The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster''s clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila''s most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential “clock kinase” phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.     

A spatial model of the plant circadian clock reveals design principles for coordinated timing

Individual plant cells possess a genetic network, the circadian clock, that times internal processes to the day‐night cycle. Mathematical models of the clock are typically either “whole‐plant” that ignore tissue or cell type‐specific clock behavior, or “phase‐only” that do not include molecular components. To address the complex spatial coordination observed in experiments, here we implemented a clock network model on a template of a seedling. In our model, the sensitivity to light varies across the plant, and cells communicate their timing via local or long‐distance sharing of clock components, causing their rhythms to couple. We found that both varied light sensitivity and long‐distance coupling could generate period differences between organs, while local coupling was required to generate the spatial waves of clock gene expression observed experimentally. We then examined our model under noisy light‐dark cycles and found that local coupling minimized timing errors caused by the noise while allowing each plant region to maintain a different clock phase. Thus, local sensitivity to environmental inputs combined with local coupling enables flexible yet robust circadian timing.     

On the persistence of memory: Soft clocks and terrestrial biosphere-atmosphere interactions

The circadian clock is considered a central “orchestrator” of gene expression and metabolism. Concomitantly, the circadian clock is considered of negligible influence in the field and beyond leaf levels, where direct physiological responses to environmental cues are considered the main drivers of diel fluctuations. I propose to bridge the gap across scales by examining current evidence on whether circadian rhythmicity in gas exchange is relevant for field settings and at the ecosystem scale. Nocturnal stomatal conductance and water fluxes appear to be influenced by a “hard” clock that may override the direct physiological responses to the environment. Tests on potential clock controls over photosynthetic carbon assimilation and daytime transpiration are scant yet, if present, could have a large impact on our current understanding and modeling of the exchanges of carbon dioxide and water between terrestrial ecosystems and the atmosphere.     

Social Experience Is Sufficient to Modulate Sleep Need of Drosophila without Increasing Wakefulness

Organisms quickly learn about their surroundings and display synaptic plasticity which is thought to be critical for their survival. For example, fruit flies Drosophila melanogaster exposed to highly enriched social environment are found to show increased synaptic connections and a corresponding increase in sleep. Here we asked if social environment comprising a pair of same-sex individuals could enhance sleep in the participating individuals. To study this, we maintained individuals of D. melanogaster in same-sex pairs for a period of 1 to 4 days, and after separation, monitored sleep of the previously socialized and solitary individuals under similar conditions. Males maintained in pairs for 3 or more days were found to sleep significantly more during daytime and showed a tendency to fall asleep sooner as compared to solitary controls (both measures together are henceforth referred to as “sleep-enhancement”). This sleep phenotype is not strain-specific as it is observed in males from three different “wild type” strains of D. melanogaster. Previous studies on social interaction mediated sleep-enhancement presumed ‘waking experience’ during the interaction to be the primary underlying cause; however, we found sleep-enhancement to occur without any significant increase in wakefulness. Furthermore, while sleep-enhancement due to group-wise social interaction requires Pigment Dispersing Factor (PDF) positive neurons; PDF positive and CRYPTOCHROME (CRY) positive circadian clock neurons and the core circadian clock genes are not required for sleep-enhancement to occur when males interact in pairs. Pair-wise social interaction mediated sleep-enhancement requires dopamine and olfactory signaling, while visual and gustatory signaling systems seem to be dispensable. These results suggest that socialization alone (without any change in wakefulness) is sufficient to cause sleep-enhancement in fruit fly D. melanogaster males, and that its neuronal control is context-specific.     

Deciphering clock cell network morphology within the biological master clock,suprachiasmatic nucleus: From the perspective of circadian wave dynamics

The biological master clock, suprachiasmatic nucleus (of rat and mouse), is composed of ~10,000 clock cells which are heterogeneous with respect to their circadian periods. Despite this inhomogeneity, an intact SCN maintains a very good degree of circadian phase (time) coherence which is vital for sustaining various circadian rhythmic activities, and it is supposedly achieved by not just one but a few different cell-to-cell coupling mechanisms, among which action potential (AP)-mediated connectivity is known to be essential. But, due to technical difficulties and limitations in experiments, so far very little information is available about the morphology of the connectivity at a cellular scale. Building upon this limited amount of information, here we exhaustively and systematically explore a large pool (~25,000) of various network morphologies to come up with some plausible network features of SCN networks. All candidates under consideration reflect an experimentally obtained ‘indegree distribution’ as well as a ‘physical range distribution of afferent clock cells.’ Then, importantly, with a set of multitude criteria based on the properties of SCN circadian phase waves in extrinsically perturbed as well as in their natural states, we select out appropriate model networks: Some important measures are, 1) level of phase dispersal and direction of wave propagation, 2) phase-resetting ability of the model networks subject to external circadian forcing, and 3) decay rate of perturbation induced “phase-singularities.” The successful, realistic networks have several common features: 1) “indegree” and “outdegree” should have a positive correlation; 2) the cells in the SCN ventrolateral region (core) have a much larger total degree than that of the dorsal medial region (shell); 3) The number of intra-core edges is about 7.5 times that of intra-shell edges; and 4) the distance probability density function for the afferent connections fits well to a beta function. We believe that these newly identified network features would be a useful guide for future explorations on the very much unknown AP-mediated clock cell connectome within the SCN.     

Kinetics and time dependence of the effect of far red light on the photoperiodic induction of flowering in wintex barley

Flowering in the long day plant Hordeum vulgare L. var. Wintex barley was enhanced by the addition of far red light to the main light portion of the photoperiod. Far red energy was provided to produce quantum flux ratios (660/730 nm) and phytochrome photoequilibria (Pfr/total phytochrome) equivalent to those reported both beneath a leaf canopy and outside a canopy at twilight. The photoperiodic requirement for long days can be completely eliminated by the addition of far red light. However, both the effect of extending the photoperiod without far red and the addition of far red to 12-hour photoperiods were suboptimal. Maximal stimulation was achieved only when far red was added to continuous light. The duration of the period of maximal apex elongation rate, as well as the reduction of the time required for floral initiation, were saturated by three inductive cycles. When far red energy was provided intermittently during 3 days of continuous light, the ability to respond varied in a circadian manner. This enhancement of flowering by far red appears to be mediated by the “high irradiance response” of phytochrome.     

Photophysiological cycles in Arctic krill are entrained by weak midday twilight during the Polar Night

Light plays a fundamental role in the ecology of organisms in nearly all habitats on Earth and is central for processes such as vision and the entrainment of the circadian clock. The poles represent extreme light regimes with an annual light cycle including periods of Midnight Sun and Polar Night. The Arctic Ocean extends to the North Pole, and marine light extremes reach their maximum extent in this habitat. During the Polar Night, traditional definitions of day and night and seasonal photoperiod become irrelevant since there are only “twilight” periods defined by the sun’s elevation below the horizon at midday; we term this “midday twilight.” Here, we characterize light across a latitudinal gradient (76.5° N to 81° N) during Polar Night in January. Our light measurements demonstrate that the classical solar diel light cycle dominant at lower latitudes is modulated during Arctic Polar Night by lunar and auroral components. We therefore question whether this particular ambient light environment is relevant to behavioral and visual processes. We reveal from acoustic field observations that the zooplankton community is undergoing diel vertical migration (DVM) behavior. Furthermore, using electroretinogram (ERG) recording under constant darkness, we show that the main migratory species, Arctic krill (Thysanoessa inermis) show endogenous increases in visual sensitivity during the subjective night. This change in sensitivity is comparable to that under exogenous dim light acclimations, although differences in speed of vision suggest separate mechanisms. We conclude that the extremely weak midday twilight experienced by krill at high latitudes during the darkest parts of the year has physiological and ecological relevance.

This study shows that ambient light cycles set an internal rhythm that controls visual sensitivity of Arctic krill during the Polar Night – the darkest part of the year, when the sun remains below the horizon all day. This demonstrates that biologically relevant photoperiods can be achieved during this time of "midday twilight".     

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.     

Circadian Regulation of Food-Anticipatory Activity in Molecular Clock–Deficient Mice

In the mammalian brain, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is considered to be the principal circadian pacemaker, keeping the rhythm of most physiological and behavioral processes on the basis of light/dark cycles. Because restriction of food availability to a certain time of day elicits anticipatory behavior even after ablation of the SCN, such behavior has been assumed to be under the control of another circadian oscillator. According to recent studies, however, mutant mice lacking circadian clock function exhibit normal food-anticipatory activity (FAA), a daily increase in locomotor activity preceding periodic feeding, suggesting that FAA is independent of the known circadian oscillator. To investigate the molecular basis of FAA, we examined oscillatory properties in mice lacking molecular clock components. Mice with SCN lesions or with mutant circadian periods were exposed to restricted feeding schedules at periods within and outside circadian range. Periodic feeding led to the entrainment of FAA rhythms only within a limited circadian range. Cry1^−/− mice, which are known to be a “short-period mutant,” entrained to a shorter period of feeding cycles than did Cry2^−/− mice. This result indicated that the intrinsic periods of FAA rhythms are also affected by Cry deficiency. Bmal1 ^−/− mice, deficient in another essential element of the molecular clock machinery, exhibited a pre-feeding increase of activity far from circadian range, indicating a deficit in circadian oscillation. We propose that mice possess a food-entrainable pacemaker outside the SCN in which canonical clock genes such as Cry1, Cry2 and Bmal1 play essential roles in regulating FAA in a circadian oscillatory manner.     

Photocontrol of Anthocyanin Synthesis: VI. Spectral Sensitivity, Irradiance Dependence, and Reciprocity Relationships

The spectral sensitivity and the irradiance dependence of anthocyanin synthesis, a “high irradiance response,” in cabbage (Brassica oleracea, cv. Red Acre) and tomato (Lycopersicon esculentum, cv. Beefsteak) seedlings exposed to continuous irradiation depend upon the length of the exposure. In cabbage, blue and red are more effective than far red when the irradiations are shorter than 12 hours and less effective than far red when the irradiations are longer than 12 hours. The irradiance dependence is negligible under red and becomes evident under blue and far red red only for exposures longer than 12 hours. Anthocyanin synthesis under intermittent light treatments, of efficiency comparable to that of continuous treatments, obeys the Bunsen-Roscoe reciprocity law and is a function of the dose (irradiance × time), rather than of the irradiance alone. The validity of the reciprocity relationships suggests that only one photoreceptor is responsible for the photocontrol of the response in the blue, red, and far red spectral regions. The characteristics of the response suggest that the photoreceptor is phytochrome, at least in cabbage.     

The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts

Background

Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype (“larks” and “owls”), clock properties measured in human fibroblasts correlated with extreme diurnal behavior.

Methodology/Principal Findings

In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer.

Conclusions/Significance

We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.