removal of the parietal eye has only slight effects on overt circadian rhythmicity, subsequent pinealectomy completely abolishes the rhythm of body temperature. However, the rhythm of locomotor activity is only slightly affected by parietalectomy plus pinealectomy. Our results demonstrate that the pineal complex is centrally involved in the generation and control of the circadian rhythm of body temperature but is only marginally involved in locomotor rhythmicity. Plasma melatonin levels are not significantly reduced by parietalectomy, whereas pinealectomy dramatically lowers the level and completely eliminates the circadian rhythm of melatonin in the circulation. Isolated parietal eye, pineal, and retina all synthesize melatonin with robust circadian rhythmicity when maintained for >/=4 d in culture, although in the intact animal all or almost all of the circulating melatonin comes from the pineal. The circadian system of I. iguana is composed of multiple circadian oscillators that reside in different tissues and have specific and different roles.

Evolution of circadian organization in vertebrates.

Circadian organization means the way in which the entire circadian system above the cellular level is put together physically and the principles and rules that determine the interactions among its component parts which produce overt rhythms of physiology and behavior. Understanding this organization and its evolution is of practical importance as well as of basic interest. The first major problem that we face is the difficulty of making sense of the apparently great diversity that we observe in circadian organization of diverse vertebrates. Some of this diversity falls neatly into place along phylogenetic lines leading to firm generalizations: i) in all vertebrates there is a "circadian axis" consisting of the retinas, the pineal gland and the suprachiasmatic nucleus (SCN), ii) in many non-mammalian vertebrates of all classes (but not in any mammals) the pineal gland is both a photoreceptor and a circadian oscillator, and iii) in all non-mammalian vertebrates (but not in any mammals) there are extraretinal (and extrapineal) circadian photoreceptors. An interesting explanation of some of these facts, especially the differences between mammals and other vertebrates, can be constructed on the assumption that early in their evolution mammals passed through a "nocturnal bottleneck". On the other hand, a good deal of the diversity among the circadian systems of vertebrates does not fall neatly into place along phylogenetic lines. In the present review we will consider how we might better understand such "phylogenetically incoherent" diversity and what sorts of new information may help to further our understanding of the evolution of circadian organization in vertebrates.

Evolution of circadian organization in vertebrates

Circadian organization means the way in which the entire circadian system above the cellular level is put together physically and the principles and rules that determine the interactions among its component parts which produce overt rhythms of physiology and behavior. Understanding this organization and its evolution is of practical importance as well as of basic interest. The first major problem that we face is the difficulty of making sense of the apparently great diversity that we observe in circadian organization of diverse vertebrates. Some of this diversity falls neatly into place along phylogenetic lines leading to firm generalizations: i) in all vertebrates there is a "circadian axis" consisting of the retinas, the pineal gland and the suprachiasmatic nucleus (SCN), ii) in many non-mammalian vertebrates of all classes (but not in any mammals) the pineal gland is both a photoreceptor and a circadian oscillator, and iii) in all non-mammalian vertebrates (but not in any mammals) there are extraretinal (and extrapineal) circadian photoreceptors. An interesting explanation of some of these facts, especially the differences between mammals and other vertebrates, can be constructed on the assumption that early in their evolution mammals passed through a "nocturnal bottleneck". On the other hand, a good deal of the diversity among the circadian systems of vertebrates does not fall neatly into place along phylogenetic lines. In the present review we will consider how we might better understand such "phylogenetically incoherent" diversity and what sorts of new information may help to further our understanding of the evolution of circadian organization in vertebrates.

Circadian rhythms in cultured mammalian retina.

Many retinal functions are circadian, but in most instances the location of the clock that drives the rhythm is not known. Cultured neural retinas of the golden hamster (Mesocricetus auratus) exhibited circadian rhythms of melatonin synthesis for at least 5 days at 27 degrees celsius. The rhythms were entrained by light cycles applied in vitro and were free-running in constant darkness. Retinas from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 hours, showed a shortened free-running period of melatonin synthesis. The mammalian retina contains a genetically programmed circadian oscillator that regulates its synthesis of melatonin.

Dermal photoreceptors regulate basking behavior in the lizard Podarcis muralis.

There is evidence that dermal photic responsiveness can be found in a wide range of animals. Behavioral responses to dermal stimulation by light have been observed in pigeon squabs and new-born rats, and more recently in a sea snake. Here we report that painting the dorsal surface of the lizard (Podarcis muralis) with opaque black paint impairs the animal's ability to position itself beneath a light source containing negligible heat. Experiments using light of different spectra and intensities show that the effect is due to light of wavelengths shorter than 600 nm and of intensity higher than 2.5 mW cm-2. These experiments demonstrate for the first time that overt behavior in a terrestrial vertebrate can be mediated by a dermal light sense.