LOV domain-containing F-box proteins: light-dependent protein degradation modules in Arabidopsis

Plants constantly survey the surrounding environment using several sets of photoreceptors. They can sense changes in the quantity (=intensity) and quality (=wavelength) of light and use this information to adjust their physiological responses, growth, and developmental patterns. In addition to the classical photoreceptors, such as phytochromes, cryptochromes, and phototropins, ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) proteins have been recently identified as blue-light photoreceptors that are important for regulation of the circadian clock and photoperiodic flowering. The ZTL/FKF1/LKP2 protein family possesses a unique combination of domains: a blue-light-absorbing LOV (Light, Oxygen, or Voltage) domain along with domains involved in protein degradation. Here, we summarize recent advances in our understanding of the function of the Arabidopsis ZTL/FKF1/LKP2 proteins. We summarize the distinct photochemical properties of their LOV domains and discuss the molecular mechanisms by which the ZTL/FKF1/LKP2 proteins regulate the circadian clock and photoperiodic flowering by controlling blue-light-dependent protein degradation.     

The involvement of a rhodopsin-like photopigment in the photoperiodic response of the Japanese quail

Summary A technique has been developed for the investigation of the photopigment involved in the photoperiodic control of reproduction in Japanese quail,Coturnix coturnix. When these photoreceptors were exposed to white or monochromatic light a clear relationship was found between light intensity and the extent of photo-induced luteinizing hormone (LH) secretion. A spectroradiometric investigation of the passage of light through the skull and brain enabled us to illuminate the hypothalamic region with equal numbers of photons at a range of wavelengths. Action spectra were then conducted and showed a photopigment with a peak sensitivity at wavelengths near 500 nm. An excellent match was obtained when the standard absorption spectrum for a rhodopsin was fitted to the action spectrum, suggesting a rhodopsin maximally sensitive at 492 nm. The absolute sensitivity of the photoreceptors was calculated at a range of wavelengths: with light at 500 nm, 2.85×10^–12 E·cm^–2·s^–1 triggered the photoperiodic response. This level of sensitivity is matched only by the rhodopsin visual pigments.Abbreviations LH luteinizing hormone - T transmission     

The Influence of Light Wavelength on Phase-Dependent Responsiveness of the Photoperiodic Clock in Migratory Blackheaded Bunting

We investigated the phase-dependent effects of light wavelength on photoperiodic clock in the migratory blackheaded bunting. Two experiments were performed, employing a skeleton paradigm (6 hours light : 6 hours darkness : 1 hour light : 11 hours darkness; 6L : 6D : 1L : 11D) at 37 ± 2 lux intensity. In the experiment 1, both 6 and 1 h light pulses were given at the same wavelength, 500 nm (green) or 650 nm (red). A group exposed to both pulses of white light served as control. In the experi-ment 2, the two light pulses were given at two different wavelengths, 6 h at 500 nm (green) and 1 h at 640 nm (red) in one group or vice-versa in the other. There was almost no photoinduction when both light pulses in experiment 1, or 1 h light pulse in experiment 2, were green. On the other hand, birds fattened and testes recrudesced when both the light pulses in experiment 1, or 1 h light pulse in experiment 2, were red. Birds receiving both pulses of white light in experiment 1 showed an intermediate response. Taken together, these results indicate that the photoperiodic clock in buntings is differentially responsive at its various circadian phases to different light wavelengths.     

The Influence of Light Wavelength on Phase-Dependent Responsiveness of the Photoperiodic Clock in Migratory Blackheaded Bunting

We investigated the phase-dependent effects of light wavelength on photoperiodic clock in the migratory blackheaded bunting. Two experiments were performed, employing a skeleton paradigm (6 hours light : 6 hours darkness : 1 hour light : 11 hours darkness; 6L : 6D : 1L : 11D) at 37 ± 2 lux intensity. In the experiment 1, both 6 and 1 h light pulses were given at the same wavelength, 500 nm (green) or 650 nm (red). A group exposed to both pulses of white light served as control. In the experi-ment 2, the two light pulses were given at two different wavelengths, 6 h at 500 nm (green) and 1 h at 640 nm (red) in one group or vice-versa in the other. There was almost no photoinduction when both light pulses in experiment 1, or 1 h light pulse in experiment 2, were green. On the other hand, birds fattened and testes recrudesced when both the light pulses in experiment 1, or 1 h light pulse in experiment 2, were red. Birds receiving both pulses of white light in experiment 1 showed an intermediate response. Taken together, these results indicate that the photoperiodic clock in buntings is differentially responsive at its various circadian phases to different light wavelengths.     

Insect photoperiodism: seeing the light

This review examines the spectral sensitivities of photoperiodic responses in insects and mites in relation to circadian‐based models for the photoperiodic clock. It concludes that there are probably a number of different photoreceptors at both the organ and molecular levels. These latter probably fall into two classes: (i) a blue‐light sensitive photoreceptor and (ii) a range of opsins (i.e. opsin proteins conjugated with a vitamin A based pigment) absorbing light at a range of wavelengths. In flesh flies (Sarcophaga spp. and possibly other higher Diptera), which are considered to exemplify the ‘external coincidence’ model, entrainment of the photoperiodic oscillator probably involves a blue‐light photoreceptor of Drosophila‐type CRYPTOCHROME (CRY1) absorbing maximally at approximately 470 nm, whereas opsins absorbing at longer wavelengths may be involved in the photo‐inductive process (diapause/nondiapause regulation) that occurs when dawn light coincides with the photo‐inducible phase. In the parasitic wasp Nasonia vitripennis, on the other hand, a species that lacks CRY1 but expresses the nonphotosensitive ‘mammalian‐type’ CRY2, and is considered to exemplify ‘internal coincidence’, entrainment of the dawn and dusk oscillators may involve opsin‐based photoreceptors absorbing light at longer wavelengths as far as the red end of the spectrum. In the Lepidoptera, which express both CRY1 and CRY2, properties of both external and internal coincidence may be evident. The presence or absence of cry1 in the genome may thus emerge as a key to the photoperiodic mechanism on its light input pathway.     

Nonvisual photoreceptors of the deep brain, pineal organs and retina

The role of the nonvisual photoreception is to synchronise periodic functions of living organisms to the environmental light periods in order to help survival of various species in different biotopes. In vertebrates, the so-called deep brain (septal and hypothalamic) photoreceptors, the pineal organs (pineal- and parapineal organs, frontal- and parietal eye) and the retina (of the "lateral" eye) are involved in the light-based entrain of endogenous circadian clocks present in various organs. In humans, photoperiodicity was studied in connection with sleep disturbances in shift work, seasonal depression, and in jet-lag of transmeridional travellers. In the present review, experimental and molecular aspects are discussed, focusing on the histological and histochemical basis of the function of nonvisual photoreceptors. We also offer a view about functional changes of these photoreceptors during pre- and postnatal development as well as about its possible evolution. Our scope in some points is different from the generally accepted views on the nonvisual photoreceptive systems. The deep brain photoreceptors are hypothalamic and septal nuclei of the periventricular cerebrospinal fluid (CSF)-contacting neuronal system. Already present in the lancelet and representing the most ancient type of vertebrate nerve cells ("protoneurons"), CSF-contacting neurons are sensory-type cells sitting in the wall of the brain ventricles that send a ciliated dendritic process into the CSF. Various opsins and other members of the phototransduction cascade have been demonstrated in telencephalic and hypothalamic groups of these neurons. In all species examined so far, deep brain photoreceptors play a role in the circadian and circannual regulation of periodic functions. Mainly called pineal "glands" in the last decades, the pineal organs actually represent a differentiated form of encephalic photoreceptors. Supposed to be intra- and extracranially outgrown groups of deep brain photoreceptors, pineal organs also contain neurons and glial elements. Extracranial pineal organs of submammalians are cone-dominated photoreceptors sensitive to different wavelengths of light, while intracranial pineal organs predominantly contain rod-like photoreceptor cells and thus scotopic light receptors. Vitamin B-based light-sensitive cryptochromes localized immunocytochemically in some pineal cells may take part in both the photoreception and the pacemaker function of the pineal organ. In spite of expressing phototransduction cascade molecules and forming outer segment-like cilia in some species, the mammalian pineal is considered by most of the authors as a light-insensitive organ. Expression of phototransduction cascade molecules, predominantly in young animals, is a photoreceptor-like characteristic of pinealocytes in higher vertebrates that may contribute to a light-percepting task in the perinatal entrainment of rhythmic functions. In adult mammals, adrenergic nerves--mediating daily fluctuation of sympathetic activity rather than retinal light information as generally supposed--may sustain circadian periodicity already entrained by light perinatally. Altogether three phases were supposed to exist in pineal entrainment of internal pacemakers: an embryological synchronization by light and in viviparous vertebrates by maternal effects (1); a light-based, postnatal entrainment (2); and in adults, a maintenance of periodicity by daily sympathetic rhythm of the hypothalamus. In addition to its visual function, the lateral eye retina performs a nonvisual task. Nonvisual retinal light perception primarily entrains genetically-determined periodicity, such as rod-cone dominance, EEG rhythms or retinomotor movements. It also influences the suprachiasmatic nucleus, the primary pacemaker of the brain. As neither rods nor cones seem to represent the nonvisual retinal photoreceptors, the presence of additional photoreceptors has been supposed. Cryptochrome 1, a photosensitive molecule identified in retinal nerve cells and in a subpopulation of retinal photoreceptors, is a good candidate for the nonvisual photoreceptor molecule as well as for a member of pacemaker molecules in the retina. When comparing various visual and nonvisual photoreceptors, transitory, "semi visual" (directional) light-perceptive cells can be detected among them, such as those in the parietal eye of reptiles. Measuring diffuse light intensity of the environment, semivisual photoreceptors also possess some directional light perceptive capacity aided by complementary lens-like structures, and screening pigment cells. Semivisual photoreception in aquatic animals may serve for identifying environmental areas of suitable illumination, or in poikilotermic terrestrial species for measuring direct solar irradiation for thermoregulation. As directional photoreceptors were identified among nonvisual light perceptive cells in the lancelet, but eyes are lacking, an early appearance of semivisual function, prior to a visual one (nonvisual --> semivisual --> visual?) in the vertebrate evolution was supposed.     

New insights into ancient seasonal life timers

Organisms must adapt to seasonal changes in the environment and time their physiology accordingly. In vertebrates, the annual change in photoperiod is often critical for entraining the neuroendocrine pathways, which drive seasonal metabolic and reproductive cycles. These cycles depend on thyroid hormone (TH), reflecting its ancestral role in metabolic control. Recent studies reveal that - in mammals and birds - TH effects are mediated by the hypothalamus. Photoperiodic manipulations alter hypothalamic TH availability by regulating the expression of TH deiodinases (DIO). In non-mammalian vertebrates, light acts through extraretinal, 'deep brain' photoreceptors, and the eyes are not involved in seasonal photoperiodic responses. In mammals, extraretinal photoreceptors have been lost, and the nocturnal melatonin signal generated from the pineal gland has been co-opted to provide the photoperiodic message. Pineal function is phased to the light-dark cycle by retinal input, and photoperiodic changes in melatonin secretion control neuroendocrine pathway function. New evidence indicates that these comparatively divergent photosensensory mechanisms re-converge in the pars tuberalis of the pituitary, lying beneath the hypothalamus. In all vertebrates studied, the pars tuberalis secretes thyrotrophin in a light- or melatonin-sensitive manner, to act on neighbouring hypothalamic DIO expressing cells. Hence, an ancient and fundamentally conserved brain thyroid signalling system governs seasonal biology in vertebrates.     

Wavelength dependency of light-induced effects on photoperiodic clock in the migratory blackheaded bunting (Emberiza melanocephala)

The effects of light wavelength on photoperiodic clock were determined in the migratory male blackheaded bunting (Emberiza melanocephala). We constructed an action spectrum for photoperiodic induction (body fattening, gain in body mass, and gonadal recrudescence) by exposing birds for 4.5 weeks to 13 h light per day (L:D = 13:11 h) of white (control), blue (450 nm), or red (640 nm) color at irradiances ranging from 0.028 to 1.4Wm(-2). The threshold light irradiance for photoinduction was about 10-fold higher for blue, compared to red and white light. Phase-dependent effects of light wavelength on the photoperiodic clock were further examined in the next two sets of skeleton photoperiods (SKPs). In the first set of SKPs, birds were exposed for four weeks to asymmetrical light periods (L:D:L:D= 6:6:1:11 h) at 0.25+/-0.01 W m(-2); two light periods applied were of the same (450nm: blue:blue, B:B; 640nm, red:red, R:R) or different (blue:red, B:R or red:blue, R:B) wavelengths, or of white:white (W:W, controls). Photoperiodic induction occurred under R:R and B:R, but not under B:B and R:B light conditions; the W:W condition induced an intermediate response. The second set of SKPs used symmetrical light periods (L:D:L:D = 1:11:1:11 h), and measured effects also on the activity rhythm. Birds were first exposed to one of the four SKPs (R:R, B:B, R:B, or B:R) for three weeks, subsequently were released into dim constant light (LLdim; approximately 0.01 Wm(-2), the night light used in an L:D cycle) for two weeks, and then were returned to respective SKPs for another three weeks. Activity was greater in the R:R compared to B:B, and in B:R compared to R:B light condition. Zugunruhe (intense nighttime activity, indicating migratory restlessness in a caged situation) developed under the R:R and B:R, but not the B:B and R:B, light condition. Under LLdim, all birds free-ran with a period >24h, the Zugunruhe had a circadian period longer than the daytime activity, and the re-entrainment to SKPs was influenced by the position of light periods relative to circadian phase of the activity rhythm. Photoperiodic induction at the end of 8 weeks was found in the R:R and B:R, but not in B:B, light conditions; in the R:B condition only one bird had initiated testes. Taken together, these results suggest that in the blackheaded bunting, the circadian photoperiodic clock is differentially responsive to light wavelengths; this responsiveness is phase-dependent, and the development of Zugunruhe reflects a true circadian function. Wavelength-dependent response of the photoperiodic clock could be part of an adaptive strategy in evolution of the seasonality in reproduction and migration among photoperiodic species under wild conditions.     

Both the anterior and posterior eyes function as photoreceptors for photoperiodic termination of diapause in the two-spotted spider mite

Photoreceptors involved in photoperiodism in insects and mites can be either the retinal photoreceptors in the visual system or nonvisual extraretinal photoreceptors. Mites with no eyes have a clear photoperiodic response, suggesting the involvement of extraretinal photoreceptors in mite photoperiodism. In mites equipped with eyes, however, it is not known whether the retinal or extraretinal photoreceptors are involved in photoperiodism. The two-spotted spider mite Tetranychus urticae possesses two pairs of eyes. Adult females of this species terminate diapause in response to long days. To investigate whether the eyes function as photoperiodic photoreceptors in T. urticae, their eyes were ablated using a laser ablation system. Mites with their eyes intact terminated diapause under long days after low temperature exposure, whereas they remained in diapause under short days. Under constant darkness, they did not terminate diapause. When all eyes were removed, the mites remained in diapause even when they were maintained under long days. In contrast, the mites showed clear photoperiodic response when only the anterior or posterior eyes were removed. These results indicate that both the anterior and posterior eyes function as photoreceptors in photoperiodic termination of diapause in T. urticae.     

Circadian rhythmicity and photoperiodism in the pitcher-plant mosquito: can the seasonal timer evolve independently of the circadian clock?

The two major rhythms of the biosphere are daily and seasonal; the two major adaptations to these rhythms are the circadian clock, mediating daily activities, and the photoperiodic timer, mediating seasonal activities. The mechanistic connection between the circadian clock and the photoperiodic timer remains unresolved. Herein, we show that the rhythmic developmental response to exotic light:dark cycles, usually used to infer a causal connection between the circadian clock and the photoperiodic timer, has evolved independently of the photoperiodic timer in the pitcher-plant mosquito Wyeomyia smithii across the climatic gradient of eastern North America from Florida to Canada and from the coastal plain to the mountains. We conclude that the photoperiodic timing of seasonal events can evolve independently of the daily circadian clock.     

Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity

The circadian clock is entrained to the daily cycle of day and night by light signals at dawn and dusk. Plants make use of both the phytochrome (phy) and cryptochrome (cry) families of photoreceptors in gathering information about the light environment for setting the clock. We demonstrate that the phytochromes phyA, phyB, phyD, and phyE act as photoreceptors in red light input to the clock and that phyA and the cryptochromes cry1 and cry2 act as photoreceptors in blue light input. phyA and phyB act additively in red light input to the clock, whereas cry1 and cry2 act redundantly in blue light input. In addition to the action of cry1 as a photoreceptor that mediates blue light input into the clock, we demonstrate a requirement of cry1 for phyA signaling to the clock in both red and blue light. Importantly, Arabidopsis cry1 cry2 double mutants still show robust rhythmicity, indicating that cryptochromes do not form a part of the central circadian oscillator in plants as they do in mammals.     

Vertebrate ancient opsin photopigment spectra and the avian photoperiodic response

In mammals, photoreception is restricted to cones, rods and a subset of retinal ganglion cells. By contrast, non-mammalian vertebrates possess many extraocular photoreceptors but in many cases the role of these photoreceptors and their underlying photopigments is unknown. In birds, deep brain photoreceptors have been shown to sense photic changes in daylength (photoperiod) and mediate seasonal reproduction. Nonetheless, the specific identity of the opsin photopigment 'sensor' involved has remained elusive. Previously, we showed that vertebrate ancient (VA) opsin is expressed in avian hypothalamic neurons and forms a photosensitive molecule. However, a direct functional link between VA opsin and the regulation of seasonal biology was absent. Here, we report the in vivo and in vitro absorption spectra (λ(max) = ~490 nm) for chicken VA photopigments. Furthermore, the spectral sensitivity of these photopigments match the peak absorbance of the avian photoperiodic response (λ(max) = 492 nm) and permits maximum photon capture within the restricted light environment of the hypothalamus. Such a correspondence argues strongly that VA opsin plays a key role in regulating seasonal reproduction in birds.     

Day-length perception and the photoperiodic regulation of flowering in Arabidopsis

The flowering of Arabidopsis plants is accelerated by long-day photoperiods, and recent genetic studies have identified elements of the photoperiodic timing mechanism. These elements comprise genes that regulate the function of the circadian clock, photoreceptors, and downstream components of light signaling pathways. These results provide evidence for the role of the circadian clock in photoperiodic time measurement and suggest that photoperiod perception may follow Pittendrigh's external coincidence model. T-cycle experiments indicated that changes in the timing of circadian rhythms, relative to dawn and dusk, correlated with altered flowering time. Thus, the perception of photoperiod maybe mediated by adjustments in the phase of the circadian cycle that arise upon re-entrainment to a different light-dark cycle. The nature of the rhythm underlying the floral response is not known, but candidate molecules have been identified.     

Tuning in to the signals controlling photoregulated gene expression in plants.

Plants have developed flexible mechanisms to respond appropriately to environmental signals. These stimuli are transduced by largely unknown signalling pathways that are likely to be modulated by endogenous developmental signals to produce an integrated response that coordinately regulates gene expression. Light is a critical environmental signal that controls many aspects of plant development via a series of photoreceptors that are able to respond to different light wavelengths. Light is also the principal energy source for photosynthesis. The photosynthetic products are carbohydrates which are translocated in the form of sucrose from the photosynthetic (source) to non-photosynthetic (sink) organs. Consequently, the control of photoregulated genes must integrate developmental inputs with signals derived from the photoreceptors, from the photosynthetic apparatus and from metabolites such as sucrose.     

Light spectrum and intensity,and the timekeeping in birds

Abstract

The important aspect of light environment is to provide time-of-day and time-of-year information to the endogenous machinery that measures time. In a 24 h day there are conspicuous alterations in light intensity and spectrum. VIBGYOR is the visible portion of spectrum covering the light wavelength range from 380-760 nm. Each wavelength can activate the select class of photoreceptors, and hence a specific colour is experienced. Photoreceptors have opsin-based molecules that can trap light and thus play a key role in the perception of light and dark signals of the day. Eyes are the main photoreceptive structure but non-mammalian vertebrates such as birds have both retinal (eyes) and extra-retinal (e.g. lateral eyes, pineal, parapineal organs and deep brain photoreceptors) structures for photoreception. These opsin-based molecules found in different regions of the eyes and brain are sensitive to different wavelengths of light, hence play an important role in regulating the circadian and seasonal rhythms by decoding dawn and dusk; the time of maximum transition in wavelength and intensity of light. The melatonin pattern is also affected by light characteristics. In birds, the wavelength is shown to affect orientation and energy expenditure as well. This review focuses on different aspects of light wavelength and intensity affecting avian physiology and behaviour.     

Experimental evidence for a non-clock role of the circadian system in spider mite photoperiodism

In the spider mite Tetranychus urticae photoperiodic time measurement proceeds accurately in orange-red light of 580 nm and above in light/dark cycles with a period length of 20 h but not in 'natural' cycles with a period length of 24 h. To explain these results it is hypothesized that the photoperiodic clock in the spider mite is sensitive to orange-red light, but the Nanda-Hamner rhythm (a circadian rhythm with a free-running period tau of 20 h involved in the photoperiodic response) is not and consequently free runs in orange-red light. To test this hypothesis a zeitgeber was sought that could entrain the Nanda-Hamner rhythm to a 24-h cycle without inducing diapause itself, in order to manipulate the rhythm independently from the orange-red sensitive photoperiodic clock. A suitable zeitgeber was found to be a thermoperiod with a 12-h warm phase and a 12-h cold phase. Combining the thermoperiod with the long-night orange-red light/dark regime, both with a cycle length of 24 h, resulted in a high diapause incidence, although neither regime was capable of inducing diapause on its own. The conclusion is that the Nanda-Hamner rhythm is necessary for the realization of the photoperiodic response, but is not part of the photoperiodic clock, because photoperiodic time measurement takes place in orange-red light whereas the rhythm is not able to 'see' the orange-red light. It is speculated that the Nanda-Hamner rhythm is involved in the timely synthesis of a substrate for the photoperiodic clock in the spider mite.     

Wavelength Dependency of Light-Induced Effects on Photoperiodic Clock in the Migratory Blackheaded Bunting (Emberiza melanocephala)

The effects of light wavelength on photoperiodic clock were determined in the migratory male blackheaded bunting (Emberiza melanocephala). We constructed an action spectrum for photoperiodic induction (body fattening, gain in body mass, and gonadal recrudescence) by exposing birds for 4.5 weeks to 13 h light per day (L:D = 13:11 h) of white (control), blue (450 nm), or red (640 nm) color at irradiances ranging from 0.028 to 1.4 W m^?2. The threshold light irradiance for photoinduction was about 10-fold higher for blue, compared to red and white light. Phase-dependent effects of light wavelength on the photoperiodic clock were further examined in the next two sets of skeleton photoperiods (SKPs). In the first set of SKPs, birds were exposed for four weeks to asymmetrical light periods (L:D:L:D = 6:6:1:11 h) at 0.25 ± 0.01 W m^?2; two light periods applied were of the same (450 nm: blue:blue, B:B; 640 nm, red:red, R:R) or different (blue:red, B:R or red:blue, R:B) wavelengths, or of white:white (W:W, controls). Photoperiodic induction occurred under R:R and B:R, but not under B:B and R:B light conditions; the W:W condition induced an intermediate response. The second set of SKPs used symmetrical light periods (L:D:L:D = 1:11:1:11 h), and measured effects also on the activity rhythm. Birds were first exposed to one of the four SKPs (R:R, B:B, R:B, or B:R) for three weeks, subsequently were released into dim constant light (LL_dim; ?0.01 W m^?2, the night light used in an L:D cycle) for two weeks, and then were returned to respective SKPs for another three weeks. Activity was greater in the R:R compared to B:B, and in B:R compared to R:B light condition. Zugunruhe (intense nighttime activity, indicating migratory restlessness in a caged situation) developed under the R:R and B:R, but not the B:B and R:B, light condition. Under LL_dim, all birds free-ran with a period >24 h, the Zugunruhe had a circadian period longer than the daytime activity, and the re-entrainment to SKPs was influenced by the position of light periods relative to circadian phase of the activity rhythm. Photoperiodic induction at the end of 8 weeks was found in the R:R and B:R, but not in B:B, light conditions; in the R:B condition only one bird had initiated testes. Taken together, these results suggest that in the blackheaded bunting, the circadian photoperiodic clock is differentially responsive to light wavelengths; this responsiveness is phase-dependent, and the development of Zugunruhe reflects a true circadian function. Wavelength-dependent response of the photoperiodic clock could be part of an adaptive strategy in evolution of the seasonality in reproduction and migration among photoperiodic species under wild conditions.     

An autonomous circadian clock in the inner mouse retina regulated by dopamine and GABA

The influence of the mammalian retinal circadian clock on retinal physiology and function is widely recognized, yet the cellular elements and neural regulation of retinal circadian pacemaking remain unclear due to the challenge of long-term culture of adult mammalian retina and the lack of an ideal experimental measure of the retinal circadian clock. In the current study, we developed a protocol for long-term culture of intact mouse retinas, which allows retinal circadian rhythms to be monitored in real time as luminescence rhythms from a PERIOD2::LUCIFERASE (PER2::LUC) clock gene reporter. With this in vitro assay, we studied the characteristics and location within the retina of circadian PER2::LUC rhythms, the influence of major retinal neurotransmitters, and the resetting of the retinal circadian clock by light. Retinal PER2::LUC rhythms were routinely measured from whole-mount retinal explants for 10 d and for up to 30 d. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the inner nuclear layer. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate) and the inner nuclear layer (dopamine, acetylcholine, GABA, glycine, and glutamate) did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36 (cx36)-containing gap junctions, indicating that PER2::LUC rhythms generation in the inner nuclear layer is likely cell autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization and casein kinase, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Light pulses reset the phase of the in vitro retinal oscillator and dopamine D1 receptor antagonists attenuated these phase shifts. Thus, dopamine and GABA act at the molecular level of PER proteins to play key roles in the organization of the retinal circadian clock.     

A Colourful Clock

Circadian rhythms are an essential property of life on Earth. In mammals, these rhythms are coordinated by a small set of neurons, located in the suprachiasmatic nuclei (SCN). The environmental light/dark cycle synchronizes (entrains) the SCN via a distinct pathway, originating in a subset of photosensitive retinal ganglion cells (pRGCs) that utilize the photopigment melanopsin (OPN4). The pRGCs are also innervated by rods and cones and, so, are both endogenously and exogenously light sensitive. Accumulating evidence has shown that the circadian system is sensitive to ultraviolet (UV), blue, and green wavelengths of light. However, it was unclear whether colour perception itself can help entrain the SCN. By utilizing both behavioural and electrophysiological recording techniques, Walmsley and colleagues show that multiple photic channels interact and enhance the capacity of the SCN to synchronize to the environmental cycle. Thus, entrainment of the circadian system combines both environmental irradiance and colour information to ensure that internal and external time are appropriately aligned.Light is sensed by three classes of retinal photoreceptors. In the outer retina, light is detected by rod and cone photoreceptors; in the inner retina a small number of photosensitive retinal ganglion cells (pRGCs) express the photopigment melanopsin, which confers photosensitivity to these neurons. The signals derived from the various photoreceptors are important for visual and nonvisual tasks. The generation of visual images is primarily a function of the classical rod and cone photoreceptors, while the classical photoreceptors together with melanopsin are involved in nonvisual tasks, such as pupillary reflexes and the synchronization of our circadian clock to the environmental light-dark cycle. The discovery of non-rod, non-cone photoreceptors [1] and the demonstration that they utilize the photopigment melanopsin [2] led to the general and unfortunate notion that melanopsin is the major—if not the only—photopigment that contributes to photoentrainment and that this sensory task is monochromatic, with no role for colour discrimination. The article of Walmsley et al. [3] addresses this misconception and presents evidence for a role for colour detection in photoentrainment.These findings are in accordance with recent publications indicating that not only melanopsin but also other photopigments contribute to entrainment [4–9]. The consensus from these studies is that rods are most important for photoentrainment at low light intensities; cone photoreceptors transduce light information to the suprachiasmatic nuclei (SCN) at intermediate and high irradiances and are able to detect sudden changes in light intensity, whilst melanopsin detects light at high irradiances and may be of specific importance for the integration of light information over longer periods of time.While it is true that the different photoreceptors are sensitive across a range of different light intensities, they are also maximally sensitive to different colours or wavelengths of light, and as a result, each class of photoreceptor has a different peak sensitivity. Rod photoreceptors have their peak sensitivity at 498 nm light (which would appear to us as green), melanopsin is maximally sensitive to 480 nm (blue) light, and most mammals express two distinct classes of cone photoreceptors, which in the majority of rodents are maximally sensitive to approximately 360 nm (UV) and approximately508 nm (green) light respectively. As a consequence, the different photoreceptive systems not only show differences in their absolute sensitivities, but in addition, they are differentially stimulated by different wavelengths of light. Theoretically, this characteristic difference in the spectral sensitivity of the photoreceptors could add to the detection of light intensities over the day-night cycle and thereby to the capacity of the SCN to adjust to it. Such a possibility was first suggested by Foster and colleagues [10] and shown for fish by Pauers and colleagues [11].Walmsley et al. make use of a sophisticated experimental design to show the functional role of colour for the circadian system. Environmental light measurements were performed in Manchester, which lies 53 degrees north of the equator, as a function of solar angle relative to the horizon. Measurements were performed between August and October of 2005. The spectral measurements showed a reduction of irradiance and an increasing amount of short-wavelength light during twilight when the sun is below the horizon (Fig 1). This is a consequence of the differential scattering of shorter wavelengths of light by particles in the atmosphere and filtering of long wavelength light by the Chappuis band of the ozone layer. Based on the known spectral sensitivities of the short- and medium-wavelength—sensitive cone opsins, the excitation of the two pigments at different solar angles was calculated. Relative to the medium-wavelength—sensitive cone opsin, excitation of the short-wavelength—sensitive cone opsin decreases with increasing elevation of the sun above the horizon. The spectral composition of light reaching the earth shows less day-to-day variability in spectral composition than in irradiance, and thus, it may have a high predictive value about the position of the sun, as originally predicted by Foster (e.g., 2001).Open in a separate windowFig 1Colour detection by the circadian system.Colour detection by the circadian system. (A) Spectral changes in light reaching the earth during twilight. At negative solar angles, short wavelength light is dominant, while at positive solar angles, long wavelength light is dominant. (B) Schematic overview of light signalling to the SCN resulting in entrainment to a light-dark cycle. Light is the main entraining signal that adjusts the endogenous period length to the day-night cycle. Electrical activity of SCN neurons is the main output signal of the SCN, which leads to temporal regulation of behavioural activity. (C) Schematic depiction of two types of light-responsive neurons observed in the SCN as shown by Walmsley and colleagues: the colour-sensitive neuron (upper traces) and the brightness-sensitive neurons (lower traces). Image credit: Hester van Diepen.The information about changes in spectral composition of light over the day were used to simulate twilight in laboratory conditions to study whether mice make use of these changes in colour as an estimation of the time of the day. Electrophysiological recordings from SCN neurons revealed that a subpopulation of light-responsive neurons is sensitive to changes in the spectral composition of daylight. These neurons were detected based on the presence of a response to changes in spectral composition of the light source, consisting of three light-emitting diodes (LEDs) with narrow band emittance at 365 nm, 460 nm, and 600 nm. These wavelengths maximally stimulate the short-wavelength, UV sensitive cone, melanopsin, and a red knock-in cone that substitutes the normal green cone and enhances discrimination between photoreceptors.In addition to being sensitive to spectral composition changes, some neurons showed colour-opponency in response to selective activation of short-wavelength—sensitive opsins versus long-wavelength—sensitive opsins or vice versa (Fig 1). Cone photoreceptors display colour-opponency, most likely by combining signals from separate classes of cone photoreceptors in an opposing way [12,13]. The SCN may make use of this antagonistic effect by determination of the relative activation of the cone photoreceptors to various wavelengths of light. Since the two classes of photoreceptors in the mammalian retina are specifically sensitive to short-wavelength and long-wavelength light, the blue-yellow colour discrimination is a reliable way in which the SCN can detect transitions from twilight to daylight. In fact, behavioural experiments in mice showed that changes in colour are required for appropriate biological timing with respect to the solar cycle.It is of utmost importance that the SCN is appropriately aligned with the environmental light-dark cycle. In rodents, the SCN consists of about 20,000 cell autonomous oscillators that are capable of producing circadian rhythms with a period deviating slightly from 24 hours. For proper function, the cells have to be mutually synchronized, and as an ensemble they should synchronize to the environmental cycle. Direct retinal input to the SCN, via the retinohypothaloamic tract (RHT), originates exclusively from pRGCs [14]. The pRGCs can be activated by rod and cone photoreceptors via synaptic connections to the outer retina [15]. Upon activation by light, photoreceptors undergo a transformational change from the inactive state to the active state, which results in a signalling cascade that ultimately leads to the generation of action potentials in the retinal ganglion cells and in the optic tract. The initial response of the classical photoreceptors is a hyperpolarization, while the conformational change of melanopsin leads to a depolarization. The present view, emerging from the various studies, is that light information reaches the SCN via all retinal photoreceptive systems. The ability of SCN neurons to not only determine the amount of light but also the wavelength of light by comparison of the relative activation of the different photoreceptors provides the SCN with additional information. The detection of changes in spectral composition may be an additive way to detect the time of the day-night cycle, as compared to irradiance detection alone. The amount of light perceived by the SCN can vary over the day, caused by covering of the sun with clouds or hiding of a mammal in its burrow [16]. The spectral composition of light during the lower light intensity time point will not change. Therefore, this perception system provides a refinement in the ability of the SCN to estimate time of day, which would not have been possible by the estimation of irradiance per se. As at least 90% of mammalian species can discriminate colour on the basis of at least two classes of cone opsins [17], it would be interesting to investigate to what degree other mammals also make use of colour to tell time of day.     

Rhodopsin 5- and Rhodopsin 6-mediated clock synchronization in Drosophila melanogaster is independent of retinal phospholipase C-β signaling

Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpA(P41) ) that disrupts canonical phototransduction due to the absence of Phospholipase C-β (PLC-β). We reveal that norpA(P41) is a true loss-of-function allele, resulting in a truncated PLC-β protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-β enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila.