Journal of Biological Rhythms recent issues

Spatial and Circadian Regulation of cry in Drosophila

Hao Zheng, , Ng, F., Yixiao Liu, , Hardin, P. E. — 2008-07-28

In Drosophila, cryptochrome (cry) encodes a blue-light photoreceptor that mediates light input to circadian oscillators and sustains oscillator function in peripheral tissues. The levels of cry mRNA cycle with a peak at ~ZT5, which is similar to the phase of Clock (Clk) mRNA cycling in Drosophila. To understand how cry spatial and circadian expression is regulated, a series of cry-Gal4 trans-genes containing different portions of cry upstream and intron 1 sequences were tested for spatial and circadian expression. In fly heads, cry upstream sequences drive constitutive expression in brain oscillator neurons, a novel group of nonoscillator cells in the optic lobe, and peripheral oscillator cells in eyes and antennae. In contrast, cry intron 1 drives rhythmic expression in eyes and antennae, but not brain oscillator neurons. These results demonstrate that intron 1 is sufficient for high-amplitude cry mRNA cycling, show that cry upstream sequences are sufficient for expression in brain oscillator neurons, and suggest that cry spatial and circadian expression are regulated by different elements.

The Blue-Light Photoreceptor CRYPTOCHROME Is Expressed in a Subset of Circadian Oscillator Neurons in the Drosophila CNS

Benito, J., Houl, J. H., Roman, G. W., Hardin, P. E. — 2008-07-28

In the fruit fly Drosophila melanogaster, CRYPTOCHROME (CRY) functions as a photoreceptor to entrain circadian oscillators to light-dark cycles and as a transcription factor to maintain circadian oscillator function in certain peripheral tissues. Given the importance of CRY to circadian clock function, we expected this protein to be expressed in all oscillator cells, yet CRY cellular distribution and subcellular localization has not been firmly established. Here we investigate CRY spatial expression in the brain using a newly developed CRY antibody and a novel set of cry deletion mutants. We find that CRY is expressed in s-LN_vs, l-LN_vs, and a subset of LN_ds and DN₁s, but not DN₂s and DN₃s. CRY is present in both the nucleus and the cytoplasm of these neurons, and its subcellular localization does not change over the circadian cycle. Although CRY is absent in DN₂s and DN₃s, cry promoter activity and/or cry mRNA accumulation can be detected in these neurons, suggesting that CRY levels are regulated posttranscriptionally. Oscillators in DN₂s and DN₃s entrain to environmental light-dark cycles, which implies that they are entrained indirectly by retinal photoreceptors, extraretinal photoreceptors, or other CRY-expressing cells.

RNA Interference of the Clock Gene period Disrupts Circadian Rhythms in the Cricket Gryllus bimaculatus

Moriyama, Y., Sakamoto, T., Karpova, S. G., Matsumoto, A., Noji, S., Tomioka, K. — 2008-07-28

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.

Circadian Regulation of Bioluminescence in the Prey-Luring Glowworm, Arachnocampa flava

Merritt, D. J., Aotani, S. — 2008-07-28

The glowworms of New Zealand and Australia are bioluminescent fly larvae that generate light to attract prey into their webs. Some species inhabit the constant darkness of caves as well as the dim, natural photophase of rain-forests. Given the diversity of light regimens experienced by glowworms in their natural environment, true circadian rhythmicity of light output could be present. Consequently the light emission characteristics of the Australian subtropical species Arachnocampa flava, both in their natural rainforest habitat and in artificial conditions in the laboratory, were established. Larvae were taken from rainforest and kept alive in individual containers. When placed in constant darkness (DD) in the laboratory they maintained free-running, cyclical light output for at least 28 days, indicating that light output is regulated by an endogenous rhythm. The characteristics of the light emission changed in DD: individuals showed an increase in the time spent glowing per day and a reduction in the maximum light output. Most individuals show a free-running period greater than 24 h. Manipulation of the photophase and exposure to skeleton photoperiods showed that light acts as both a masking and an entraining agent and suggests that the underlying circadian rhythm is sinusoidal in the absence of light-based masking. Manipulation of thermoperiod in DD showed that temperature cycles are an alternative entraining agent. Exposure to a period of daily feeding in DD failed to entrain the rhythm in the laboratory. The endogenous regulation of luminescence poses questions about periodicity and synchronization of bioluminescence in cave glowworms.

Genetic Differences in Human Circadian Clock Genes among Worldwide Populations

2008-07-28

The daily biological clock regulates the timing of sleep and physiological processes that are of fundamental importance to human health, performance, and well-being. Environmental parameters of relevance to biological clocks include (1) daily fluctuations in light intensity and temperature, and (2) seasonal changes in photoperiod (day length) and temperature; these parameters vary dramatically as a function of latitude and locale. In wide-ranging species other than humans, natural selection has genetically optimized adaptiveness along latitudinal clines. Is there evidence for selection of clock gene alleles along latitudinal/photoperiod clines in humans? A number of polymorphisms in the human clock genes Per2, Per3, Clock, and AANAT have been reported as alleles that could be subject to selection. In addition, this investigation discovered several novel polymorphisms in the human Arntl and Arntl2 genes that may have functional impact upon the expression of these clock transcriptional factors. The frequency distribution of these clock gene polymorphisms is reported for diverse populations of African Americans, European Americans, Ghanaians, Han Chinese, and Papua New Guineans (including 5 subpopulations within Papua New Guinea). There are significant differences in the frequency distribution of clock gene alleles among these populations. Population genetic analyses indicate that these differences are likely to arise from genetic drift rather than from natural selection.

The Impact of Sleep Timing and Bright Light Exposure on Attentional Impairment during Night Work

Santhi, N., Aeschbach, D., Horowitz, T. S., Czeisler, C. A. — 2008-07-28

The prevalence of hazardous incidents induced by attentional impairment during night work and ensuing commute times is attributable to circadian misalignment and increased sleep pressure. In a 10-day shift work simulation protocol (4 day shifts and 3 night shifts), the efficacies of 2 countermeasures against nighttime (2300 to 0700 h) attentional impairment were compared: (1) Morning Sleep (0800 to 1600 h; n = 18) in conjunction with a phase-delaying light exposure (2300 to 0300 h), and (2) Evening Sleep (1400 to 2200 h; n = 17) in conjunction with a phase-advancing light exposure (0300 to 0700 h). Analysis of the dim light salivary melatonin onset indicated a modest but significant circadian realignment in both sleep groups (evening sleep: 2.27 ± 0.6 h phase advance, p < 0.01; morning sleep: 4.98 ± 0.43 h phase delay, p < 0.01). Daytime sleep efficiency and total sleep time did not differ between them or from their respective baseline sleep (2200 to 0600 h; p > 0.05). However, on the final night shift, the evening sleep subjects had 37% fewer episodes of attentional impairment (long response times: 22 ± 4 vs. 35 ± 4; p = 0.02) and quicker responses (p < 0.01) on the Psychomotor Vigilance Task than their morning sleep counterparts. Their response speed recovered to near daytime levels (p = 0.47), whereas those of the morning sleep subjects continued to be slower than their daytime levels (p = 0.008). It is concluded that partial circadian realignment to night work in combination with reduced homeostatic pressure contributed to the greater efficacy of a schedule of Evening Sleep with a phase-advancing light exposure as a countermeasure against attentional impairment, over a schedule of Morning Sleep with a phase-delaying light exposure. These results have important implications for managing patients with shift work disorder.

An Endogenous Circadian Rhythm in Sleep Inertia Results in Greatest Cognitive Impairment upon Awakening during the Biological Night

Scheer, F. A. J. L., Shea, T. J., Hilton, M. F., Shea, S. A. — 2008-07-28

Sleep inertia is the impaired cognitive performance immediately upon awakening, which decays over tens of minutes. This phenomenon has relevance to people who need to make important decisions soon after awakening, such as on-call emergency workers. Such awakenings can occur at varied times of day or night, so the objective of the study was to determine whether or not the magnitude of sleep inertia varies according to the phase of the endogenous circadian cycle. Twelve adults (mean, 24 years; 7 men) with no medical disorders other than mild asthma were studied. Following 2 baseline days and nights, subjects underwent a forced desynchrony protocol composed of seven 28-h sleep/wake cycles, while maintaining a sleep/wakefulness ratio of 1:2 throughout. Subjects were awakened by a standardized auditory stimulus 3 times each sleep period for sleep inertia assessments. The magnitude of sleep inertia was quantified as the change in cognitive performance (number of correct additions in a 2-min serial addition test) across the first 20 min of wakefulness. Circadian phase was estimated from core body temperature (fitted temperature minimum assigned 0°). Data were segregated according to: (1) circadian phase (60° bins); (2) sleep stage; and (3) 3rd of the night after which awakenings occurred (i.e., tertiary 1, 2, or 3). To control for any effect of sleep stage, the circadian rhythm of sleep inertia was initially assessed following awakenings from Stage 2 (62% of awakening occurred from this stage; n = 110). This revealed a significant circadian rhythm in the sleep inertia of cognitive performance (p = 0.007), which was 3.6 times larger during the biological night (circadian bin 300°, ~2300—0300 h in these subjects) than during the biological day (bin 180°, ~1500—1900 h). The circadian rhythm in sleep inertia was still present when awakenings from all sleep stages were included (p = 0.004), and this rhythm could not be explained by changes in underlying sleep drive prior to awakening (changes in sleep efficiency across circadian phase or across the tertiaries), or by the proportion of the varied sleep stages prior to awakenings. This robust endogenous circadian rhythm in sleep inertia may have important implications for people who need to be alert soon after awakening.

Emergence of Circadian and Photoperiodic System Level Properties from Interactions among Pacemaker Cells

Beersma, D. G. M., van Bunnik, B. A. D., Hut, R. A., Daan, S. — 2008-07-28

Daily patterns of behavior and physiology in animals in temperate zones often differ substantially between summer and winter. In mammals, this may be a direct consequence of seasonal changes of activity of the suprachiasmatic nucleus (SCN). The purpose of this study was to understand such variation on the basis of the interaction between pacemaker neurons. Computer simulation demonstrates that mutual electrical activation between pacemaker cells in the SCN, in combination with cellular electrical activation by light, is sufficient to explain a variety of circadian phenomena including seasonal changes. These phenomena are: self-excitation, that is, spontaneous development of circadian rhythmicity in the absence of a light-dark cycle; persistent rhythmicity in constant darkness, and loss of circadian rhythmicity in pacemaker output in constant light; entrainment to light-dark cycles; aftereffects of zeitgeber cycles with different periods; adjustment of the circadian patterns to day length; generation of realistic phase response curves to light pulses; and relative independence from day-to-day variation in light intensity. In the model, subsets of cells turn out to be active at specific times of day. This is of functional importance for the exploitation of the SCN to tune specific behavior to specific times of day. Thus, a network of on-off oscillators provides a simple and plausible construct that behaves as a clock with readout for time of day and simultaneously as a clock for all seasons.

Human Tau in an Ultradian Light-Dark Cycle

Burgess, H. J., Eastman, C. I. — 2008-07-28

Genome Streamlining Results in Loss of Robustness of the Circadian Clock in the Marine Cyanobacterium Prochlorococcus marinus PCC 9511

2008-05-16

The core oscillator of the circadian clock in cyanobacteria consists of 3 proteins, KaiA, KaiB, and KaiC. All 3 have previously been shown to be essential for clock function. Accordingly, most cyanobacteria possess at least 1 copy of each kai gene. One exception is the marine genus Prochlorococcus, which we suggest here has suffered a stepwise deletion of the kaiA gene, together with significant genome streamlining. Nevertheless, natural Prochlorococcus populations and laboratory cultures are strongly synchronized by the alternation of day and night, displaying 24-h rhythms in DNA replication, with a temporal succession of G1, S, and G2-like cell cycle phases. Using quantitative real-time PCR, we show here that in Prochlorococcus marinus PCC 9511, the mRNA levels of the clock genes kaiB and kaiC, as well as a few other selected genes including psbA, also displayed marked diel variations when cultures were kept under a light-dark rhythm. However, both cell cycle and psbA gene expression rhythms damped very rapidly under continuous light. In the closely related Synechococcus sp. WH8102, which possesses all 3 kai genes, cell cycle rhythms persisted over several days, in agreement with established cyanobacterial models. These data indicate a correlation between the loss of kaiA and a loss of robustness in the endogenous oscillator of Prochlorococcus and raise questions about how a basic KaiBC system may function and through which mechanism the daily "lights-on" and "lights-off" signal could be mediated.

Behavioral Rhythmicity of Mice Lacking AhR and Attenuation of Light-Induced Phase Shift by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Mukai, M., Lin, T.-M., Peterson, R. E., Cooke, P. S., Tischkau, S. A. — 2008-05-16

Transcription factors belonging to the Per/Arnt/Sim (PAS) domain family are highly conserved and many are involved in circadian rhythm regulation. One member of this family, aryl hydrocarbon receptor (AhR), is an orphan receptor whose physiological role is unknown. Recent findings have led to the hypothesis that AhR has a role in circadian rhythm, which is the focus of the present investigation. First, time-of-day-dependent mRNA expression of AhR and its signaling target, cytochrome p4501A1 (Cyp1a1), was determined in C57BL/6J mice by quantitative RT-PCR. Circadian expression of AhR and Cyp1a1 was observed both in the suprachiasmatic nucleus (SCN) and liver. Next, the circadian phenotype of mice lacking AhR (AhRKO) was investigated using behavioral monitoring. Intact AhRKO mice had robust circadian rhythmicity with a similar tau under constant conditions compared to wild-type mice, but a significant difference in tau was observed between genotypes in ovariectomized female mice. Time to reentrainment following 6-h advances or delays of the light/dark cycle was not significantly different between genotypes. However, mice exposed to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 µg/kg of body weight) displayed decreased phase shifts in response to light and had altered expression of Per1 and Bmal1. These results suggest that chronic activation of AhR may affect the ability of the circadian timekeeping system to adjust to alterations in environmental lighting by affecting canonical clock genes. Further studies are necessary to decipher the mechanism of how AhR agonists could disrupt light-induced phase shifts. If AhR does have a role in circadian rhythm, it may share redundant roles with other PAS domain proteins and/or the role of AhR may not be exhibited in the behavioral activity rhythm, but could be important elsewhere in the peripheral circadian system.

Behavioral Responses of Vipr2-/- Mice to Light

Hughes, A.T.L., Piggins, H.D. — 2008-05-16

Vasoactive intestinal polypeptide and its receptor, VPAC₂ , play important roles in the functioning of the dominant circadian pacemaker, located in the hypothalamic suprachiasmatic nuclei (SCN). Mice lacking VPAC₂ receptors (Vipr2^–/–) show altered circadian rhythms and impaired synchronization to environmental lighting cues. However, light can increase phosphoprotein and immediate early gene expression in the Vipr2^–/– SCN demonstrating that the circadian clock is readily responsive to light in these mice. It is not clear whether these neurochemical responses to light can be transduced to behavioral changes as seen in wild-type (WT) animals. In this study we investigated the diurnal and circadian wheel-running profile of WT (C57BL/6J) and Vipr2^–/– mice under a 12-h light:12-h complete darkness (LD) lighting schedule and in constant darkness (DD) and used 1-h light pulses to shift the activity of mice in DD. Unlike WT mice, Vipr2^–/– mice show grossly altered locomotor patterns making the analysis of behavioral responses to light problematic. However, analyses of both the onset and the offset of locomotor activity reveal that in a subset of these mice, light can reset the offset of behavioral rhythms during the subjective night. This suggests that the SCN clock of Vipr2^–/– mice and the rhythms it generates are responsive to photic stimulation and that these responses can be integrated to whole animal behavioral changes.

Decline of the Presynaptic Network, Including GABAergic Terminals, in the Aging Suprachiasmatic Nucleus of the Mouse

2008-05-16

Biological rhythms, and especially the sleep/wake cycle, are frequently disrupted during senescence. This draws attention to the study of aging-related changes in the hypothalamic suprachiasmatic nucleus (SCN), the master circadian pacemaker. The authors here compared the SCN of young and old mice, analyzing presynaptic terminals, including the gamma-aminobutyric acid (GABA)ergic network, and molecules related to the regulation of GABA, the main neurotransmitter of SCN neurons. Transcripts of the 3 subunit of the GABA_A receptor and the GABA-synthesizing enzyme glutamic acid decarboxylase isoform 67 (GAD67) were analyzed with real-time RT-PCR and GAD67 protein with Western blotting. These parameters did not show significant changes between the 2 age groups. Presynaptic terminals were identified in confocal microscopy with synaptophysin immunofluorescence, and the GABAergic subset of those terminals was revealed by the colocalization of GAD67 and synaptophysin. Quantitative analysis of labeled synaptic endings performed in 2 SCN subregions, where retinal afferents are known to be, respectively, very dense or very sparse, revealed marked aging-related changes. In both subregions, the evaluated parameters (the number of and the area covered by presynaptic terminals and by their GABAergic subset) were significantly decreased in old versus young mice. No significant differences were found between SCN tissue samples from animals sacrificed at different times of day, in either age group. Altogether, the data point out marked reduction in the synaptic network of the aging biological clock, which also affects GABAergic terminals. Such alterations could underlie aging-related SCN dysfunction, including low-amplitude output during senescence.

The Development of Day-Night Differences in Sleep and Wakefulness in Norway Rats and the Effect of Bilateral Enucleation

Gall, A. J., Todd, W. D., Ray, B., Coleman, C. M., Blumberg, M. S. — 2008-05-16

The suprachiasmatic nucleus exhibits circadian rhythmicity in fetal and infant rats, but little is known about the consequences of this rhythmicity for infant behavior. Here, in experiment 1, the authors measured sleep and wakefulness in rats during the day and night in postnatal day (P)2, P8, P15, and P21 subjects. As early as P2, day-night differences in sleep-wake activity were detected. Nocturnal wakefulness began to emerge around P15 and was reliably expressed by P21. The authors hypothesized that the process of photic entrainment over the 1st postnatal week, which depends on the development of connectivity between the retinohypothalamic tract (RHT) and the SCN, influences the later emergence of nocturnal wakefulness. To test this hypothesis, in experiment 2 infant rats were enucleated bilaterally at P3 and P11, that is, before and after photic entrainment. Whereas pups enucleated at P11 and tested at P21 exhibited increased wakefulness at night, identical to sham controls, pups enucleated at P3 and tested at P21 exhibited the opposite pattern of increased wakefulness during the day. Pups tested at P28 and P35 exhibited this same pattern of increased daytime wakefulness. All together, these results suggest that prenatal and postnatal experience modulates the development of species-typical circadian sleep-wake patterns. Moreover, the authors suggest that visual system stimulation, via the RHT's connections with the SCN, exerts an organizational influence on the developing circadian system and, consequently, contributes to the emergence of nocturnality in this species.

A Melatonin-Independent Seasonal Timer Induces Neuroendocrine Refractoriness to Short Day Lengths

Butler, M. P., Turner, K. W., Zucker, I. — 2008-05-16

The duration of nocturnal pineal melatonin secretion transduces effects of day length (DL) on the neuroendocrine axis of photoperiodic rodents. Long DLs support reproduction, and short DLs induce testicular regression, followed several months later by spontaneous recrudescence; gonadal regrowth is thought to reflect development of tissue refractoriness to melatonin. In most photoperiodic species, pinealectomy does not diminish reproductive competence in long DLs. Turkish hamsters (Mesocricetus brandti) deviate from this norm: elimination of melatonin secretion in long-day males by pinealectomy or constant light treatment induces testicular regression and subsequently recrudescence; the time course of these gonadal transitions is similar to that observed in males transferred from long to short DLs. In the present study, long-day Turkish hamsters that underwent testicular regression and recrudescence in constant light subsequently were completely unresponsive to the antigonadal effects of short DLs. Other hamsters that manifested testicular regression and recrudescence in short DLs were unresponsive to the antigonadal effects of pinealectomy or constant light. Long-term suppression of melatonin secretion induces a physiological state in Turkish hamsters similar or identical to the neuroendocrine refractoriness produced by short-day melatonin signals (i.e., neural refractoriness to melatonin develops in the absence of circulating melatonin secretion). A melatonin-independent interval timer, which would remain operative in the absence of melatonin during hibernation, may determine the onset of testicular recrudescence in the spring. In this respect, Turkish hamsters differ from most other photoperiodic rodents.

A Physiological Model of a Circannual Oscillator

MacGregor, D. J., Lincoln, G. A. — 2008-05-16

Recent evidence based on studies in hypothalamo-pituitary disconnected Soay sheep suggests that the generation of circannual rhythms may be localizable to specific tissues or physiological systems. Now, the authors present a physiological model of a circannual rhythm generator centered in the pituitary gland based on the interaction between melatonin-responsive cells in the pars tuberalis that act to decode photoperiod, and lactotroph cells of the adjacent pars distalis that secrete prolactin. The model produces a self-sustained, circannual rhythm in endocrine output that the authors explore by mathematical modeling. The circannual oscillation requires a delayed negative feedback mechanism. The authors highlight specific features of the pituitary dynamics as a guide to future research on circannual rhythms.

The Endogenous Circadian Pacemaker Imparts a Scale-Invariant Pattern of Heart Rate Fluctuations across Time Scales Spanning Minutes to 24 Hours

Kun Hu, , Scheer, F. A. J. L., Buijs, R. M., Shea, S. A. — 2008-05-16

Heartbeat fluctuations in mammals display a robust temporal structure characterized by scale-invariant/fractal patterns. These scale-invariant patterns likely confer physiological advantage because they change with cardiovascular disease and these changes are associated with reduced survival. Models of physical systems imply that to produce scale-invariant patterns, factors influencing the system at different time scales must be coupled via a network of feedback interactions. A similar cardiac control network is hypothesized to be responsible for the scale-invariant pattern in heartbeat dynamics, although the essential network components have not been determined. Here is shown that scale-invariant cardiac control occurs across time scales from minutes to ~24 h, and that lesioning the mammalian circadian pacemaker (suprachiasmatic nucleus; SCN) completely abolishes the scale-invariant pattern at time scales >~4 h. At time scales <~4 h, the scale invariance persisted following SCN lesion but with a different pattern. These results indicate previously unrecognized multiscale influences of the SCN on heart rate fluctuations that cannot be explained by a simple pacemaker of 24-h rhythmicity. The conclusion is that the SCN serves as a major node in the cardiac control network and imparts scale-invariant cardiac control across a wide range of time scales with strongest effects between ~4 and 24 h. These results demonstrate that experimental manipulations (e.g., SCN lesion) can be used to begin to model and understand the origin of scale-invariant behavior in a neurophysiological system.

A Gentler Method to Raise Melatonin Levels in Birds

Goymann, W., Trappschuh, M., Fusani, L. — 2008-05-16

The Clockwork Orange Drosophila Protein Functions as Both an Activator and a Repressor of Clock Gene Expression

Richier, B., Michard-Vanhee, C., Lamouroux, A., Papin, C., Rouyer, F. — 2008-03-28

The Drosophila clock relies on transcriptional feedback loops that generate daily oscillations of the clock gene expression at mRNA and protein levels. In the evening, the CLOCK (CLK) and CYCLE (CYC) basic helix-loop-helix (bHLH) PAS-domain transcription factors activate the expression of the period (per) and timeless (tim) genes. Posttranslational modifications delay the accumulation of PER and TIM, which inhibit CLK/CYC activity in the late night. We show here that a null mutant of the clockwork orange (cwo) gene encoding a bHLH orange-domain putative transcription factor displays long-period activity rhythms. cwo loss of function increases cwo mRNA levels but reduces mRNA peak levels of the 4 described CLK/CYC targets, inducing an almost complete loss of their cycling. In addition, the absence of CWO induces alterations of PER and CLK phosphorylation cycles. Our results indicate that, in vivo, CWO modulates clock gene expression through both repressor and activator transcriptional functions.

Electrical Silencing of PDF Neurons Advances the Phase of non-PDF Clock Neurons in Drosophila

Wu, Y., Cao, G., Nitabach, M. N. — 2008-03-28

Drosophila clock neurons exhibit self-sustaining cellular oscillations that rely in part on rhythmic transcriptional feedback loops. We have previously determined that electrical silencing of the pigment dispersing factor (PDF)-expressing lateral-ventral (LN_V) pacemaker subset of fly clock neurons via expression of an inward-rectifier K⁺ channel (Kir2.1) severely disrupts free-running rhythms of locomotor activity—most flies are arrhythmic and those that are not exhibit weak short-period rhythms—and abolishes LN_V molecular oscillation in constant darkness. PDF is known to be an important LN_V output signal. Here we examine the effects of electrical silencing of the LN_V pacemakers on molecular rhythms in other, nonsilenced, subsets of clock neurons. In contrast to previously described cell-autonomous abolition of free-running molecular rhythms, we find that electrical silencing of the LN_V pacemakers via Kir2.1 expression does not impair molecular rhythms in LN_D, DN1, and DN2 subsets of clock neurons. However, free-running molecular rhythms in these non-LN_V clock neurons occur with advanced phase. Electrical silencing of LN_Vs phenocopies PDF null mutation (pdf ⁰¹ ) at both behavioral and molecular levels except for the complete abolition of free-running cellular oscillation in the LN_Vs themselves. LN_V electrically silenced or pdf 01 flies exhibit weak free-running behavioral rhythms with short period, and the molecular oscillation in non-LN_V neurons phase advances in constant darkness. That LN_V electrical silencing leads to the same behavioral and non-LN_V molecular phenotypes as pdf 01 suggests that persistence of LN_V molecular oscillation in pdf 01 flies has no functional effect, either on behavioral rhythms or on non-LN_V molecular rhythms. We thus conclude that functionally relevant signals from LN_Vs to non-LN_V clock neurons and other downstream targets rely both on PDF signaling and LN_V electrical activity, and that LN_Vs do not ordinarily send functionally relevant signals via PDF-independent mechanisms.

Photoperiodic Induction of Diapause Requires Regulated Transcription of timeless in the Larval Brain of Chymomyza costata

Stehlik, J., Zavodska, R., Shimada, K., Sauman, I., Kostal, V. — 2008-03-28

Photoperiodic signal stimulates induction of larval diapause in Chymomyza costata. Larvae of NPD strain (npd-mutants) do not respond to photoperiod. Our previous results indicated that the locus npd could code for the timeless gene and its product might represent a molecular link between circadian and photoperiodic clock systems. Here we present results of tim mRNA (real time-PCR) and TIM protein (immunohistochemistry) analyses in the larval brain. TIM protein was localized in 2 neurons of each brain hemisphere of the 4-d-old 3rd instar wild-type larvae. In a marked contrast, no TIM neurons were detected in the brain of 4-day-old 3rd instar npd -mutant larvae and the level of tim transcripts was approximately 10-fold lower in the NPD than in the wild-type strain. Daily changes in tim expression and TIM presence appeared to be under photoperiodic control in the wild-type larvae. Clear daily oscillations of tim transcription were observed during the development of 3rd instars under the short-day conditions. Daily oscillations were less apparent under the long-day conditions, where a gradual increase of tim transcript abundance appeared as a prevailing trend. Analysis of the genomic structure of tim gene revealed that npd-mutants carry a 1855 bp-long deletion in the 5'-UTR region. This deletion removed the start of transcription and promoter regulatory motifs E-box and TER-box. The authors hypothesize that this mutation was responsible for dramatic reduction of tim transcription rates, disruption of circadian clock function, and disruption of photoperiodic calendar function in npd-mutant larvae of C. costata.

Reorganization of the Suprachiasmatic Nucleus Coding for Day Length

Naito, E., Watanabe, T., Tei, H., Yoshimura, T., Ebihara, S. — 2008-03-28

In mammals, the suprachiasmatic nucleus (SCN), the circadian pacemaker, receives light information via the retina and functions in the entrainment of circadian rhythms and in phasing the seasonal responses of behavioral and physiological functions. To better understand photoperiod-related alterations in the SCN physiology, we analyzed the clock gene expression in the mouse SCN by performing in situ hybridization and real-time monitoring of the mPer1::luc bioluminescence. Under long photoperiod (LP) conditions, the expression rhythms of mPer1 and Bmal1 in the caudal SCN phase-led those in the rostral SCN; further, within the middle SCN, the rhythms in the ventrolateral (VL)—like subdivision advanced compared with those in the dorsomedial (DM)—like subdivision. The mPer1::luc rhythms in the entire coronal slice obtained from the middle SCN exhibited 2 peaks with a wide peak width under LP conditions. Imaging analysis of the mPer1::luc rhythms in several subdivisions of the rostral, middle, caudal, and horizontal SCN revealed wide regional variations in the peak time in the rostral half of the SCN under LP conditions. These variations were not due to alterations in the waveform of a single SCN neuronal rhythm. Our results indicate that LP conditions induce phase changes in the rhythms in multiple regions in the rostral half of the SCN; this leads to different circadian waveforms in the entire SCN, coding for day length.

Interferon-{gamma} Alters Electrical Activity and Clock Gene Expression in Suprachiasmatic Nucleus Neurons

2008-03-28

The proinflammatory cytokine interferon (IFN-) is an immunomodulatory molecule released by immune cells. It was originally described as an antiviral agent but can also affect functions in the nervous system including circadian activity of the principal mammalian circadian pacemaker, the suprachiasmatic nucleus. IFN- and the synergistically acting cytokine tumor necrosis factor- acutely decrease spontaneous excitatory postsynaptic activity and alter spiking activity in tissue preparations of the SCN. Because IFN- can be released chronically during infections, the authors studied the long-term effects of IFN- on SCN neurons by treating dispersed rat SCN cultures with IFN- over a 4-week period. They analyzed the effect of the treatment on the spontaneous spiking pattern and rhythmic expression of the "clock gene," Period 1. They found that cytokine-treated cells exhibited a lower average spiking frequency and displayed a more irregular firing pattern when compared with controls. Furthermore, long-term treatment with IFN- in cultures obtained from a transgenic Per1-luciferase rat significantly reduced the Per1-luc rhythm amplitude in individual SCN neurons. These results show that IFN- can alter the electrical properties and circadian clock gene expression in SCN neurons. The authors hypothesize that IFN- can modulate circadian output, which may be associated with sleep and rhythm disturbances observed in certain infections and in aging.

Circadian Rhythms of Photorefractory Siberian Hamsters Remain Responsive to Melatonin

2008-03-28

Short day lengths increase the duration of nocturnal melatonin (Mel) secretion, which induces the winter phenotype in Siberian hamsters. After several months of continued exposure to short days, hamsters spontaneously revert to the spring-summer phenotype. This transition has been attributed to the development of refractoriness of Mel-binding tissues, including the suprachiasmatic nucleus (SCN), to long-duration Mel signals. The SCN of Siberian hamsters is required for the seasonal response to winter-like Mel signals, and becomes refractory to previously effective long-duration Mel signals restricted to this area. Acute Mel treatment phase shifts circadian locomotor rhythms of photosensitive Siberian hamsters, presumably by affecting circadian oscillators in the SCN. We tested whether seasonal refractoriness of the SCN to long-duration Mel signals also renders the circadian system of Siberian hamsters unresponsive to Mel. Males manifesting free-running circadian rhythms in constant dim red light were injected with Mel or vehicle for 5 days on a 23.5-h T-cycle beginning at circadian time 10. Mel injections caused significantly larger phase advances in activity onset than did the saline vehicle, but the magnitude of phase shifts to Mel did not differ between photorefractory and photosensitive hamsters. Similarly, when entrained to a 16-h light/8-h dark photocycle, photorefractory and photosensitive hamsters did not differ in their response to Mel injected 4 h before the onset of the dark phase. Activity onset in Mel-injected hamsters was masked by light but was revealed to be significantly earlier than in vehicle-injected hamsters upon transfer to constant dim red light. The acute effects of melatonin on circadian behavioral rhythms are preserved in photorefractory hamsters.

Nocturnal Behavior and Rhythmic Period Gene Expression in a Lancelet, Branchiostoma lanceolatum

Schomerus, C., Korf, H.-W., Laedtke, E., Moret, F., Zhang, Q., Wicht, H. — 2008-03-28

The authors here present the first anatomical, molecular biological, and ethological data on the organization of the circadian system of a lancelet, Branchiostoma lanceolatum, a close invertebrate relative of vertebrates. B. lanceolatum was found to be a nocturnal animal and, since its rhythmic activity persisted under constant darkness, it also appears to possess an endogenous, circadian oscillator. The authors cloned a homolog of the clock gene Period (Per), which plays a central (inhibitory) role in the biochemical machinery of the circadian oscillators of both vertebrates and protostomians. This gene from B. lanceolatum was designated as amphiPer. Both the sequence of its cDNA and that of the predicted protein are more similar to those of the Per paralogs of vertebrates than to those of the single protostomian Per gene. A strong expression of amphiPer was found in a small cell group in the anterior neural tube. The amphiPer mRNA levels fluctuated in a rhythmic manner, being high early in the day and low late at night. The authors' data suggest a homology of the amphiPer expessing cells to the suprachiasmatic nucleus of vertebrates.

The Challenge to Science Management

Wagstaff, Q. A. — 2008-03-28

Casein kinase I{varepsilon} Does Not Rescue double-time Function in Drosophila Despite Evolutionarily Conserved Roles in the Circadian Clock

Sekine, T., Yamaguchi, T., Hamano, K., Young, M. W., Shimoda, M., Saez, L. — 2008-02-07

double-time (dbt) is a casein kinase gene involved in cell survival, proliferation, and circadian rhythms in the fruit fly, Drosophila melanogaster. Genetic and biochemical studies have shown that dbt and its mammalian ortholog casein kinase I (hckI) regulate the circadian phosphorylation of period (per), thus controlling per subcellular localization and stability. Mutations in these kinases can shorten the circadian period in both mammals and Drosophila. Since similar activities in circadian clock have been described for these kinases, we investigated whether the expression of mammalian casein kinase I can replace the activity of dbt in flies. Global expression of the full-length dbt rescued lethality of the null mutant dbt^revVIII and rescued flies showed normal locomotor activity rhythms. Global expression of dbt also restored the locomotor activity rhythm of the arrhythmic genotype, dbt^ar/dbt^revVIII. In contrast, global expression of hckI or hckI did not rescue lethality or locomotor activity of dbt mutants. Furthermore dbt overexpression in wild-type clock cells had only a small effect on period length, whereas hckI expression in clock cells greatly lengthened period to ~30.5 hours and increased the number of arrhythmic flies. These results indicate that hckI cannot replace the activity of dbt in flies despite the high degree of similarity in primary sequence and kinase function. Moreover, expression of hckI in flies appears to interfere with dbt activity. Thus, caution should be used in interpreting assays that measure activity of mammalian casein kinase mutants in Drosophila, or that employ vertebrate CKI in studies of dPER phosphorylations.

Interactions of GABAA Receptor Activation and Light on Period mRNA Expression in the Suprachiasmatic Nucleus

Ehlen, J. C., Novak, C. M., Karom, M. C., Gamble, K. L., Albers, H. E. — 2008-02-07

Activation of -aminobutyric acid (GABA)_A receptors in the suprachiasmatic nucleus (SCN) resets the circadian clock during the day and inhibits the ability of light to reset the clock at night. Light in turn acts during the day to inhibit the phase-resetting effects of GABA. Some evidence suggests that Period mRNA changes in the SCN are responsible for these interactions between light and GABA. Here, the hypothesis that light and the GABA_A receptor interact by altering the expression of Period 1 and/or Period 2 mRNA in the SCN is tested. The GABA_A agonist muscimol was injected near the SCN just prior to a light pulse, during the mid-subjective day and the early and late subjective night. Changes in Period 1 and Period 2 mRNA were measured in the SCN by in situ hybridization. Light-induced Period 1 mRNA was inhibited by GABA_A receptor activation in the early and late subjective night, while Period 2 mRNA was only inhibited during the late night. During the subjective day, light had no effect on the ability of muscimol to suppress Period 1 mRNA hybridization signal. Thus, light and GABA_A receptor activation inhibit each other's ability to induce behavioral phase shifts throughout the subjective day and night. However, only in the late night are these behavioral effects correlated with changes in Period gene expression. Together, our data support the hypothesis that the interacting effects of light and GABA are the result of the opposing actions of these stimuli on Period mRNA, but only during the subjective night.

The Circadian Clock Protein BMAL1 Is Necessary for Fertility and Proper Testosterone Production in Mice

2008-02-07

Although it is well established that the circadian clock regulates mammalian reproductive physiology, the molecular mechanisms by which this regulation occurs are not clear. The authors investigated the reproductive capacity of mice lacking Bmal1 (Arntl, Mop3), one of the central circadian clock genes. They found that both male and female Bmal1 knockout (KO) mice are infertile. Gross and microscopic inspection of the reproductive anatomy of both sexes suggested deficiencies in steroidogenesis. Male Bmal1 KO mice had low testosterone and high luteinizing hormone serum concentrations, suggesting a defect in testicular Leydig cells. Importantly, Leydig cells rhythmically express BMAL1 protein, suggesting peripheral control of testosterone production by this clock protein. Expression of steroidogenic genes was reduced in testes and other steroidogenic tissues of Bmal1 KO mice. In particular, expression of the steroidogenic acute regulatory protein (StAR) gene and protein, which regulates the rate-limiting step of steroidogenesis, was decreased in testes from Bmal1 KO mice. A direct effect of BMAL1 on StAR expression in Leydig cells was indicated by in vitro experiments showing enhancement of StAR transcription by BMAL1. Other hormonal defects in male Bmal1 KO mice suggest that BMAL1 also has functions in reproductive physiology outside of the testis. These results enhance understanding of how the circadian clock regulates reproduction.

Photic Sensitivity Ranges of Hamster Pupillary and Circadian Phase Responses Do Not Overlap

Hut, R. A., Oklejewicz, M., Rieux, C., Cooper, H. M. — 2008-02-07

Mammalian retinal photoreceptors form an irradiance detection system that drives many nonvisual responses to light such as pupil reflex and resetting of the circadian clock. To understand the role of pupil size in circadian light responses, pupil diameter was pharmacologically manipulated and the effect on behavioral phase shifts at different irradiance levels was studied in the Syrian hamster. Dose-response curves for steady-state pupil size and for behavioral phase shifts were constructed for 3 pupil conditions (dilated, constricted, and control). Retinal irradiance was calculated from corneal irradiance, pupil size, retinal surface area, and absorption of ocular media. The sensitivity of photic responses to retinal irradiance is approximately 1.5 log units higher than to corneal irradiance. When plotted against corneal irradiance, pharmacological pupil constriction reduces the light sensitivity of the circadian system, but pupil dilation has no effect. As expected, when plotted against retinal irradiance all dose-response curves superimposed, confirming that the circadian system responds to photon flux on the retina. Pupil dilation does not increase the circadian response to increasing irradiance, since the response of the circadian system attains saturation at irradiance levels lower than those required to induce pupil constriction. The main finding shows that due to the different response sensitivities, the effect of pupil constriction on the light sensitivity of the circadian system in the hamster under natural conditions is virtually negligible. We further suggest the existence of distinct modulating mechanisms for the differential retinal irradiance sensitivity of the pupil system and the circadian system, which enables the different responses to be tuned to their specific tasks while using similar photoreceptive input.

Duration of Melatonin Regulates Seasonal Changes in Song Control Nuclei of the House Sparrow, Passer domesticus: Independence from Gonads and Circadian Entrainment

Cassone, V. M., Bartell, P. A., Earnest, B. J., Kumar, V. — 2008-02-07

Avian behavior and physiology are temporally regulated by a complex circadian clock on both a daily and an annual basis. The circadian secretion of the hormone melatonin is a critical component of the regulation of circadian/daily processes in passerine birds, but there is little evidence that the gland regulates annual changes in primary reproductive function. Here it is shown that locomotor rhythms of house sparrows, Passer domesticus, which are made arrhythmic by either pinealectomy or maintenance in constant light, can be synchronized by daily administration of melatonin of different durations to simulate the melatonin profiles indicative of long and short photoperiods. Pinealectomized male sparrows maintained in constant darkness were entrained by both melatonin regimens. In both cases, testes were regressed and the song control nuclei were small. Intact male house sparrows maintained in constant light were also entrained to both melatonin regimens. However, sparrows that received a long duration melatonin cycle exhibited small song control nuclei, while sparrows that received short duration melatonin or no melatonin at all exhibited large song control nuclei. The data indicate that seasonal changes in melatonin duration contribute to the regulation of song control nuclei.

Circadian and Masking Control of Migratory Restlessness in Gambel's White-Crowned Sparrow (Zonotrichia leucophrys gambelii)

Coverdill, A. J., Bentley, G. E., Ramenofsky, M. — 2008-02-07

Avian migration is a seasonal activity that requires intricate timing on both an annual and daily basis. With increasing evidence for endogenous regulation of daily activities in migrant species, we tested whether a circadian oscillator may be involved with the expressions of daily locomotor activities and specific behaviors of the long-distance migrant, Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii). Our previous studies have identified both daytime and nighttime behavioral patterns under a photoperiod of 18L:6D. In 2 separate trials, birds in the vernal migratory life-history stage were exposed to constant dim light, (DD)_dim, and constant bright light, LL, while locomotor activity and behavioral observations were collected. Under (DD)_dim, the daytime behaviors that included active and quiescent components observed under 18L:6D were lost as migratory restlessness, the intense nighttime activity, persisted nonstop for 36.4 h. Furthermore, the specific behaviors of migratory restlessness that are normally confined to the dark phase of 18L:6D, beak-up and beak-up flight, were expressed also during the subjective day of (DD)_dim. Birds exposed to LL retained similar patterns of activity to the 18L:6D condition for 3 days, after which they became arrhythmic. Behavioral observations of intense locomotor activity observed during the subjective night of LL revealed no beak-up and beak-up flight. Thus, the complete expression of migratory restlessness that includes beak-up and beak-up flight may not be regulated by a circadian oscillator but instigated by very low light intensity. Locomotor activity and associated daytime behaviors appear to be influenced by a circadian oscillator, given their persistent patterns under LL. Therefore, we suggest that the separate components of migratory behavior are regulated differentially by a circadian oscillator and ambient lighting conditions.

Relative Photorefractoriness, Prolactin, and Reproductive Regression in a Flexibly Breeding Sonoran Desert Passerine, the Rufous-Winged Sparrow, Aimophila carpalis

Small, T. W., Sharp, P. J., Bentley, G. E., Deviche, P. — 2008-02-07

We tested the hypothesis that adult male rufous-winged sparrows, Aimophila carpalis, exhibit relative photorefractoriness. This condition results in partial loss of sensitivity to photoperiod as a reproductive stimulus after prolonged exposure to long photoperiods and is similar to the mammalian condition called photoperiodic memory. Captive birds were exposed either to 8 h of light/16 h of dark per day (8L) or to 16L for 11 weeks and were then exposed either to 8L, 13L, 14L, or 16L. Testicular diameter, plasma luteinizing hormone (LH), and plasma prolactin (PRL) were measured to assess reproductive system activity in response to photostimulation. In free-living birds, testicular diameter, plasma LH, and PRL were compared in birds caught in September in a year when birds were breeding and in a year when birds were not breeding to further evaluate the role of PRL in the termination of seasonal breeding. Testes completely developed after transfer from 8L to 14L or to 16L and partially developed after transfer from 8L to 13L. However, after 11 weeks of 16L exposure, transfer to 14L caused partial regression and transfer to 13L caused complete regression of the testes. Plasma LH increased in all birds that were transferred from 8L to a longer photoperiod. PRL showed a weak response to longer photoperiod treatment and was elevated in birds after chronic 16L exposure in comparison to birds exposed to chronic 8L. These data indicate that male rufous-winged sparrows lose sensitivity to photoperiod after long photoperiod exposure consistent with the relative photorefractoriness and photoperiodic memory models. Lower PRL in birds that developed testes on 13L and 14L compared to birds that regressed testes on 13L and 14L are consistent with the hypothesis that PRL regulates relative photorefractoriness. However, PRL does not appear to regulate interannual differences in the timing of testicular regression.

Photoperiodic Regulation of Behavioral Responses to Bacterial and Viral Mimetics: A Test of the Winter Immunoenhancement Hypothesis

Baillie, S. R., Prendergast, B. J. — 2008-02-07

Siberian hamsters (Phodopus sungorus) exhibit changes in immune function following adaptation to short photoperiods, including a marked attenuation of energetically expensive thermoregulatory and behavioral responses to gram-negative bacterial infections. Whether this seasonal attenuation of the immune response is idiosyncratic to gram-negative infections or is representative of innate immune responses in general is not known. If seasonal attenuation of responsiveness to infection is indeed driven primarily by anticipation of energetic constraints, then one would predict that responsiveness to all pathogens would be diminished during short days. If, on the other hand, seasonal changes in responsiveness to infection reflect anticipation of specific pathogens that are common at different phases of the annual cycle, then one would expect short photoperiods to attenuate responsiveness to some pathogens and long photoperiods to attenuate responsiveness to others. To resolve this issue, we exposed male Siberian hamsters to either long or short photoperiods for 11 weeks, then examined their behavioral sickness responses to compounds that represent the minimally immunogenic components of gram-negative bacterial (lipopolysaccharide), gram-positive bacterial (muramyl dipeptide), and viral (polyinosinepolycytidylic acid) organisms. Hamsters exhibited anorexic, anhedonic, ponderal, and/or thermoregulatory sickness behaviors to all 3 pathogen mimetics, but in all cases in which sickness responses were evident, they were attenuated in short days. Energetically costly behavioral responses to several distinct classes of infectious organisms are attenuated in anticipation of winter. The data are not consistent with a pathogen-specific seasonal modulation of innate immune responses.

Circadian Behavioral Rhythms in Cry1/Cry2 Double-Deficient Mice Induced by Methamphetamine

Honma, S., Yasuda, T., Yasui, A., van der Horst, G. T. J., Honma, K.-i. — 2008-02-07

Mammalian Peripheral Circadian Oscillators Are Temperature Compensated

Reyes, B. A., Pendergast, J. S., Yamazaki, S. — 2008-02-07