Journal of Biological Rhythms recent issues

Maroli Krishnayya Chandrashekaran (1937-2009)

Sharma, V. K. — Thu, 19 Nov 2009 11:24:28 PST

Circadian Expression of Clock Genes in Two Mosquito Disease Vectors: cry2 Is Different

Thu, 19 Nov 2009 11:24:28 PST

Different mosquito species show a full range of activity patterns, including diurnal, crepuscular, and nocturnal behaviors. Although activity and blood-feeding rhythms are controlled by the circadian clock, it is not yet known whether such species-specific differences in behavior are controlled directly by core clock genes or instead reflect differences in how the information of the central clock is translated into output signals. The authors have analyzed the circadian expression of clock genes in two important mosquito vectors of tropical diseases, Aedes aegypti and Culex quinquefasciatus . Although these two species show very different locomotor activity patterns and are estimated to have diverged more than 22 million years ago, they show conserved circadian expression patterns for all major cycling clock genes except mammalian-like cryptochrome2 (cry2). The results indicate that different mechanisms for cry2 regulation may exist for the two species. The authors speculate that the correlation between the differences in behavior between Ae. aegypti and Cx. quinquefasciatus and their corresponding cry2 mRNA profiles suggests a potential role for this clock gene in controlling species-specific rhythmic behavior. However, further work is needed to establish that this is the case as the different cry2 expression patterns might reflect differences between the Aedes and Culex lineages that are not directly related to changes in behavior.

Synergic Entrainment of Drosophila's Circadian Clock by Light and Temperature

Yoshii, T., Vanin, S., Costa, R., Helfrich-Forster, C. — Thu, 19 Nov 2009 11:24:28 PST

Daily light and temperature cycles are considered the most important zeitgebers for circadian clocks in many organisms. The influence of each single zeitgeber on the clock has been well studied, but little is known about any synergistic effects of both zeitgebers on the clock. In nature, light and temperature show characteristic daily oscillations with the temperature rising during the light phase and reaching its maximum in the late afternoon. Here, we studied behavioral and molecular rhythms in Drosophila melanogaster under simulated natural low light-dark (LD) and temperature (T) cycles that typically occur during the September equinox. Wild-type flies were either subjected to simulated LD or T cycles alone or to a combination of both. Behavioral rhythms and molecular rhythms in the different clock neurons were assessed under the 3 different conditions. Although behavioral rhythms entrained to all conditions, the rhythms were most robust under the combination of LD and T cycles. The clock neurons responded differently to LD and T cycles. Some were not entrained by T cycles alone; others were only slightly entrained by LD cycles alone. The amplitude of the molecular cycling was not different between LD alone and T cycles alone; but LD alone could set the pacemaker neurons to similar phases, whereas T cycles alone could not. The combination of the 2 zeitgebers entrained all clock neurons not only with similar phase but also enhanced the amplitude of Timeless cycling in the majority of cells. Our results show that the 2 zeitgebers synergistically entrain behavioral and molecular rhythms of Drosophila melanogaster.

Diurnal Variations in Natural and Drug Reward, Mesolimbic Tyrosine Hydroxylase, and Clock Gene Expression in the Male Rat

Thu, 19 Nov 2009 11:24:28 PST

The impact of the circadian timing system upon behavior and physiology is pervasive, and previous evidence suggests a circadian modulation of drug-seeking behavior and responsiveness to drugs of abuse. To further characterize daily rhythms in reward and to extend these observations to natural reinforcers, diurnal variation in the rewarding value of sex and systemic amphetamine was assessed via the conditioned place preference paradigm. To identify potential mechanisms for rhythmicity in reward, levels of tyrosine hydroxylase (TH) and core clock proteins (Period1 and Bmal1) were examined across the day in the ventral tegmental area (VTA) and the nucleus accumbens (NAcc). During an initial training period, male rat sexual performance varied diurnally with a nadir near the light-to-dark transition. Diurnal rhythms also were evident for both mating and amphetamine-related reward. However, the rhythms for these particular stimuli exhibited differences in their pattern of timing, with sex reward showing a peak during the middark period and amphetamine reward exhibiting high points during the late night and midday with a nadir prior to the light-to-dark transition. A diurnal variation also was seen for the locomotor-activating effect of acute amphetamine administration with a peak during the late night. Western blot analyses revealed that Period1 and Bmal1 protein levels were rhythmic in the NAcc but not in the VTA. By contrast, TH protein levels were rhythmic in both the NAcc and VTA, but the peaks differed with that in the NAcc coinciding with the peak of sex reward and that in the VTA associated with the peak in amphetamine reward. Thus, it appears that both natural and drug-related reward vary in a diurnal fashion but differ in the timing of their peak and nadir levels. The phase relationships between reward rhythms and mesolimbic TH protein levels suggest that an increased capacity for the release of dopamine in the NAcc may underlie the rhythms in sex-related reward, while amphetamine-related reward occurs at a time when the likelihood of evoked NAcc DA release is relatively low.

Correlation with Behavioral Activity and Rest Implies Circadian Regulation by SCN Neuronal Activity Levels

Houben, T., Deboer, T., van Oosterhout, F., Meijer, J. H. — Thu, 19 Nov 2009 11:24:28 PST

The SCN of the hypothalamus contains a major pacemaker, which exhibits 24-h rhythms in electrical impulse frequency. Although it is known that SCN electrical activity is high during the day and low during the night, the precise relationship between electrical activity and behavioral rhythms is almost entirely unknown. The authors performed long-term recordings of SCN multiple unit activity with the aid of implanted microelectrodes in parallel with the drinking activity in freely moving mice. The animals were kept in a 12h:12h light-dark cycle (LD 12:12) and in short-day (LD 8:16) and long-day photoperiods (LD 16:8). Onsets and offsets of behavioral activity occurred when SCN discharge was around half-maximum value. Of the onsets 80%, and of the offsets 62%, occurred when SCN electrical activity differed less than 15% from the half-maximum electrical activity levels. Transitions between rest and activity could be described by a sigmoid shaped probability curve with Hill coefficients of 7.0 for onsets and 5.7 for offsets. The similarity in the onset and offset levels shows an absence of hysteresis in the control of behavioral activity by the SCN. Exposure to short- or long-day photoperiods induced significant alterations in the waveform of electrical activity but did not affect SCN electrical activity levels at which behavioral transitions occurred. In all photoperiods, the SCN signal was skewed with more rapid discharge changes during onsets (19% per hour) than offsets (11% per hour). The precision of the circadian system appears optimized, as transitions between behavioral activity and rest occur when the change in SCN electrical activity is maximal, both during the declining and rising phase. The authors conclude that transitions in behavioral state can be described by a probability function around half-maximum electrical activity levels and that the parameters of the SCN, predicting onset and offset of behavior, are remarkably insensitive to environmental conditions.

Circadian Trafficking of Calbindin-ir in Fibers of SCN Neurons

LeSauter, J., Bhuiyan, T., Shimazoe, T., Silver, R. — Thu, 19 Nov 2009 11:24:28 PST

Calbindin-D28K (CalB)—containing cells form a distinct cluster within the core of the hamster suprachiasmatic nucleus (SCN). These cells are directly retinorecipient but lack detectable rhythms in clock gene expression or electrical activity. In studies exploring SCN connectivity using double-label immunochemistry, we previously reported an absence of contacts among CalB fibers and vasopressin (VP) cells in animals sacrificed during the day. Here, we explored circadian variations in CalB-immunoreactivity (-ir) and re-examined the connections between CalB and other SCN cell types at zeitgeber times (ZT) 4 and 14. The results reveal a circadian rhythm of CalB-ir in fibers of SCN cells with high expression during the night and subjective night and low expression during the day and subjective day. This circadian difference is not seen in the other brain regions studied. Significantly more appositions were detected between CalB fibers and VP cells during the night than during the day, while circadian variation in numbers of contacts was not seen between CalB fibers and vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), or gastrin-releasing peptide (GRP) cells. There was no detectable variation in appositions from any peptidergic fiber type onto CalB cells. The present findings suggest that CalB cells relay photic information to VP oscillator cells of the SCN shell in a temporally gated manner.

Millisecond Light Pulses Make Mice Stop Running, then Display Prolonged Sleep-Like Behavior in the Absence of Light

Morin, L.P., Studholme, K.M. — Thu, 19 Nov 2009 11:24:28 PST

Masking, measured as a decrease in nocturnal rodent wheel running, is a visual system response to rod/cone and retinal ganglion cell photoreception. Here, the authors show that a few milliseconds of light are sufficient to initiate masking, which continues for many minutes without additional photic stimulation. C57J/B6 mice were tested using flash stimuli previously shown to elicit large circadian rhythm phase shifts. Ten flashes, 2 msec each and equally distributed over 5 min, activate locomotor suppression that endures for an additional 25 to 35 min in the dark and does not differ in magnitude or duration from that elicited by 5-min saturating light pulse. Locomotor activity by mice without access to running wheels is also suppressed by light flashes. The effects of various light flash patterns on mouse locomotor suppression are similar to those previously described for hamster phase shifts. Video analysis of active mice indicates that light flashes initiated at ZT13 rapidly induce an interval of behavioral quiescence that lasts about 10 min at which time the animals assume a typical sleep posture that is maintained for an additional 25 min. Thus, the period coincident with light-induced wheel running suppression appears to consist of two distinct behavioral states, one interval during which locomotor quiescence is initiated and maintained, followed by a second interval characterized by behavioral sleep. Given this sequence effected by light stimulation, we suggest that it be referred to as "photosomnolence," the term reflecting upon both the nature of the stimulus and the associated behavioral change.

Photoperiod History Differentially Impacts Reproduction and Immune Function in Adult Siberian Hamsters

Prendergast, B. J., Pyter, L. M. — Thu, 19 Nov 2009 11:24:28 PST

Seasonal changes in numerous aspects of mammalian immune function arise as a result of the annual variation in environmental day length (photoperiod), but it is not known if absolute photoperiod or relative change in photoperiod drives these changes. This experiment tested the hypothesis that an individual’s history of exposure to day length determines immune responses to ambiguous, intermediate-duration day lengths. Immunological (blood leukocytes, delayed-type hypersensitivity reactions [DTH]), reproductive, and adrenocortical responses were assessed in adult Siberian hamsters (Phodopus sungorus) that had been raised initially in categorically long (15-h light/day; 15L) or short (9L) photoperiods and were subsequently transferred to 1 of 7 cardinal experimental photoperiods between 9L and 15L, inclusive. Initial photoperiod history interacted with contemporary experimental photoperiods to determine reproductive responses: 11L, 12L, and 13L caused gonadal regression in hamsters previously exposed to 15L, but elicited growth in hamsters previously in 9L. In hamsters with a 15L photoperiod history, photoperiods ≤ 11L elicited sustained enhancement of DTH responses, whereas in hamsters with a 9L photoperiod history, DTH responses were largely unaffected by increases in day length. Enhancement and suppression of blood leukocyte concentrations occurred at 13L in hamsters with photoperiod histories of 15L and 9L, respectively; however, prior exposure to 9L imparted marked hysteresis effects, which suppressed baseline leukocyte concentrations. Cortisol concentrations were only enhanced in 15L hamsters transferred to 9L and, in common with DTH, were unaffected by photoperiod treatments in hamsters with a 9L photoperiod history. Photoperiod history acquired in adulthood impacts immune responses to photoperiod, but manifests in a markedly dissimilar fashion as compared to the reproductive system. Prior photoperiod exposure has an enduring impact on the ability of the immune system to respond to subsequent changes in day length.

A Mathematical Model of Homeostatic Regulation of Sleep-Wake Cycles by Hypocretin/Orexin

Postnova, S., Voigt, K., Braun, H. A. — Thu, 19 Nov 2009 11:24:28 PST

We introduce a physiology-based mathematical model of sleep-wake cycles, suggesting a novel mechanism of homeostatic regulation of sleep. In this model, the homeostatic process is determined by the neuropeptide hypocretin/ orexin, which is a cotransmitter of the lateral hypothalamus. Hypocretin/ orexin neurons are silent during sleep and active during wakefulness. Firing of these neurons is sustained by reciprocal excitatory synaptic connections with local glutamate interneurons. This feedback loop has been simulated with a minimal but physiologically plausible model. It includes 2 simplified Hodgkin-Huxley type neurons that are connected via glutamate synapses, one of which additionally contains hypocretin/orexin as the functionally relevant cotransmitter. During the active state (wakefulness), the synaptic efficacy of hypocretin/orexin declines as a result of the ongoing firing. It recovers during the silent (sleep) state. We demonstrate that these homeostatic changes can account for typical alterations of sleep-wake transitions, for example, introduced by napping, sleep deprivation, or alarm clock. In combination with a circadian input, the model mimics the transitions between silent and firing states in agreement with sleep-wake cycles. These simulation results support the concept of state-dependent alterations of hypocretin/orexin effects as an important homeostatic process in sleep-wake regulation, although additional mechanisms can be involved.

Victor Bruce (1920-2009)

Feldman, J. — Tue, 15 Sep 2009 09:21:42 PDT

Basis of Robustness and Resilience in the Suprachiasmatic Nucleus: Individual Neurons Form Nodes in Circuits that Cycle Daily

Butler, M. P., Silver, R. — Tue, 15 Sep 2009 09:21:42 PDT

How the cellular elements of the SCN are synchronized to each other is not well understood. We explore circadian oscillations manifest at the level of the cell, the tissue, and the whole animal to better understand intra-SCN synchrony and master clock function of the nucleus. At each level of analysis, responses to variations in operating environment (robustness), and following damage to components of the system (resilience), provide insight into the mechanisms whereby the SCN orchestrates circadian timing. Tissue level rhythmicity reveals circuits associated with an orderly spatiotemporal daily pattern of activity that is not predictable from their cellular elements. Specifically, in stable state, some SCN regions express low amplitude or undetectable rhythms in clock gene expression while others produce high amplitude oscillations. Within the SCN, clock gene expression follows a spatially ordered, repeated pattern of activation and inactivation. This pattern of activation is plastic and subserves responses to changes in external and internal conditions. Just as daily rhythms at the cellular level depend on sequential expression and interaction of clock genes, so too do rhythms at the SCN tissue level depend on sequential activation of local nodes. We hypothesize that individual neurons are organized into nodes that are themselves sequentially activated across the volume of the SCN in a cycle that repeats on a daily basis. We further propose that robustness is expressed in the ability of the SCN to sustain rhythmicity over a wide range of internal and external conditions, and that this reflects plasticity of the underlying nodes and circuits. Resilience is expressed in the ability of SCN cells to oscillate and to sustain activity-related rhythms at the behavioral level. Importantly, other aspects of pacemaker function remain to be examined.

Analysis of the Drosophila Clock Promoter Reveals Heterogeneity in Expression between Subgroups of Central Oscillator Cells and Identifies a Novel Enhancer Region

Gummadova, J. O., Coutts, G. A., Glossop, N. R. J. — Tue, 15 Sep 2009 09:21:42 PDT

The CLOCK-CYCLE (CLK-CYC) heterodimer lies at the heart of the circadian oscillator mechanism in Drosophila, yet little is known about the identity of transcription factors that regulate the expression of Clk and/or cyc. Here, the authors have used a transgenic approach to isolate regions of the Clk locus that are necessary for expression in central oscillator neurons in the adult fly brain. This analysis shows that central clock cells can be subdivided into 2 distinct groups based on Clk gene regulation. Expression in the lateral neuron (LN), dorsal neuron 1 anterior (DN1a) and 2 (DN2) clusters requires cis-elements located in a 122 base-pair (bp) region (–206 to –84) of the Clk promoter. Expression in the remaining dorsal neurons, 1 posterior (DN1p) and 3 (DN3) and the lateral posterior neurons (LPN), requires regulatory elements located in the –856 to –206 region. In addition, expression in photoreceptors of the compound eye is enhanced by cis-elements located in a 3rd region of the Clk locus (–1982 to –856). This region also enhances expression in nonoscillator cells in the brain including the Kenyon cells, but expression in these neurons is suppressed by regulatory sites located further upstream of –1982. The authors’ analysis reveals clear heterogeneity in Clk gene expression in the adult brain and provides a necessary focus to isolate novel transcription factors that bind at the Clk locus to regulate expression in different oscillator neuron subgroups. These results also suggest that the DN1a/DN2 neurons may have more molecular commonality with the LNs than they do with the DN1p/DN3/LPN neurons. Finally, this analysis has generated new transgenic lines that will enable genes to be misexpressed in subgroups of central oscillator cells that have previously been resistant to discrete genetic manipulation. Hence, these lines provide important new tools to facilitate a more complete dissection of the neural network that regulates output rhythms in physiology and behavior.

Genetic Analysis of Ectopic Circadian Clock Induction in Drosophila

Kilman, V. L., Allada, R. — Tue, 15 Sep 2009 09:21:42 PDT

Cell-autonomous feedback loops underlie the molecular oscillations that define circadian clocks. In Drosophila the transcription factor Clk activates multiple clock components of feedback loops many of which feed back and regulate Clk expression or activity. Previously the authors evoked similar molecular oscillations in putatively naïve neurons in Drosophila by ectopic expression of a single gene, Clk, suggesting a master regulator function. Using molecular oscillations of the core clock component PERIOD (PER), the authors observed dramatic and widespread molecular oscillations throughout the brain in flies expressing ectopic Clk. Consistent with the master regulator hypothesis, they found that Clk is uniquely capable of inducing ectopic clocks as ectopic induction of other clock components fails to induce circadian rhythms. Clk also induces oscillations even when expression is adult restricted, suggesting that ectopic clocks can even be induced in differentiated cells. However, if transgene expression is discontinued, PER expression disappears, indicating that Clk must be continually active to sustain ectopic clock function. In some cases Clk-mediated PER induction was observed without apparent synchronous cycling, perhaps due to desynchronization of rhythms between clocks or truly cell autonomous arrhythmic PER expression, indicating that additional factors may be necessary for coherent rhythms in cells ectopically expressing Clk. To determine minimal requirements for circadian clock induction by Clk, the authors determined the genetic requirements of ectopic clocks. No ectopic clocks are induced in mutants of Clk’s heterodimeric partner cyc. In addition, noncycling PER is observed when ectopic Clk is induced in a cry^b mutant background. While other factors may contribute, these results indicate that persistent Clock induction is uniquely capable of broadly inducing ectopic rhythms even in adults, consistent with a special role at the top of a clock gene hierarchy.

The Clock Gene period Plays an Essential Role in Photoperiodic Control of Nymphal Development in the Cricket Modicogryllus siamensis

Sakamoto, T., Uryu, O., Tomioka, K. — Tue, 15 Sep 2009 09:21:42 PDT

Photoperiodic regulation of development is a common strategy for insects in the temperate zone to adapt to the seasonally changing environment. Although the circadian clock is generally thought to be involved, the underlying time measurement mechanism is still elusive. Here, we demonstrate that the circadian clock gene period (per) plays an essential role in the photoperiodic regulation of nymphal development in the cricket Modicogryllus siamensis. Nymphal development of this cricket depends on photoperiods, being accelerated by long days and slowed down by short days. We examined the role of per in the nymphal photoperiodic response as well as circadian rhythm generation using parental RNA interference (pRNAi). per mRNA levels in nymphal heads showed a rhythmic expression with the pattern dependent on photoperiods, and pRNAi significantly suppressed the per mRNA level with no significant rhythmicity in the early nymphal stage. Irrespective of photoperiods, nymphs treated with per pRNAi showed adult emergence patterns neither of intact nymphs nor of DsRed2 pRNAi nymphs kept under long days or under short days but similar to those kept under constant dark conditions. Most per pRNAi adults showed arrhythmic or aberrant circadian locomotor activity. These results suggest that the photoperiodic time measurement requires the normal circadian clock that is controlled by the per gene.

Circadian Modulation of Melanopsin-Driven Light Response in Rat Ganglion-Cell Photoreceptors

Weng, S., Wong, K. Y., Berson, D. M. — Tue, 15 Sep 2009 09:21:42 PDT

Intrinsically photosensitive retinal ganglion cells (ipRGCs) project to the suprachiasmatic nucleus (SCN) and are essential for normal photic entrainment of global circadian rhythms in physiology and behavior. The effect of light on the central clock is dependent on circadian phase, and the retina itself contains intrinsic circadian oscillators that can alter its sensitivity to light. This raises the possibility that the ipRGCs, and hence the photoentraining signals in the retinohypothalamic tract, are subject to circadian modulation. Although the ipRGC photopigment melanopsin reportedly exhibits circadian variations in expression, there has been no direct test of the hypothesis that ipRGC sensitivity is under circadian control. Here, the authors provide such a test by measuring the sensitivity of intrinsic photoresponses of rat ipRGCs at 4 circadian times (CTs) using multielectrode array recording. There was little if any circadian modulation in the threshold of intrinsic ipRGC photoresponses. However, very bright light evoked significantly more spiking early in the subjective night (CT12-13) than at other circadian phases. Thus, the gain of the melanopsin-driven response is slightly increased in the early night, at roughly the circadian phase when melanopsin synthesis is thought to be elevated. However, this gain change is probably too modest to contribute much to shape the phase response curve (PRC) for behavioral photoentrainment.

Twilight and Photoperiod Affect Behavioral Entrainment in the House Mouse (Mus musculus)

Comas, M., Hut, R.A. — Tue, 15 Sep 2009 09:21:42 PDT

The effect of twilight transitions on entrainment of C57BL/6JOlaHsd mice (Mus musculus) was studied using light-dark cycles of different photoperiods (6, 12, and 18 h) and twilight transitions of different durations (0, 1, and 2 h). Phase angle differences of the onset, center of gravity, and offset of activity, activity duration (), as well as free-running period () in continuous darkness were analyzed. The main finding was that for all conditions the onset of activity was close to dusk or lights-off except for the short photoperiod with 2 h of twilight where activity onset was on average 5.3 (SEM 1.07) h after lights-off. This finding contrasts with the results of Boulos and Macchi for Syrian hamsters where a 5.9-h earlier activity onset was observed when similar photoperiod and twilight conditions are compared with a rectangular LD cycle. The authors suggest the opposite effects of 2 h of twilight in the 2 species may be related to their different free-running periods under DD conditions following entrainment to short photoperiod with 2-h twilight conditions. Since the authors observed larger variation in phase angle of entrainment in longer twilight conditions, twilight does not necessarily form a stronger zeitgeber.

Circannual Phase Response Curves to Short and Long Photoperiod in the European Hamster

Monecke, S., Saboureau, M., Malan, A., Bonn, D., Masson-Pevet, M., Pevet, P. — Tue, 15 Sep 2009 09:21:42 PDT

This study investigated in male European hamsters (Cricetus cricetus ) whether entrainment of circannual rhythms follows the principles of the nonparametric entrainment model. In 2 experiments the times of the year when long (LP) or short photoperiod (SP) are able to synchronize the reproductive cycle were determined, by recording phase response curves (PRCs). A total of 28 groups of 10 hamsters were synchronized by SP, before being subjected to 2 converse experiments: a) 14 groups were transferred to constant LP, only interrupted by SP for 1 month (SP-pulse), the pulse being increasingly delayed between groups by 2 weeks or 1 month steps; and b) the remaining 14 groups stayed in constant SP interrupted by LP for 1 month (LP-pulse) at different phases of the cycle. In a 3rd experiment 5 groups of 10 European hamsters were subjected to constant LP interrupted by 1-month SP-pulses in regular non-365-day zeitgeber intervals (circannual T-cycles) differing between groups (c). The reproductive state was checked every 2 or 4 weeks. The PRCs revealed that an SP-pulse had a very strong phase-resetting capability of —180° to at least +81° in subjective summer (a). During subjective winter when the animals hibernate, a SP-pulse had only weak effectiveness (a) whereas an LP-pulse advanced the circannual clock by up to +41° (b). In the latter conditions a further advance of up to +156° was achieved by the decrease in photoperiod at the return to SP conditions, which terminated the reproductive phase already after 4 to 5 weeks. In different circannual T-cycles the animals entrained for at least 2 cycles (c). In conclusion, 1) the circannual rhythm of European hamsters can be entrained by one photoperiodic signal per cycle, 2) the decrease in photoperiod is most important for its synchronization, and 3) as in circadian clocks the resetting of circannual clocks follows the principles of the nonparametric entrainment model.

Controlled Patterns of Daytime Light Exposure Improve Circadian Adjustment in Simulated Night Work

Dumont, M., Blais, H., Roy, J., Paquet, J. — Tue, 15 Sep 2009 09:21:42 PDT

Circadian misalignment between the endogenous circadian signal and the imposed rest-activity cycle is one of the main sources of sleep and health troubles in night shift workers. Timed bright light exposure during night work can reduce circadian misalignment in night workers, but this approach is limited by difficulties in incorporating bright light treatment into most workplaces. Controlled light and dark exposure during the daytime also has a significant impact on circadian phase and could be easier to implement in real-life situations. The authors previously described distinctive light exposure patterns in night nurses with and without circadian adaptation. In the present study, the main features of these patterns were used to design daytime light exposure profiles. Profiles were then tested in a laboratory simulation of night work to evaluate their efficacy in reducing circadian misalignment in night workers. The simulation included 2 day shifts followed by 4 consecutive night shifts (2400-0800 h). Healthy subjects (15 men and 23 women; 20-35 years old) were divided into 3 groups to test 3 daytime light exposure profiles designed to produce respectively a phase delay (delay group, n = 12), a phase advance (advance group, n = 13), or an unchanged circadian phase (stable group, n = 13). In all 3 groups, light intensity was set at 50 lux during the nights of simulated night work. Salivary dim light melatonin onset (DLMO) showed a significant phase advance of 2.3 h (± 1.3 h) in the advance group and a significant phase delay of 4.1 h (± 1.3 h) in the delay group. The stable group showed a smaller but significant phase delay of 1.7 h (± 1.6 h). Urinary 6-sulfatoxymelatonin (aMT6s) acrophases were highly correlated to salivary DLMOs. Urinary aMT6s acrophases were used to track daily phase shifts. They showed that phase shifts occurred rapidly and differed between the 3 groups by the 3rd night of simulated night work. These results show that significant phase shifts can be achieved in night workers by controlling daytime light exposure, with no nighttime intervention.

Does the Morning and Evening Oscillator Model Fit Better for Flies or Mice?

Helfrich-Forster, C. — Wed, 22 Jul 2009 15:08:43 PDT

The morning and evening dual oscillator model can explain the adaptation of animals to different photoperiods and other phenomena as bimodal activity patterns, aftereffects, and internal desynchronization of the activity rhythm into 2 free-running components. This review summarizes evidence for and against the existence of morning and evening oscillator cells in the core circadian pacemaker centers of mice and fruit flies.

Period Gene Expression in Four Neurons Is Sufficient for Rhythmic Activity of Drosophila melanogaster under Dim Light Conditions

Rieger, D., Wulbeck, C., Rouyer, F., Helfrich-Forster, C. — Wed, 22 Jul 2009 15:08:43 PDT

The clock gene expressing lateral neurons (LN) is crucial for Drosophila 's rhythmic locomotor activity under constant conditions. Among the LN, the PDF expressing small ventral lateral neurons (s-LN_v) are thought to control the morning activity of the fly (M oscillators) and to drive rhythmic activity under constant darkness. In contrast, a 5th PDF-negative s-LN_v and the dorsal lateral neurons (LN_d) appeared to control the fly's evening activity (E oscillators) and to drive rhythmic activity under constant light. Here, the authors restricted period gene expression to 4 LN—the 5th s-LN_v and 3 LN_d— that are all thought to belong to the E oscillators and tested them in low light conditions. Interestingly, such flies showed rather normal bimodal activity patterns under light moonlight and constant moonlight conditions, except that the phase of M and E peaks was different. This suggests that these 4 neurons behave as ''M'' and ''E'' cells in these conditions. Indeed, they found by PER and TIM immunohistochemistry that 2 LN_d advanced their phase upon moonlight as predicted for M oscillators, whereas the 5th s-LN_v and 1 LN_d delayed their activity upon moonlight as predicted for E oscillators. Their results suggest that the M or E characteristic of clock neurons is rather flexible. M and E oscillator function may not be restricted to certain anatomically defined groups of clock neurons but instead depends on the environmental conditions.

Clock Gene Modulation by TNF-{alpha} Depends on Calcium and p38 MAP Kinase Signaling

Petrzilka, S., Taraborrelli, C., Cavadini, G., Fontana, A., Birchler, T. — Wed, 22 Jul 2009 15:08:43 PDT

A 24-h treatment with the cytokine tumor necrosis factor- (TNF-) suppresses transcription of E-box—driven clock genes (D-site albumin promoter binding protein, Dbp; Tyrotroph embryonic factor, Tef ; Hepatic leukemia factor, Hlf; Period homolog to Drosophila 1/2/3, Per1, Per2, and Per3) by yet unknown molecular mechanisms. The attenuation of clock genes has been suggested as a putative cause for the development of sickness behavior syndrome in infectious and autoimmune diseases. Here, the authors studied the effect of TNF- at early time points (<3 h) on intracellular signaling events and clock gene expression in fibroblasts. Interaction of TNF- with TNFR1 (Tnfrsf1a , CD120a, p55), but not TNFR2 (Tnfrsf1b, CD120b , p75), leads to fast downregulation of gene expression of Dbp and upregulation of negative regulators of the molecular clock, Per1 and Per2, Cryptochrome-1 (Cry1), and Differentiated embryo chondrocytes-1 (Dec1). Since the decrease of Dbp is also observed in cells deficient for Per1/Per2, Cry1/Cry2 , or Dec1, these genes are unlikely to be responsible for inhibition of Dbp. The early effect of TNF- on the clock gene Per1 is dependent on p38, mitogen-activated protein kinase (MAPK), and/or calcium signaling, whereas the effect on Dbp is independent of p38 MAPK, but also involves calcium signaling. Both genes remain unaffected by the NF-B and AP-1 pathway. Taken collectively these data show p38 MAPK- and calcium-dependent TNFR1-mediated transient increase of the negative regulator Per1 and an independent decrease of Dbp.

Metabolic Rhythms of the Cyanobacterium Cyanothece sp. ATCC 51142 Correlate with Modeled Dynamics of Circadian Clock

Cerveny, J., Nedbal, L. — Wed, 22 Jul 2009 15:08:43 PDT

These experiments aim to reveal the dynamic features that occur during the metabolism of the unicellular, nitrogen fixing cyanobacterium Cyanothece sp. when exposed to diverse circadian forcing patterns (LD 16:8, LD 12:12, LD 8:16, LD 6:6). The chlorophyll concentration grew rapidly from subjective morning when first illuminated to around noon, then remained stable from later in the afternoon and throughout the night. The optical density measured at 735 nm was stable during the morning chlorophyll accumulation, then increased in the early afternoon toward a peak, followed at dusk by a rapid decline toward the late night steady state. The authors propose that these dynamics largely reflect accumulation and subsequent consumption of glycogen granules. This hypothesis is consistent with the sharp peak of respiration that coincides with the putative hydrocarbon catabolism. In the long-day regimen (LD 16:8), these events may mark the transition from the aerobic photosynthetic metabolism to microaerobic nitrogen metabolism that occurs at dusk, and thus cannot be triggered by the darkness that comes later. Rather, control is likely to originate in the circadian clock signaling an approaching night. To explore the dynamics of the link between respiration and circadian oscillations, the authors extrapolated an earlier model of the KaiABC oscillator from Synechococcus elongatus to Cyanothece sp. The measured peak of respiratory activity at dusk correlated strongly in its timing and time width with the modeled peak in accumulation of the KaiB₄ complex, which marks the late afternoon phase of the circadian clock. The authors propose a hypothesis that high levels of KaiB₄ (or of its Cyanothece sp. analog) trigger the glycogen catabolism that is reflected in the experiments in the respiratory peak. The degree of the correlation between the modeled KaiB₄ dynamics and the dynamics of experimentally measured peaks of respiratory activity was further tested during the half-circadian regimen (LD 6:6). The model predicted an irregular pattern of the KaiABC oscillator, quite unlike mechanical or electrical clock pacemakers that are strongly damped when driven at double their endogenous frequency. This highly unusual dynamic pattern was confirmed experimentally, supporting strongly the validity of the circadian model and of the proposed direct link to respiration.

Chronic Ethanol Intake Alters Circadian Phase Shifting and Free-Running Period in Mice

Seggio, J. A., Fixaris, M. C., Reed, J. D., Logan, R. W., Rosenwasser, A. M. — Wed, 22 Jul 2009 15:08:43 PDT

Chronic alcohol intake is associated with widespread disruptions in sleep and circadian rhythms in both human alcoholics and in experimental animals. Recent studies have demonstrated that chronic and acute ethanol treatments alter fundamental properties of the circadian pacemaker—including free-running period and responsiveness to photic and nonphotic phase-shifting stimuli—in rats and hamsters. In the present work, the authors extend these observations to the C57BL/6J mouse, an inbred strain characterized by very high levels of voluntary ethanol intake and by reliable and stable free-running circadian activity rhythms. Mice were housed individually in running-wheel cages under conditions of either voluntary or forced ethanol intake, whereas controls were maintained on plain water. Forced ethanol intake significantly attenuated photic phase delays (but not phase advances) and shortened free-running period in constant darkness, but voluntary ethanol intake failed to affect either of these parameters. Thus, high levels of chronic ethanol intake, beyond those normally achieved under voluntary drinking conditions, are required to alter fundamental circadian pacemaker properties in C57BL/6J mice. These observations may be related to the relative ethanol insensitivity displayed by this strain in several other phenotypic domains, including ethanol-induced sedation, ataxia, and withdrawal. Additional experiments will investigate chronobiological sensitivity to ethanol in a range of inbred strains showing diverse ethanol-related phenotypes.

Sleep Timing and Circadian Phase in Delayed Sleep Phase Syndrome

Chang, A.-M., Reid, K. J., Gourineni, R., Zee, P. C. — Wed, 22 Jul 2009 15:08:43 PDT

Delayed sleep phase syndrome (DSPS) is a circadian rhythm sleep disorder in which the timing of the sleep episode occurs later than desired and is associated with difficulty falling asleep, problems awakening on time (e.g., to meet work or school obligations), and daytime sleepiness. The phase relationship between the timing of sleep and endogenous circadian rhythms is critical to the initiation and maintenance of sleep, and significant alteration leads to impairment of sleep quality and duration. The aim of this retrospective study was to determine the phase relationship between sleep-wake times and physiological markers of circadian timing in clinic patients with DSPS. Objective and subjective measures of sleep timing and circadian phase markers (core body temperature and melatonin) were measured in patients with DSPS and compared with age-matched controls. As expected, significant delays in the timing of the major sleep episode and circadian phase of body temperature and melatonin rhythms were seen in the DSPS group when allowed to sleep at their own habitual schedules, but the phase relationship between sleep-wake times and circadian phase was similar between the 2 groups. These results suggest that the symptoms of insomnia and excessive daytime sleepiness in DSPS patients living under entrained real-life conditions cannot be explained by an alteration in the phase relationship between sleep-wake patterns and other physiological circadian rhythms.

Clustering Predicted by an Electrophysiological Model of the Suprachiasmatic Nucleus

Diekman, C. O., Forger, D. B. — Wed, 22 Jul 2009 15:08:43 PDT

Despite the wealth of experimental data on the electrophysiology of individual neurons in the suprachiasmatic nuclei (SCN), the neural code of the SCN remains largely unknown. To predict the electrical activity of the SCN, the authors simulated networks of 10,000 GABAergic SCN neurons using a detailed model of the ionic currents within SCN neurons. Their goal was to understand how neuronal firing, which occurs on a time scale faster than a second, can encode a set phase of the circadian (24-h) cycle. The authors studied the effects of key network properties including: 1) the synaptic density within the SCN, 2) the magnitude of postsynaptic currents, 3) the heterogeneity of circadian phase in the neuronal population, 4) the degree of synaptic noise, and 5) the balance between excitation and inhibition. Their main result was that under a wide variety of conditions, the SCN network spontaneously organized into (typically 3) groups of synchronously firing neurons. They showed that this type of clustering can lead to the silencing of neurons whose intracellular clocks are out of circadian phase with the rest of the population. Their results provide clues to how the SCN may generate a coherent electrical output signal at the tissue level to time rhythms throughout the body.

The Evolution of the Cyanobacterial Posttranslational Clock from a Primitive "Phoscillator"

Simons, M. J.P. — Fri, 22 May 2009 14:01:21 PDT

Cyanobacteria were among the 1st organisms to evolve on earth. The molecular circadian clock proteins of cyanobacteria and their phylogenetics have recently been elucidated. This allows for a conjecture on the evolution of 1 of the 1st circadian clocks. A scenario has now been created by combining known in vitro and in vivo properties of the 3 clock proteins of cyanobacteria (KaiA, KaiB, and KaiC). This scenario describes the evolution of the cyanobacterial clock in gradual steps: evolving from a masking mechanism, toward an hourglass, into a clock.

HSP90, a Capacitor of Behavioral Variation

Hung, H.-C., Kay, S. A., Weber, F. — Fri, 22 May 2009 14:01:21 PDT

Many aspects of behavior such as aggression, courtship, sexual orientation, and the sleep-wake cycle are determined by specific genes. Although point mutations in these genes predictably change characteristics of behavior, substantial variation can be observed among a population as well as during the lifetime of individuals. The origin of variation in behavior, however, is largely unknown. Here the authors investigated the role of HSP90 for the circadian control of behavior in Drosophila. They found that a partial loss of HSP90 function, either by mutagenesis or by pharmacological inhibition, did not affect the circadian clock itself, but the translation of molecular oscillations into behavioral rhythms. In HSP90-deficient flies behavioral activity was no longer stringently coupled to molecular oscillations giving rise to a large variation in individual behavioral activity patterns. The results show that HSP90 is a potent capacitor of behavioral variation, analogous to its role in morphology. Decreased HSP90 activity not only increases behavioral variability among a population, but interestingly also during the lifetime of individuals.

A Phylogenetically Conserved DNA Damage Response Resets the Circadian Clock

Gamsby, J. J., Loros, J. J., Dunlap, J. C. — Fri, 22 May 2009 14:01:21 PDT

The mammalian circadian clock influences the timing of many biological processes such as the sleep/wake cycle, metabolism, and cell division. Environmental cues such as light exposure can influence the timing of this system through the posttranslational modification of key components of the core molecular oscillator. We have previously shown that DNA damage can reset the circadian clock in a time-of-day—dependent manner in the filamentous fungus Neurospora crassa through the modulation of negative regulator FREQUENCY levels by PRD-4 (homologue of mammalian Chk2). We show that DNA damage, generated with either the radiomimetic drug methyl methane sulfonate or UV irradiation, in mouse embryonic fibroblasts isolated from PER2::LUC transgenic mice or in the NIH3T3 cell line, elicits similar responses. In addition to induction of phase advances, DNA damage caused a decrease in luciferase signal in PER2::LUC mouse embryonic fibroblast cells that is indicative of PER2 degradation. Finally, we show that the activity of the BMAL1 promoter is enhanced during DNA damage. These findings provide further evidence that the DNA damage-mediated response of the clock is conserved from lower eukaryotes to mammals.

Ryanodine-Sensitive Intracellular Ca2+ Channels in Rat Suprachiasmatic Nuclei Are Required for Circadian Clock Control of Behavior

Fri, 22 May 2009 14:01:21 PDT

Electrophysiological and calcium mobilization experiments have suggested that the intracellular calcium release channel ryanodine receptors (RyRs) are involved in the circadian rhythmicity of the suprachiasmatic nucleus (SCN). In the present report the authors provide behavioral evidence that RyRs play a specific and major role in the output of the molecular circadian clock in SCN neurons. They measured the circadian rhythm of drinking and locomotor behaviors in dim red light before, during, and after administration of an activator (ryanodine 0.1 µM) or an inhibitor (ryanodine 100 µM) of the RyRs. Drugs were delivered directly into the SCN by cannulas connected to osmotic minipumps. Control treatments included administration of artificial cerebrospinal fluid, KCl (20 mM), tetrodotoxin (1 µM), and anysomicin (5 µg/µl). Activation of RyRs induced a significant shortening of the endogenous period, whereas inhibition of these Ca²⁺ release channels disrupted the circadian rhythmicity. After the pharmacological treatments the period of rhythmicity returned to basal values and the phase of activity onset was predicted from a line projected from the activity onset of basal recordings. These results indicate that changes in overt rhythms induced by both doses of ryanodine did not involve an alteration in the clock mechanism. The authors conclude that circadian modulation of RyRs is a key element of the output pathway from the molecular circadian clock in SCN neurons in rats.

Chronotype Influences Diurnal Variations in the Excitability of the Human Motor Cortex and the Ability to Generate Torque during a Maximum Voluntary Contraction

Tamm, A. S., Lagerquist, O., Ley, A. L., Collins, D. F. — Fri, 22 May 2009 14:01:21 PDT

The ability to generate torque during a maximum voluntary contraction (MVC) changes over the day. The present experiments were designed to determine the influence of an individual's chronotype on this diurnal rhythm and on cortical, spinal, and peripheral mechanisms that may be related to torque production. After completing a questionnaire to determine chronotype, 18 subjects (9 morning people, 9 evening people) participated in 4 data collection sessions (at 09:00, 13:00, 17:00, and 21:00) over 1 day. We used magnetic stimulation of the cortex, electrical stimulation of the tibial nerve, electromyographic (EMG) recordings of muscle activity, and isometric torque measurements to evaluate the excitability of the motor cortex, the spinal cord, and the torque-generating capacity of the triceps surae (TS) muscles. We found that for morning people, cortical excitability was highest at 09:00, spinal excitability was highest at 21:00, and there were no significant differences in TS EMG or torque produced during MVCs over the day. In contrast, evening people showed parallel increases in cortical and spinal excitability over the day, and these were associated with increased TS EMG and MVC torque. There were no differences at the level of the muscle over the day between morning and evening people. We propose that the simultaneous increases in cortical and spinal excitability increased central nervous system drive to the muscles of evening people, thus increasing torque production over the day. These differences in cortical excitability and performance of a motor task between morning and evening people have implications for maximizing human performance and highlight the influence of chronotype on an individual's diurnal rhythms.

Under Cover of Darkness: Nocturnal Life of Diurnal Birds

Mukhin, A., Grinkevich, V., Helm, B. — Fri, 22 May 2009 14:01:21 PDT

Songbirds are generally considered diurnal, although many species show periodic nocturnal activity during migration seasons. From a breeding-range perspective, such migratory species appear to be diurnal because they are observed to nest and feed their young during the day. But are they really exclusively diurnal? The authors tested how a passerine long-distance migrant, the Eurasian reed warbler, schedules movements during the breeding period by tracking birds in 2 experimental situations: 1) Birds experienced simulated nest loss and were monitored during their search for alternative locations, and 2) birds were translocated to reed beds at distances from 2 to 21 km and tracked during homing. The simulated unpredictable events disrupted normal breeding, forced birds to move over relatively long distances, and triggered rapid change in diel activity. In all but 1 case, birds resorted to nocturnality to find their way home and to search for new places to breed. Nocturnality during the breeding season indicates that songbird schedules are far more flexible than previously assumed. The reasons for nocturnal movements are poorly understood. Among the presumed advantages, the reduced predation pressure at night stands out because it is advantageous for movements on local as well as global scales. Predation may be particularly relevant for inhabitants of fragmented habitats, which encounter unfavorable conditions when crossing gaps in their preferred habitat. Therefore, similar selection pressures around the year may have favored the evolution of a general circadian mechanism for switches to nocturnality. Furthermore, the novel finding of homing and dispersal at night may give leads toward understanding the still enigmatic navigational abilities of songbirds.

Subjective Well-Being Is Modulated by Circadian Phase, Sleep Pressure, Age, and Gender

Fri, 22 May 2009 14:01:21 PDT

Subjective well-being largely depends on mood, which shows circadian rhythmicity and can be linked to rhythms in many physiological circadian markers, such as melatonin and cortisol. In healthy young volunteers mood is influenced by an interaction of circadian phase and the duration of time awake. The authors analyzed this interaction under differential sleep pressure conditions to investigate age and gender effects on subjective well-being. Sixteen healthy young (8 women, 8 men; 20-35 years) and 16 older volunteers (8 women, 8 men; 55-75 years) underwent a 40-h sleep deprivation (high sleep pressure) and a 40-h nap protocol (low sleep pressure) in a balanced crossover design under constant routine conditions. Mood, tension, and physical comfort were assessed by visual analogue scales during scheduled wakefulness, and their average formed a composite score of well-being. Significant variations in well-being were determined by the factors "age," "sleep pressure," and "circadian phase." Well-being was generally worse under high than low sleep pressure. Older volunteers felt significantly worse than the young under both experimental conditions. Significant interactions were found between "sleep pressure" and "age," and between "sleep pressure" and "gender." This indicated that older volunteers and women responded with a greater impairment in well-being under high compared with low sleep pressure. The time course of well-being displayed a significant circadian modulation, particularly in women under high sleep pressure conditions. The results demonstrate age- and/or gender-related modifications of well-being related to sleep deprivation and circadian phase and thus point to specific biological components of mood vulnerability.

Small-World Network Models of Intercellular Coupling Predict Enhanced Synchronization in the Suprachiasmatic Nucleus

Vasalou, C., Herzog, E. D., Henson, M. A. — Fri, 22 May 2009 14:01:21 PDT

The suprachiasmatic nucleus (SCN) of the hypothalamus is a multioscillator system that drives daily rhythms in mammalian behavior and physiology. Based on recent data implicating vasoactive intestinal polypeptide (VIP) as the key intercellular synchronizing agent, we developed a multicellular SCN model to investigate the effects of cellular heterogeneity and intercellular connectivity on circadian behavior. A 2-dimensional grid was populated with 400 model cells that were heterogeneous with respect to their uncoupled rhythmic behavior (intrinsic and damped pacemakers with a range of oscillation periods) and VIP release characteristics (VIP producers and nonproducers). We constructed small-world network architectures in which local connections between VIP producing cells and their 4 nearest neighbors were augmented with random connections, resulting in long-range coupling across the grid. With only 10% of the total possible connections, the small-world network model was able to produce similar phase synchronization indices as a mean-field model with VIP producing cells connected to all other cells. Partial removal of random connections decreased the synchrony among neurons, the amplitude of VIP and cAMP response element binding protein oscillations, the mean period of intrinsic periods across the population, and the percentage of oscillating cells. These results indicate that small-world connectivity provides the optimal compromise between the number of connections and control of circadian amplitude and synchrony. This model predicts that small decreases in long-range VIP connections in the SCN could have dramatic effects on period and amplitude of daily rhythms, features commonly described with aging.