Phosphorylation of Rat Melanopsin at Ser-381 and Ser-398 by Light/Dark and Its Importance for Intrinsically Photosensitive Ganglion Cells (ipRGCs) Cellular Ca2+ Signaling

The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca²⁺ response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca²⁺ response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.     

Melanopsin as a Sleep Modulator: Circadian Gating of the Direct Effects of Light on Sleep and Altered Sleep Homeostasis in Opn4−/− Mice

Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4^−/−) mice under various light–dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7–10 Hz) and gamma (40–70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4^−/− mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-h1-h schedule revealed that the failure to respond to light in Opn4^−/− mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4^−/− mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4^−/− mice slept 1 h less during the 12-h light period of a LD 1212 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4^−/− mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4^−/− mice. In mice, melanopsin's contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.     

Melanopsin as a Sleep Modulator: Circadian Gating of the Direct Effects of Light on Sleep and Altered Sleep Homeostasis in Opn4?/? Mice

Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4^−/−) mice under various light–dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7–10 Hz) and gamma (40–70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4^−/− mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-h∶1-h schedule revealed that the failure to respond to light in Opn4^−/− mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4^−/− mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4^−/− mice slept 1 h less during the 12-h light period of a LD 12∶12 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4^−/− mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4^−/− mice. In mice, melanopsin''s contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.     

Phaseshifting of the photoperiodic flowering response rhythm in Pharbitis nil by red-light pulses

The control by light of the flowering response rhythm in the short-day plant Pharbitis nil Choisy cv. Violet was examined by giving a single pulse of light at various times between 1 and 6 h after a 24-h light period. When the first circadian cycle of the rhythm was monitored, it was found that a pulse of red light given at 1, 2 or 3 h into a 72-dark period caused a 1-h delay of the phase of the response rhythm, while a pulse at 6 h caused a 2-h delay. These results support the hypothesis that, when red-light pulses are given at hourly intervals, they are as effective as continuous light in preventing the onset of dark timing because they repeatedly return the rhythm to the circadian time at which it is apparently suspended in continuous light. The perception of and response to continuous light and red-light pulses are also briefly discussed.     

Circadian gene expression patterns of melanopsin and pinopsin in the chick pineal gland

The directly light-sensitive chick pineal gland contains at least two photopigments. Pinopsin seems to mediate the acute inhibitory effect of light on melatonin synthesis, whereas melanopsin may act by phase-shifting the intrapineal circadian clock. In the present study we have investigated, by means of quantitative RT-PCR, the daily rhythm of photopigment gene expression as monitored by mRNA levels. Under a 12-h light/12-h dark cycle, the mRNA levels of both pigments were 5-fold higher in the transitional phase from light to dark than at night, both in vivo and in vitro. Under constant darkness in vivo and in vitro, the peak of pinopsin mRNA levels was attenuated, whereas that of melanopsin was not. Thus, whereas the daily rhythm of pinopsin gene expression is dually regulated by light plus the intrapineal circadian oscillator, that of melanopsin appears to depend solely on the oscillator.     

Circadian clock resetting by sleep deprivation without exercise in Syrian hamsters: dark pulses revisited

Circadian rhythms in Syrian hamsters can be phase shifted by procedures that stimulate wheel running ("exercise") in the mid-subjective day (the hamster's usual sleep period). The authors recently demonstrated that keeping hamsters awake by gentle handling, without continuous running, is sufficient to mimic this effect. Here, the authors assessed whether wakefulness, independent of wheel running, also mediates phase shifts to dark pulses during the midsubjective day in hamsters free-running in constant light (LL). With running wheels locked during a 3 h dark pulse on day 3 of LL, hamsters (N = 16) averaged only 43+/-15 min of spontaneous wake time and phase shifted only 24+/-43 min. When wheels were open during a dark pulse, two hamsters remained awake, ran continuously, and showed phase advance shifts of 7.3 h and 8.7 h, respectively, whereas the other hamsters were awake <60 min and shifted only 45+/-38 min. No animals stayed awake for 3 h without running. Additional time in LL (10 and 20 days) did not potentiate the waking or phase shift response to dark pulses. When all hamsters were sleep deprived with wheels locked during a dark pulse, phase advance shifts averaged 261+/-110 min and ranged up to 7.3 h. These shifts are large compared to those previously observed in response to the 3 h sleep deprivation procedure. Additional tests revealed that this potentiated shift response is dependent on LL prior to sleep deprivation but not LL after sleep deprivation. A final sleep deprivation test showed that a small part of the potentiation may be due to suppression of spontaneous wheel running by LL. These results indicate that some correlate of waking, other than continuous running, mediates the phase-shifting effect of dark pulses in the mid-subjective day. The mechanism by which LL potentiates shifting remains to be determined. The lack of effect of subsequent LL on the magnitude of shifts to sleep deprivation in the dark suggests that LL reduces responsivity to light by processes that take >3 h of dark to reverse.     

Slow fluorescence fluctuations following high light to low light or dark transitions in Chlamydomonas reinhardi.

Slow fluorescence transients in Chlamydomonas reinhardi arise after transitions from high light intensities to low light or dark conditions. Characteristics of the newly described transient phenomena include: (a) A slow biphasic decrease in fluorescence yield occurs in the dark, followed by an even slower, hour long, increase in fluorescence. (b) A similar, but faster, fluorescence yield decrease and subsequent increase also occurs during low intensity illumination periods separating high light intervals, or after transitions from high intensity to low intensity light. (c) Short (several seconds) flashes of light given during a dark period have no effect on the dark fluorescence decay, regardless of the flash frequency. Such flash regimes accurately monitor the dark decline of the M2 level by tracing the parallel decay of flash-generated P2 (Kautsky) peaks. However, flashes during a low light illumination period do influence the decay kinetics. Frequent flashes allow decay similar to that occurring in dark, but less frequent flashes inhibit the decrease in fluorescence yield.     

Light-induced tyrosine phosphorylation of BIT in the rat suprachiasmatic nucleus

Circadian changes of protein tyrosine phosphorylation in the hypothalamic suprachiasmatic nucleus have been studied using rats maintained under 12-h light/ 12-h dark cycles as well as constant dark conditions. We found that tyrosine phosphorylation of BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs), a transmembrane glycoprotein of 90-95 kDa, was higher in the light period than in the dark period and was increased after light exposure in the dark period. Similar changes in tyrosine phosphorylation were observed under constant dark conditions, but its amplitude was weaker than that in 12-h light/12-h dark cycles. As the tyrosine-phosphorylated form of BIT is able to bind to the Src homology 2 domain of a protein tyrosine phosphatase, SHP-2, we examined association of these proteins in suprachiasmatic nucleus extracts and found that SHP-2 was coprecipitated with BIT in parallel with its tyrosine phosphorylation. These results suggest that tyrosine phosphorylation of BIT might be involved in light-induced entrainment of the circadian clock.     

The role of the main photophase on dark-time measurement used for diapause determination in the Indian meal moth, Plodia interpunctella

Abstract.  The Indian meal moth Plodia interpunctella Hubner (Lepidoptera: Pyralidae) measures night length and enters diapause as a last-instar larva. To examine the role of photophase on dark-time measurement, the main LD 7 : 17 h photoperiod is disrupted by various lengths of darkness at 25 °C. When the light phase is not disrupted, the incidence of diapause is 76%. As the dark pulse disrupting a 7-h photophase becomes longer, the incidence of diapause decreases. To detect the dynamic kinetics of the time-measuring process, the main scotophase of 17 h is scanned by a 2-h light pulse. When the dark pulse in a 7-h photophase is fixed at 1 h after dawn and its duration is varied systematically from 1 to 3 h, or when the end of the dark pulse is fixed at 1 h before dusk, diapause is prevented completely by a 2-h light pulse inserted in the middle of 17-h darkness. These results are compared with those of a single night interruption of a 17-h scotophase with a 2-h light pulse but with an intact 7-h photophase. The disruption of a 7-h photophase by a dark pulse shifts the descending and ascending slopes of the response curve to some extent toward dawn and dusk, respectively, indicating that the dark pulse tends to shorten the critical length of dark time for diapause induction. When the main photophase (7 h) is interrupted by a 1-h dark pulse at 3–4 h after dawn, the 2-h scanning light pulse in the main scotophase (17 h) appears to act effectively as a dusk signal in the early scotophase. However, those in the mid- and late scotophase do not define the critical night length from dusk as sharply as for the critical night length from a 2-h light pulse to dawn. The results indicate the importance of photophase in the dark-time measurement.     

Response of the human circadian system to millisecond flashes of light

Ocular light sensitivity is the primary mechanism by which the central circadian clock, located in the suprachiasmatic nucleus (SCN), remains synchronized with the external geophysical day. This process is dependent on both the intensity and timing of the light exposure. Little is known about the impact of the duration of light exposure on the synchronization process in humans. In vitro and behavioral data, however, indicate the circadian clock in rodents can respond to sequences of millisecond light flashes. In a cross-over design, we tested the capacity of humans (n = 7) to respond to a sequence of 60 2-msec pulses of moderately bright light (473 lux) given over an hour during the night. Compared to a control dark exposure, after which there was a 3.5±7.3 min circadian phase delay, the millisecond light flashes delayed the circadian clock by 45±13 min (p<0.01). These light flashes also concomitantly increased subjective and objective alertness while suppressing delta and sigma activity (p<0.05) in the electroencephalogram (EEG). Our data indicate that phase shifting of the human circadian clock and immediate alerting effects can be observed in response to brief flashes of light. These data are consistent with the hypothesis that the circadian system can temporally integrate extraordinarily brief light exposures.     

State-dependent effects of light-dark cycle on somatosensory and visual cortex EEG in rats

Somatosensory (SSctx) and visual cortex (Vctx) EEG were evaluated in rats under a 12:12-h light-dark (LD) cycle and under constant light (LL) or constant dark (DD) in each sleep or wake state. Under LD conditions during light period, relative Vctx EEG slow-wave activity (SWA) was higher than that of the SSctx, whereas during dark period, relative Vctx EEG SWA was lower than in the SSctx. These effects were state specific, occurring only during non-rapid eye movement sleep (NREMS). Under LL conditions, the duration of REMS and NREMS during the period that would have been dark if the LD cycle had continued (subjective dark period) was greater than under LD conditions. DD conditions had little effect on the duration of NREMS and REMS. SSctx and Vctx EEG SWA were suppressed by LL during the subjective dark period; however, the degree of Vctx SWA suppression was smaller than that of the SSctx. DD conditions during the subjective light period enhanced SSctx SWA, whereas Vctx SWA was suppressed. Under LL conditions during the subjective dark period, Vctx EEG power was higher than that of the SSctx across a broad frequency range during NREMS, REMS, and wakefulness. During DD, SSctx EEG power during NREMS was higher than that of the Vctx in the delta wave band, whereas SSctx power during REMS and wakefulness was higher than that of the Vctx in frequencies higher than 8 Hz. We concluded that the SSctx and Vctx EEGs are differentially affected by light during subsequent sleep. Results provide support for the notion that regional sleep intensity is dependent on prior regional afferent input.     

Capacity of cytochrome and alternative path in coupled and uncoupled root respiration of Pisum and Plantago

A critical duration of darkness must be exceeded for the photoperiodic induction of flowering in short-day plants. This requires detection of the light/dark transition at dusk and the coupling of this information to a time-measuring system.
Lowering the P_fr/P_tot, ratio photochemically at the end of the day did not accelerate the onset of dark timing in Pharbitis nil Choisy cv. Violet. Time-measurement was initiated when, with no change in spectral quality, the irradiance fell below a threshold value. Thus, if the light/dark transition at dusk is sensed by a reduction in P_fr, this reduction can be achieved as rapidly through thermal reactions as through photochemical ones. When given at hourly intervals during a 6-h extension of a 24-h main light period in white light, pulses of red light were as effective as continuous red light in delaying the onset of timing; pulses every 2 or 3 h were less effective. The effectiveness of intermittent red light indicates that phytochrome is the photoreceptor and the requirement for frequent exposures suggests that P_fr is lost rapidly in the dark. However, the red light pulses could not be reversed by far-red light, which argues against this hypothesis. An alternative explanation is that the perception of light as being continuous occurs only when "new" P_fr is regenerated sufficiently frequently.
The nature of the coupling of the dusk signal to the time-measuring system is discussed and it is suggested that the effect of each red light pulse is to delay the phase of the photoperiodic rhythm by 1–3 h.     

Sleep‐like behavior and 24‐h rhythm disruption in the Tc1 mouse model of Down syndrome

Down syndrome is a common disorder associated with intellectual disability in humans. Among a variety of severe health problems, patients with Down syndrome exhibit disrupted sleep and abnormal 24‐h rest/activity patterns. The transchromosomic mouse model of Down syndrome, Tc1, is a trans‐species mouse model for Down syndrome, carrying most of human chromosome 21 in addition to the normal complement of mouse chromosomes and expresses many of the phenotypes characteristic of Down syndrome. To date, however, sleep and circadian rhythms have not been characterized in Tc1 mice. Using both circadian wheel‐running analysis and video‐based sleep scoring, we showed that these mice exhibited fragmented patterns of sleep‐like behaviour during the light phase of a 12:12‐h light/dark (LD) cycle with an extended period of continuous wakefulness at the beginning of the dark phase. Moreover, an acute light pulse during night‐time was less effective in inducing sleep‐like behaviour in Tc1 animals than in wild‐type controls. In wheel‐running analysis, free running in constant light (LL) or constant darkness (DD) showed no changes in the circadian period of Tc1 animals although they did express subtle behavioural differences including a reduction in total distance travelled on the wheel and differences in the acrophase of activity in LD and in DD. Our data confirm that Tc1 mice express sleep‐related phenotypes that are comparable with those seen in Down syndrome patients with moderate disruptions in rest/activity patterns and hyperactive episodes, while circadian period under constant lighting conditions is essentially unaffected.     

Running wheel size influences circadian rhythm period and its phase shift in mice

Running wheels are widely used in studies on biological rhythms. In mice wheel diameters have ranged from 11 cm to 23 cm. We provided mice with running wheels of two different sizes: 15 cm diameter and 11 cm diameter. The amount of running in the 12-h light:12-h dark condition and the endogenous period of wheel running in constant darkness was determined over 40 days. On the 1st day in constant darkness all animals were exposed to a 15-min light pulse at circadian time 13. The animals in the small wheel ran significantly less both in 12 h light: 12 h dark and constant darkness, and showed a longer endogenous period in constant darkness compared to animals in the large wheel. Moreover, after the light pulse at circadian time 13, mice in the small wheel showed a significantly smaller phase delay in running wheel activity than mice in the larger wheels. The data suggest that the magnitude of a photic phase shift depends on the amount and timing of activity the animals display in relation to this stimulus. It can be concluded that technical features of the running wheel can influence the circadian period of wheel running.     

Sleep and EEG spectra in the pigeon (Columba livia) under baseline conditions and after sleep deprivation

Summary Sleep in adult domestic pigeons was studied by continuous 24-h recording of the EEG, EMG and EOG. Vigilance states were scored on the basis of behavioral observations, visual scoring of the polygraph records, and EEG power spectra.The animals showed a clear nocturnal preference for sleep. Throughout the dark period, EEG slow-wave activity was at a uniform level, whereas REM sleep (REMS) showed an increasing trend.EEG power density values differed significantly between the vigilance states. In general the values were highest in nonREM sleep (NREMS), intermediate in waking (W) and lowest in REMS.Twenty-four hour sleep deprivation reduced W and increased REMS, effects that are well documented in mammals. Unlike in mammals, EEG slow-wave activity remained unchanged, whereas EOG activity in W and NREMS was enhanced.Abbreviations EEG electroencephalogram - EMG electromyogram - EOG electrooculogram - SD sleep deprivation - L light - D dark - LD light dark - NREMS non rapid eye movement sleep - REMS REM sleep     

Response of the Sleep-Wake Rhythm to an 8-Hour Advance of the Light-Dark Cycle in the Rat

We have studied the effects of an 8-h advance of the environmental light-dark (LD) cycle on the sleep-wake rhythm in the rat. Electroencephalograms and electromyograms were recorded simultaneously on chart paper through a two-channel telemetry system for 3 days before phase shift (baseline) and 8 days during and after phase shift. Phase advance of the LD cycle led to an increase in both non-rapid eye movement (NREM) and REM sleep. The amount of NREM sleep in the light period correlated positively with that in the preceding dark period for 4 days after phase advance. The duration of REM sleep in the light period correlated negatively with that in the preceding dark period. The results suggest that homeostatic control of the amount of NREM sleep between the preceding dark period and the following light period is disturbed by phase advance of the LD cycle.     

Response of the Sleep-Wake Rhythm to an 8-Hour Advance of the Light-Dark Cycle in the Rat

We have studied the effects of an 8-h advance of the environmental light-dark (LD) cycle on the sleep-wake rhythm in the rat. Electroencephalograms and electromyograms were recorded simultaneously on chart paper through a two-channel telemetry system for 3 days before phase shift (baseline) and 8 days during and after phase shift. Phase advance of the LD cycle led to an increase in both non-rapid eye movement (NREM) and REM sleep. The amount of NREM sleep in the light period correlated positively with that in the preceding dark period for 4 days after phase advance. The duration of REM sleep in the light period correlated negatively with that in the preceding dark period. The results suggest that homeostatic control of the amount of NREM sleep between the preceding dark period and the following light period is disturbed by phase advance of the LD cycle.     

Effect of the 33-kDa protein on the S-state transitions in photosynthetic oxygen evolution

The effect of the extrinsic 33-kDa protein on the photosynthetic oxygen evolution was studied by comparing spinach Photosystem II particles depleted of the 33-kDa protein with those reconstituted with the protein. The light-intensity dependence of the oxygen-evolution activity under continuous illumination suggests that a dark step, but not a light step, in the oxygen-evolving reaction is accelerated by the 33-kDa protein. Consistently, the pattern of oxygen yield with a series of short saturating flashes, which showed a maximum on the third flash and a damped oscillation with a period of 4, was not much affected by the removal and rebinding of the 33-kDa protein, when the dark interval between the flashes was long enough, i.e., longer than 0.5 s. The millisecond kinetics of oxygen release after the third flash was retarded by the removal of the 33-kDa protein and stimulated by its rebinding, suggesting that the transition from S₃ to S₀ is accelerated by the 33-kDa protein. The stability of the S₂ and S₃ states in darkness was higher in the absence of the 33-kDa protein than its presence.