Circadian photoreception in Drosophila: functions of cryptochrome in peripheral and central clocks.

In Drosophila melanogaster, disruption of night by even short light exposures results in degradation of the clock protein TIMELESS (TIM), leading to shifts in the fly molecular and behavioral rhythms. Several lines of evidence indicate that light entrainment of the brain clock involves the blue-light photoreceptor cryptochrome (CRY). In cryptochrome-depleted Drosophila (cry(b)), the entrainment of the brain clock by short light pulses is impaired but the clock is still entrainable by light-dark cycles, probably due to light input from the visual system. Whether cryptochrome and visual transduction pathways play a role in entrainment of noninnervated, directly photosensitive peripheral clocks is not known and the subject of this study. The authors monitored levels of the clock protein TIM in the lateral neurons (LNs) of larval brains and in the renal Malpighian tubules (MTs) of flies mutant for the cryptochrome gene (cry(b)) and in mutants that lack signaling from the visual photopigments (norpA(P41)). In cry(b) flies, light applied during the dark period failed to induce degradation of TIM both in MTs and in LNs, yet attenuated cycling of TIM was observed in both tissues in LD. This cycling was abolished in LNs, but persisted in MTs, of norpA(P41);cry(b) double mutants. Furthermore, the activity of the tim gene in the MTs of cry(b) flies, reported by luciferase, seemed stimulated by lights-on and suppressed by lights-off, suggesting that the absence of functional cryptochrome uncovered an additional light-sensitive pathway synchronizing the expression of TIM in this tissue. In constant darkness, cycling of TIM was abolished in MTs; however, it persisted in LNs of cry(b) flies. The authors conclude that cryptochrome is involved in TIM-mediated entrainment of both central LN and peripheral MT clocks. Cryptochrome is also an indispensable component of the endogenous clock mechanism in the examined peripheral tissue, but not in the brain. Thus, although neural and epithelial cells share the core clock mechanism, some clock components and light-entrainment pathways appear to have tissue-specific roles.     

Adult Emergence Rhythm of Fruit Flies Drosophila melanogaster under Seminatural Conditions

In insects, the role of circadian clocks in the temporal regulation of adult emergence rhythm under natural conditions has not previously been reported. Here we present the results of a study aimed at examining the time course and waveform of emergence rhythm in the fruit fly Drosophila melanogaster under seminatural condition (SN). We studied this rhythm in wild-type and clock mutant flies under SN in parallel with laboratory condition (LAB) to examine (1) how the rhythm differs between SN and LAB, (2) what roles the circadian clock protein PERIOD and the circadian photoreceptor CRYPTOCHROME (CRY) play in the regulation of emergence rhythm under SN, and (3) whether there is seasonality in the rhythm. Under SN, wild-type flies displayed tightly gated emergence, peaking at "dawn" and gradually tapering down toward the evening, with little or no emergence by night, while in LAB, flies emerged throughout the light phase of light-dark (LD) cycles. The period loss-of-function mutant (per ( 0 )) flies were arrhythmic in LAB but displayed weak rhythmic emergence under SN. Under SN, cry mutants displayed less robust rhythm with wider gates, greater variance in peak timing, and enhanced nighttime emergence compared to controls. Furthermore, flies showed seasonal variation in emergence rhythm, coupled either to light or to humidity/temperature depending on the severity of environmental conditions. These results suggest that adult emergence rhythm of Drosophila is more robust in nature, is coupled to environmental cycles, and shows seasonal variations.     

Cryptochrome-positive and -negative clock neurons in Drosophila entrain differentially to light and temperature

The blue-light photoreceptive protein Cryptochrome (CRY) plays an important role in the light synchronization of the Drosophila circadian clock. Previously, we found that among the approximately 150 clock neurons, many but not all neurons express CRY. We speculated that the CRY-positive pacemaker neurons may be especially important for light entrainment, whereas the CRY-negative neurons may be important for other environmental cues, for example, temperature. To investigate this hypothesis, we tested the entrainability of the clock neurons to out-of-phase light and temperature cycles. When light-dark or light-dim light cycles were shifted by 12 h with respect to temperature cycles, behavioral rhythms of wild-type flies were re-entrained by the light cycles. In this condition, we found that TIMELESS (TIM) level was strongly influenced by the temperature cycles in many CRY-negative clock neurons, suggesting that the CRY-negative neurons have higher sensitivity to temperature. Under the same conditions, cry-null mutants entrained to the temperature cycles or very slowly re-entrained to light-dark cycles. Our results suggest that there are 2 types of clock neurons having differential sensitivities to light and temperature, and CRY is a key component for the preferential entrainment to light.     

Photic resetting and entrainment in CLOCK-deficient mice

Mice lacking the CLOCK protein have a relatively subtle circadian phenotype, including a slightly shorter period in constant darkness, differences in phase resetting after 4-hour light pulses in the early and late night, and a variably advanced phase angle of entrainment in a light-dark (LD) cycle. The present series of experiments was conducted to more fully characterize the circadian phenotype of Clock(-/-) mice under various lighting conditions. A phase-response curve (PRC) to 4-hour light pulses in free-running mice was conducted; the results confirm that Clock(-/-) mice exhibit very large phase advances after 4-hour light pulses in the late subjective night but have relatively normal responses to light at other phases. The abnormal shape of the PRC to light may explain the tendency of CLOCK-deficient mice to begin activity before lights-out when housed in a 12-hour light:12-hour dark lighting schedule. To assess this relationship further, Clock(-/-) and wild-type control mice were entrained to skeleton lighting cycles (1L:23D and 1L:10D:1L:12D). Comparing entrainment under the 2 types of skeleton photoperiods revealed that exposure to 1-hour light in the morning leads to a phase advance of activity onset (expressed the following afternoon) in Clock(-/-) mice but not in the controls. Constant light typically causes an intensity-dependent increase in circadian period in mice, but this did not occur in CLOCK-deficient mice. The failure of Clock(-/-) mice to respond to the period-lengthening effect of constant light likely results from the increased functional impact of light falling in the phase advance zone of the PRC. Collectively, these experiments reveal that alterations in the response of CLOCK-deficient mice to light in several paradigms are likely due to an imbalance in the shape of the PRC to light.     

Rhythm defects caused by newly engineered null mutations in Drosophila's cryptochrome gene

Much of the knowledge about cryptochrome function in Drosophila stems from analyzing the cryb mutant. Several features of this variant's light responsiveness imply either that CRYb retains circadian-photoreceptive capacities or that additional CRY-independent light-input routes subserve these processes. Potentially to resolve these issues, we generated cry knock-out mutants (cry0's) by gene replacement. They behaved in an anomalously rhythmic manner in constant light (LL). However, cry0 flies frequently exhibited two separate circadian components in LL, not observed in most previous cryb analyses. Temperature-dependent circadian phenotypes exhibited by cry(0) flies suggest that CRY is involved in core pacemaking. Further locomotor experiments combined cry0 with an externally blinding mutation (norpAP24), which caused the most severe decrements of circadian photoreception observed so far. cryb cultures were shown previously to exhibit either aperiodic or rhythmic eclosion in separate studies. We found cry0 to eclose in a solidly periodic manner in light:dark cycles or constant darkness. Furthermore, both cry0 and cryb eclosed rhythmically in LL. These findings indicate that the novel cry0 type causes more profound defects than does the cryb mutation, implying that CRYb retains residual activity. Because some norpAP24 cry0 individuals can resynchronize to novel photic regimes, an as-yet undetermined light-input route exists in Drosophila.     

Drosophila CRY is a deep brain circadian photoreceptor

cry (cryptochrome) is an important clock gene, and recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cry(b), inhibits circadian photoresponses. Restricting CRY expression to specific fly tissues shows that CRY expression is needed in a cell-autonomous fashion for oscillators present in different locations. CRY overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted CRY expression also rescues all circadian defects of cry(b) behavior. As wild-type pacemaker neurons express CRY, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pace-maker and a behavioral output system.     

Effects of the duper mutation on circadian responses to light

The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)) by 2 hours when expressed on a tau mutant (τ(ss)) background and by 1 hour on a wild-type background. We have examined the effects of this mutation on phase response curves and entrainment. In contrast to wild types, duper hamsters entrained to 14L:10D with a positive phase angle. Super duper hamsters (expressing duper on a τ(ss) background) showed weak entrainment, while τ(ss) animals either completely failed to entrain or showed sporadic entrainment with episodes of relative coordination. As previously reported, wild-type and τ(ss) hamsters show low amplitude resetting in response to 15-minute light pulses after short-term (10 days) exposure to DD. In contrast, super duper hamsters show high amplitude resetting. This effect is attributable to the duper allele, as hamsters carrying duper on a wild-type background also show large phase shifts. Duper mutants that were born and raised in DD also showed high amplitude resetting in response to 15-minute light pulses, indicating that the effect of the mutation on PRC amplitude is not an aftereffect of entrainment to 14L:10D. Hamsters that are heterozygous for duper do not show amplified resetting curves, indicating that for this property, as for determination of free-running period, the mutant allele is recessive. In a modified Aschoff type II protocol, super duper and duper hamsters show large phase shifts as soon as the second day of DD. Despite the amplification of the PRC in super duper hamsters, the induction of Period1 gene expression in the SCN by light is no greater in these mutants than in wild-type animals. Period2 expression in the SCN did not differ between super duper and wild-type hamsters exposed to light at CT15, but albumin site D-binding protein (Dbp) mRNA showed higher basal levels and greater light induction in the SCN of super duper compared to wild-type animals. These results indicate that the duper mutation alters the amplitude of the circadian oscillator and further distinguish it from the tau mutation.     

Circadian modulation of light-induced locomotion responses in Drosophila melanogaster

The relationship between light and the circadian system has long been a matter of discussion. Many studies have focused on entrainment of light with the internal biological clock. Light also functions as an environmental stimulus that affects the physiology and behaviour of animals directly. In this study, we used light as an unexpected stimulus for flies at different circadian times. We found that wildtype flies showed circadian changes in light-induced locomotion responses. Elevation of locomotor activity by light occurred during the subjective night, and performance in response to light pulses declined to trough during the subjective day. Moreover, arrhythmic mutants lost the rhythm of locomotion responses to light, with promotion of activity by light in timeless(01)mutants and inhibition of activity by light in Clock(ar)mutants. However, neither ablation of central oscillators nor disturbance of the functional clock inside compound eyes was sufficient to disrupt the rhythm of light responses. We show that, compound eyes, which have been identified as the control point for normal masking (promotion of activity by light), are sufficient but not necessary for paradoxical masking (suppression of activity by light) under high light intensity. This, taken together with the clear difference of light responses in wildtype flies, suggests that two different masking mechanisms may underlie the circadian modulation of light-induced locomotion responses.     

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.     

Temperature entrainment of the circadian cuticle deposition rhythm in Drosophila melanogaster

The cuticle deposition rhythm, which is observed in the apodeme of the furca in the thorax, is controlled by a peripheral circadian clock in the epidermal cells and entrained to light-dark (LD) cycles via CRYPTOCHROME (CRY) in Drosophila melanogaster. In the present study, we examined the effects of temperature (TC) cycles and the combination of LD and TC cycles on entrainment of the cuticle deposition rhythm. The rhythm was entrained to TC cycles, whose period was 28 h. In T = 21 and 24 h, the rhythm was entrained to TC cycles in some individuals. CRY is not necessary for temperature entrainment of the cuticle deposition rhythm because the rhythm in cry(b) (lacking functional CRY) was entrained to TC cycles. Temperature entrainment of the rhythm was achieved even when the thoraxes or furcae were cultured in vitro, suggesting that the mechanism for temperature entrainment is independent of the central clock in the brain and the site of the thermoreception resides in the epidermal cells. When LD and TC cycles with different periods were applied, the rhythm was entrained to LD cycles with a slight influence of TC cycles. Thus, the LD cycle is a stronger zeitgeber than the TC cycle. The variance of the number of the cuticle layers decreased in the flies kept under LD and TC cycles with the same period in which the thermophase coincided with the photophase. Therefore, we conclude that LD and TC cycles synergistically entrain the rhythm. Synergistic effects of LD and TC cycles on entrainment were also observed even when the thoraxes were cultured in vitro, suggesting that the light and temperature information is integrated within the peripheral circadian system.     

Drosophila cryb mutation reveals two circadian clocks that drive locomotor rhythm and have different responsiveness to light

Cryptochrome (CRY) is a blue-light-absorbing protein involved in the photic entrainment of the circadian clock in Drosophila melanogaster. We have investigated the locomotor activity rhythms of flies carrying cryb mutant and revealed that they have two separate circadian oscillators with different responsiveness to light. When kept in constant light conditions, wild-type flies became arrhythmic, while cryb mutant flies exhibited free-running rhythms with two rhythmic components, one with a shorter and the other with a longer free-running period. The rhythm dissociation was dependent on the light intensities: the higher the light intensities, the greater the proportion of animals exhibiting the two oscillations. External photoreceptors including the compound eyes and the ocelli are the likely photoreceptors for the rhythm dissociation, since rhythm dissociation was prevented in so1;cryb and norpAP41;cryb double mutant flies. Immunohistochemical analysis demonstrated that the PERIOD expression rhythms in ventrally located lateral neurons (LNvs) occurred synchronously with the shorter period component, while those in the dorsally located per-expressing neurons showed PER expression most likely related to the longer period component, in addition to that synchronized to the LNvs. These results suggest that the Drosophila locomotor rhythms are driven by two separate per-dependent clocks, responding differentially to constant light.     

PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry^b. The period mutant per^L manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per^L flies, because a per^L; cry^b strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.     

PER-TIM Interactions with the Photoreceptor Cryptochrome Mediate Circadian Temperature Responses in Drosophila

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry^b. The period mutant per^L manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per^L flies, because a per^L; cry^b strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.     

Light pulses suppress responsiveness within the mouse photic entrainment pathway

We have characterized a decrease in photic responsiveness of the mammalian circadian entrainment pathway caused by light stimulation. Phase delays of the running-wheel activity rhythm were used to quantify the photic responsiveness of the circadian system in mice (C57BL/6J). In an initial experiment, the authors measured the responsiveness to single "saturating" light pulses ("white" fluorescent light; approximately 1876 [microW; 15 min). In two additional experiments, the authors measured responses to this stimulus at several time points following a saturating pulse at CT 14 or CT 16. Data from these experiments were analyzed in two manners. Experiment 2 was analyzed assuming that the phase of the circadian pacemaker was unchanged by an initial pulse, and Experiment 3 was analyzed assuming that the initial pulse induced an instantaneous phase delay. Results reveal a significant reduction in responsivity to light that persists for at least 2 h and possibly up to 4 h after the initial stimulus. Immediately after the stimulus, the responsiveness of the photic entrainment pathway was reduced to levels < or = 12% of normal. After 2 h, the responsiveness was < or = 42% of normal, and by 4 h, responsiveness had recovered to levels that were < or = 60% of normal (levels not statistically different from controls). By the following circadian cycle, responsiveness was more completely recovered, although the magnitude of some phase delays remained < or = 85% of normal. These major reductions in the magnitude of phase delays (and phase response curve amplitude) caused by saturating light pulses confound interpretations of two-pulse experiments designed to measure the rate of circadian phase delays. In addition, the time course for this reduced responsiveness may reflect the time course of cellular and molecular events that underlie light-induced resetting of the mammalian circadian pacemaker.     

Anterior paraventricular thalamus modulates light-induced phase shifts in circadian rhythmicity in rats

The reciprocal connections between the paraventricular thalamic nucleus (PVT) and the suprachiasmatic nuclei suggest that PVT may participate in the regulation of circadian rhythms. We studied in rats the effect of lesions of the anterior and midposterior regions of the PVT on phase shifts of drinking circadian rhythm induced by light pulses at circadian times 6, 12, and 23, as well as the phase shifts produced by electrical or glutamatergic stimulation of the anterior PVT at the same circadian times. Lesion of the anterior PVT abolishes the advances induced by light during late subjective night, whereas midposterior PVT lesions did not affect the phase shifts. Electrical stimulation or glutamate injections in the anterior PVT mimic the phase-shifting effects of light pulses. These results indicate the participation of the anterior PVT as a modulator of entrainment of circadian rhythms to light.     

A fly's eye view of circadian entrainment

The Drosophila circadian clock is an ideal model system for teasing out the molecular mechanisms of circadian behavior and the means by which animals synchronize to day-night cycles. The clock that drives behavioral rhythms, located in the lateral neurons in the central brain, consists of a feedback loop of the circadian genes period (per) and timeless (tim). The molecular cycle, roughly 24 h long, is constantly reset by the environment. This review focuses on the main input pathways of the dominant circadian zeitgeber, light. Light acts directly on the clock primarily through cryptochrome (cry), a deep brain blue-light photoreceptor. CRY activation causes rapid TIM degradation, which is a predicted means of resetting the clock both on a daily basis at dawn and on an acute basis following an entraining light pulse during the night hours. In the absence of cry, the clock can still be driven by photic input through the visual system, though the mechanisms underlying this entrainment are unclear. Temperature can also entrain the clock, although the mechanisms by which this occurs are also unclear.     

The estrous cycle in golden hamsters: a circadian pacemaker times preovulatory gonadotropin release

In a population of cycling female hamsters entrained to an LD 6:18 light cycle (lights 1000-1600 hours), preovulatory release of luteinizing hormone and follicle-stimulating hormone occurred in some animals at 1300-1400 hours and in others at 1900 hours. In every case peak release was phase-locked (2-3-hour positive phase angle) to the circadian rhythm of locomotor activity. The pattern of entrainment of gonadotropin release on LD 6:18 is fully explicable in terms of the hamster's phase response curve to light. We conclude that periodic gonadotropin release in cycling females is timed by a circadian oscillator (biological clock) that is probably the same oscillator driving the circadian rhythm of locomotor activity.