NEURAL BASIS AND BIOLOGICAL FUNCTION OF MASKING BY LIGHT IN MAMMALS: SUPPRESSION OF MELATONIN AND LOCOMOTOR ACTIVITY

Light influences mammalian circadian rhythms in two different ways: (1) It entrains endogenous oscillators (clocks), which regulate physiology and behavior; and (2) it affects directly and often immediately physiology and behavior (these effects are also referred to as masking). Masking effects of light on pineal melatonin, locomotor activity, and the sleep-wake cycle in mammals and man are reviewed. They seem to represent a universal response in this group. The review reveals that the mechanism of photic inhibition of melatonin is fairly well understood, whereas only little is known about the influence of light on other circadian rhythm outputs, such as locomotor activity. (Chronobiology International, 18(5), 737–758, 2001)     

Biological rhythms in the context of light at night (LAN)

In mammals including man, the most important zeitgeber for endogenous rhythms is the environmental light/dark cycle. Mammals perceive light through the eyes and that perception is relayed to the suprachiasmatic nucleus (SCN) by means of neuronal signals. The SCN, in turn, innervates the pineal gland, resulting in the production and release of melatonin almost exclusively during night-time hours. Thus, besides object recognition, eyes serve as the sensory organ for detecting the presence or absence of light. The way that light entrains the SCN is still a matter of intense research. It has been shown, for example, that the light intensities required for affecting melatonin rhythms are much higher than the intensities needed for object identification. On the other hand, even in rodents who completely lack the "classical" photoreceptors of the retina, their endogenous rhythms still can be synchronized by normal light/dark cycles. These two observations led to the hypothesis that there must be photoreceptors, apart from the known (object-identifying) retinal photoreceptors, which are responsible for the entrainment of internal rhythms. Very recently, a number of reports showed that in fact a completely new type of retinal photoreceptor, located in ganglion cells, may be responsible for entraining the SCN. It contains a photopigment, melanopsin, which shares homologies with rhodopsin, but also is evolutionarily older. Compared to rods or cones, the melanopsin-containing neurons are rare, but evenly distributed within the retina, indicating that they serve as a global, integrating light sensor. These ganglion cells apparently project directly into the SCN. Taken together, these new developments in photo-chronobiology open new areas of research. It will be of special interest, for example, to determine how the photosensitive ganglion cells and their dendrites integrate the environmental light stimuli.     

NEURAL BASIS AND BIOLOGICAL FUNCTION OF MASKING BY LIGHT IN MAMMALS: SUPPRESSION OF MELATONIN AND LOCOMOTOR ACTIVITY

Light influences mammalian circadian rhythms in two different ways: (1) It entrains endogenous oscillators (clocks), which regulate physiology and behavior; and (2) it affects directly and often immediately physiology and behavior (these effects are also referred to as masking). Masking effects of light on pineal melatonin, locomotor activity, and the sleep-wake cycle in mammals and man are reviewed. They seem to represent a universal response in this group. The review reveals that the mechanism of photic inhibition of melatonin is fairly well understood, whereas only little is known about the influence of light on other circadian rhythm outputs, such as locomotor activity. (Chronobiology International, 18(5), 737-758, 2001)     

Temperature can entrain egg laying rhythm of Drosophila but may not be a stronger zeitgeber than light

In Drosophila multiple circadian oscillators and behavioral rhythms are known to exist, yet most previous studies that attempted to understand circadian entrainment have focused on the activity/rest rhythm and to some extent the adult emergence rhythm. Egg laying behavior of Drosophila females also follows circadian rhythmicity and has been seen to deviate substantially from the better characterized rhythms in a few aspects. Here we report the findings of our study aimed at evaluating how circadian egg laying rhythm in fruit flies Drosophila melanogaster entrains to time cues provided by light and temperature. Previous studies have shown that activity/rest rhythm of flies entrains readily to light/dark (LD) and temperature cycles (TC). Our present study revealed that egg laying rhythm of a greater percentage of females entrains to TC compared to LD cycles. Therefore, in the specific context of our study this result can be taken to suggest that egg laying clocks of D. melanogaster entrains to TC more readily than LD cycles. However, when TC were presented along with out-of-phase LD cycles, the rhythm displayed two peaks, one occurring close to lights-off and the other near the onset of low temperature phase, indicating that upon entrainment by TC, LD cycles may be able to exert a greater influence on the phase of the rhythm. These results suggest that temperature and light associatively entrain circadian egg laying clocks of Drosophila.     

Melatonin is a redundant entraining signal in the rat circadian system

The role of melatonin in maintaining proper function of the circadian system has been proposed but very little evidence for such an effect has been provided. To ascertain the role, the aim of the study was to investigate impact of long-term melatonin absence on regulation of circadian system. The parameters of behavior and circadian clocks of rats which were devoid of the melatonin signal due to pinealectomy (PINX) for more than one year were compared with those of intact age-matched controls. PINX led to a decrease in spontaneous locomotor activity and a shortening of the free-running period of the activity rhythm driven by the central clock in the suprachiasmatic nuclei (SCN) in constant darkness. However, the SCN-driven rhythms in activity and feeding were not affected and remained well entrained in the light/dark cycle. In contrast, in these conditions PINX had a significant effect on amplitudes of the clock gene expression rhythms in the duodenum and also partially in the liver. These results demonstrate the significant impact of long-term melatonin absence on period of the central clock in the SCN and the amplitudes of the peripheral clocks in duodenum and liver and suggest that melatonin might be a redundant but effective endocrine signal for these clocks.     

Melanopsin-dependent photo-perturbation reveals desynchronization underlying the singularity of mammalian circadian clocks

Singularity behaviour in circadian clocks--the loss of robust circadian rhythms following exposure to a stimulus such as a pulse of bright light--is one of the fundamental but mysterious properties of clocks. To quantitatively perturb and accurately measure the dynamics of cellular clocks, we synthetically produced photo-responsiveness within mammalian cells by exogenously introducing the photoreceptor melanopsin and continuously monitoring the effect of photo-perturbation on the state of cellular clocks. Here we report that a critical light pulse drives cellular clocks into singularity behaviour. Our theoretical analysis consistently predicts and subsequent single-cell level observation directly proves that desynchronization of individual cellular clocks underlies singularity behaviour. Our theoretical framework also explains why singularity behaviours have been experimentally observed in various organisms, and it suggests that desynchronization is a plausible mechanism for the observable singularity of circadian clocks. Importantly, these in vitro and in silico findings are further supported by in vivo observations that desynchronization underlies the multicell-level amplitude decrease in the rat suprachiasmatic nucleus induced by critical light pulses.     

Circadian regulation of bird song, call, and locomotor behavior by pineal melatonin in the zebra finch

As both a photoreceptor and pacemaker in the avian circadian clock system, the pineal gland is crucial for maintaining and synchronizing overt circadian rhythms in processes such as locomotor activity and body temperature through its circadian secretion of the pineal hormone melatonin. In addition to receptor presence in circadian and visual system structures, high-affinity melatonin binding and receptor mRNA are present in the song control system of male oscine passeriform birds. The present study explores the role of pineal melatonin in circadian organization of singing and calling behavior in comparison to locomotor activity under different lighting conditions. Similar to locomotor activity, both singing and calling behavior were regulated on a circadian basis by the central clock system through pineal melatonin, since these behaviors free-ran with a circadian period and since pinealectomy abolished them in constant environmental conditions. Further, rhythmic melatonin administration restored their rhythmicity. However, the rates by which these behaviors became arrhythmic and the rates of their entrainment to rhythmic melatonin administration differed among locomotor activity, singing and calling under constant dim light and constant bright light. Overall, the study demonstrates a role for pineal melatonin in regulating circadian oscillations of avian vocalizations in addition to locomotor activity. It is suggested that these behaviors might be controlled by separable circadian clockworks and that pineal melatonin entrains them all through a circadian clock.     

Temperature synchronization of the Drosophila circadian clock

BACKGROUND: Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. RESULTS: We show here that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins PERIOD and TIMELESS in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. We show that expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. Further, we show that the phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. We initiated the further genetic dissection of temperature-entrainment and isolated the novel Drosophila mutation nocte, which is defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. CONCLUSIONS: We conclude that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. Our data suggest that it involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte.     

Mechanism of all-trans-retinal toxicity with implications for stargardt disease and age-related macular degeneration

Compromised clearance of all-trans-retinal (atRAL), a component of the retinoid cycle, increases the susceptibility of mouse retina to acute light-induced photoreceptor degeneration. Abca4(-/-)Rdh8(-/-) mice featuring defective atRAL clearance were used to examine the one or more underlying molecular mechanisms, because exposure to intense light causes severe photoreceptor degeneration in these animals. Here we report that bright light exposure of Abca4(-/-)Rdh8(-/-) mice increased atRAL levels in the retina that induced rapid NADPH oxidase-mediated overproduction of intracellular reactive oxygen species (ROS). Moreover, such ROS generation was inhibited by blocking phospholipase C and inositol 1,4,5-trisphosphate-induced Ca(2+) release, indicating that activation occurs upstream of NADPH oxidase-mediated ROS generation. Because multiple upstream G protein-coupled receptors can activate phospholipase C, we then tested the effects of antagonists of serotonin 2A (5-HT(2A)R) and M(3)-muscarinic (M(3)R) receptors and found they both protected Abca4(-/-)Rdh8(-/-) mouse retinas from light-induced degeneration. Thus, a cascade of signaling events appears to mediate the toxicity of atRAL in light-induced photoreceptor degeneration of Abca4(-/-)Rdh8(-/-) mice. A similar mechanism may be operative in human Stargardt disease and age-related macular degeneration.     

A role for cyclic AMP in entrainment of the circadian oscillator in Xenopus retinal photoreceptors by dopamine but not by light

The circadian oscillator in Xenopus retinal photoreceptor layers can be reset in similar ways by light and agonists of D2-like dopamine receptors. Treatments that increase cyclic AMP levels act on this oscillator in an opposite fashion, mimicking darkness in the induction of phase shifts. Light and dopamine have each been reported to inhibit adenylate cyclase in photoreceptors. Together, these data suggest that the transduction pathways for entrainment by dopamine and/or light include suppression of cyclic AMP or a cyclic AMP-sensitive step. In these studies, we examined this hypothesis by measuring the effects of treatment with a cyclic AMP analogue on the phase shifts induced in photoreceptor melatonin rhythms by light or a D2 receptor agonist (quinpirole). When photoreceptor layers were treated simultaneously with 8-(4-chlorophenylthio)cyclic AMP (8-CPT-cAMP) and quinpirole at any of three different phases of the circadian cycle, the resulting phase shifts of the melatonin rhythm were always the same as those caused by 8-CPT-cAMP alone. This indicates that there is a cyclic AMP-sensitive step in the dopamine entrainment pathway. In contrast, light pulses did reset the oscillator in the presence of elevated cyclic AMP. This suggests a separate cyclic AMP-insensitive transduction pathway for entrainment by light. Quinpirole reduced basal levels of cyclic AMP in photoreceptors, but light did not. These data suggest that cyclic AMP plays a role in the entrainment pathway activated by dopamine but not in the entrainment pathway activated by light.     

Melatonin Signaling Modulates Clock Genes Expression in the Mouse Retina

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT₁ and MT₂) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.     

Influence of the photocycle and thermocycle on rhythms of plasma thyroxine and plasma and ocular melatonin in late metamorphic stages of the bullfrog tadpole, Rana catesbeiana

The diel fluctuations in plasma thyroxine (T(4)) and plasma and ocular melatonin entrain to the light/dark (LD) cycle in the bullfrog tadpole, although the phase of the rhythms changes during development. Previous studies on the rhythmicity of these hormones were conducted under various LD cycles, but with a constant temperature, raising the question of the role of the natural thermocycle in determining the phase of the rhythms, and the changes that occur in the hormone levels and rhythms during late metamorphosis. To study this question, tadpoles were acclimated to simulated natural conditions of 14.5L:9.5D with a corresponding thermocycle in which the thermophase was 28 degrees C and the cryophase was 18 degrees C, or to the same thermocycle under constant light (24L). On both photoregimens, the diel fluctuations changed between prometamorphosis and metamorphic climax. However, more statistically significant rhythms, as indicated by the cosinor, occurred on 14.5L:9.5D than on 24L. At climax on the LD cycle, all hormones peaked around the same time in the late scotocryophase, whereas on 24L, plasma T(4) peaked in the thermophase and plasma and ocular melatonin peaks occurred some distance from each other early in the cryophase. The earlier peaks of plasma and ocular melatonin on 24L were due to a transient rise in these hormones at the onset of the cryophase, which was not sustained in the absence of an LD cycle. On 14.5L:9.5D with a corresponding thermocycle, the hormone rhythms had nearly the same phases as was found in previous work on 12L:12D at a constant temperature of 22 degrees C, allowing for minor phase shifting due to the photocycle differences, indicating that in this species laboratory studies on constant temperature give valid results even in the absence of a thermocycle. The findings show that the phases of the hormone rhythms are determined by the LD cycle although the onset of the cryophase, in the absence of a photocycle, may exert some influence on the nighttime rise in melatonin. The developmental rise in plasma T(4), and drop in plasma melatonin, occurred on both 14.5L:9.5D and 24L, indicating, taken together with previous work, that these climactic changes were independent of temperature and light cycling.     

Differential contribution of rod and cone circadian clocks in driving retinal melatonin rhythms in Xenopus

Background

Although an endogenous circadian clock located in the retinal photoreceptor layer governs various physiological events including melatonin rhythms in Xenopus laevis, it remains unknown which of the photoreceptors, rod and/or cone, is responsible for the circadian regulation of melatonin release.

Methodology/Principal Findings

We selectively disrupted circadian clock function in either the rod or cone photoreceptor cells by generating transgenic Xenopus tadpoles expressing a dominant-negative CLOCK (XCLΔQ) under the control of a rod or cone-specific promoter. Eyecup culture and continuous melatonin measurement revealed that circadian rhythms of melatonin release were abolished in a majority of the rod-specific XCLΔQ transgenic tadpoles, although the percentage of arrhythmia was lower than that of transgenic tadpole eyes expressing XCLΔQ in both rods and cones. In contrast, whereas a higher percentage of arrhythmia was observed in the eyes of the cone-specific XCLΔQ transgenic tadpoles compare to wild-type counterparts, the rate was significantly lower than in rod-specific transgenics. The levels of the transgene expression were comparable between these two different types of transgenics. In addition, the average overall melatonin levels were not changed in the arrhythmic eyes, suggesting that CLOCK does not affect absolute levels of melatonin, only its temporal expression pattern.

Conclusions/Significance

These results suggest that although the Xenopus retina is made up of approximately equal numbers of rods and cones, the circadian clocks in the rod cells play a dominant role in driving circadian melatonin rhythmicity in the Xenopus retina, although some contribution of the clock in cone cells cannot be excluded.     

S20098 AFFECTS THE FREE-RUNNING RHYTHMS OF BODY TEMPERATURE AND ACTIVITY AND DECREASES LIGHT-INDUCED PHASE DELAYS OF CIRCADIAN RHYTHMS OF THE RAT

Mammalian endogenous circadian rhythms are entrained to the environmental day-night cycle by light exposure. Melatonin is involved in this entrainment by signaling the day-night information to the endogenous circadian pacemaker. Furthermore, melatonin is known to affect the circadian rhythm of body temperature directly. A striking property of the endogenous melatonin signal is its synthesis pattern, characterized by long-term elevated melatonin levels throughout the night. In the present study, the influence of prolonged treatment with the melatonin agonist S20098 during the activity phase of free-running rats was examined. This was achieved by giving S20098 in the food. The free-running body temperature and activity rhythms were studied. The present study shows that enhancement of the melatonin signal, using S20098, affected the free-running rhythm by gradual phase advances of the start of the activity phase, consequently causing an increase in length of the activity phase. A well-known feature of circadian rhythms is its time-dependent sensitivity for light. Light pulse exposure of an animal housed under continuous dark conditions can cause a phase shift of the circadian pacemaker. Therefore, in a second experiment, the influence of melatonin receptor stimulation on the sensitivity of the pacemaker to light was examined by giving the melatonin agonist S20098 in the food during 1 day prior to exposure to a 60-min light pulse of 0, 1.5, 15, or 150 lux given at circadian time (CT) 14. S20098 pretreatment caused a diminished lightpulse- induced phase shift when a light pulse of low light intensity (1.5 lux) was given. S20098 treatment via the food was sufficient to exert chronobiotic activity, and S20098 treatment resulting in prolonged overstimulation of melatonin receptors is able to attenuate the effect of light on the circadian timing system. (Chronobiology International, 18(5), 781–799, 2001)     

Divergent photic thresholds in the non-image-forming visual system: entrainment, masking and pupillary light reflex

Light is the principal cue that entrains the circadian timing system, but the threshold of entrainment and the relative contributions of the retinal photoreceptors—rods, cones and intrinsically photosensitive retinal ganglion cells—are not known. We measured thresholds of entrainment of wheel-running rhythms at three wavelengths, and compared these to thresholds of two other non-image-forming visual system functions: masking and the pupillary light reflex (PLR). At the entrainment threshold, the relative spectral sensitivity and absolute photon flux suggest that this threshold is determined by rods. Dim light that entrained mice failed to elicit either masking or PLR; in general, circadian entrainment is more sensitive by 1–2 log units than other measures of the non-image-forming visual system. Importantly, the results indicate that dim light can entrain circadian rhythms even when it fails to produce more easily measurable acute responses to light such as phase shifting and melatonin suppression. Photosensitivity to one response, therefore, cannot be generalized to other non-image-forming functions. These results also impact practical problems in selecting appropriate lighting in laboratory animal husbandry.     

The evolution of irradiance detection: melanopsin and the non-visual opsins

Circadian rhythms are endogenous 24 h cycles that persist in the absence of external time cues. These rhythms provide an internal representation of day length and optimize physiology and behaviour to the varying demands of the solar cycle. These clocks require daily adjustment to local time and the primary time cue (zeitgeber) used by most vertebrates is the daily change in the amount of environmental light (irradiance) at dawn and dusk, a process termed photoentrainment. Attempts to understand the photoreceptor mechanisms mediating non-image-forming responses to light, such as photoentrainment, have resulted in the discovery of a remarkable array of different photoreceptors and photopigment families, all of which appear to use a basic opsin/vitamin A-based photopigment biochemistry. In non-mammalian vertebrates, specialized photoreceptors are located within the pineal complex, deep brain and dermal melanophores. There is also strong evidence in fish and amphibians for the direct photic regulation of circadian clocks in multiple tissues. By contrast, mammals possess only ocular photoreceptors. However, in addition to the image-forming rods and cones of the retina, there exists a third photoreceptor system based on a subset of melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). In this review, we discuss the range of vertebrate photoreceptors and their opsin photopigments, describe the melanopsin/pRGC system in some detail and then finally consider the molecular evolution and sensory ecology of these non-image-forming photoreceptor systems.     

S20098 AFFECTS THE FREE-RUNNING RHYTHMS OF BODY TEMPERATURE AND ACTIVITY AND DECREASES LIGHT-INDUCED PHASE DELAYS OF CIRCADIAN RHYTHMS OF THE RAT

Mammalian endogenous circadian rhythms are entrained to the environmental day-night cycle by light exposure. Melatonin is involved in this entrainment by signaling the day-night information to the endogenous circadian pacemaker. Furthermore, melatonin is known to affect the circadian rhythm of body temperature directly. A striking property of the endogenous melatonin signal is its synthesis pattern, characterized by long-term elevated melatonin levels throughout the night. In the present study, the influence of prolonged treatment with the melatonin agonist S20098 during the activity phase of free-running rats was examined. This was achieved by giving S20098 in the food. The free-running body temperature and activity rhythms were studied. The present study shows that enhancement of the melatonin signal, using S20098, affected the free-running rhythm by gradual phase advances of the start of the activity phase, consequently causing an increase in length of the activity phase. A well-known feature of circadian rhythms is its time-dependent sensitivity for light. Light pulse exposure of an animal housed under continuous dark conditions can cause a phase shift of the circadian pacemaker. Therefore, in a second experiment, the influence of melatonin receptor stimulation on the sensitivity of the pacemaker to light was examined by giving the melatonin agonist S20098 in the food during 1 day prior to exposure to a 60-min light pulse of 0, 1.5, 15, or 150 lux given at circadian time (CT) 14. S20098 pretreatment caused a diminished lightpulse- induced phase shift when a light pulse of low light intensity (1.5 lux) was given. S20098 treatment via the food was sufficient to exert chronobiotic activity, and S20098 treatment resulting in prolonged overstimulation of melatonin receptors is able to attenuate the effect of light on the circadian timing system. (Chronobiology International, 18(5), 781-799, 2001)