Journal of Biological Rhythms

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.

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.

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.

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.

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.

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 (α), as well as free-running period (τ) 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.

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.

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.