Loss of dexras1 Alters Nonphotic Circadian Phase Shifts and Reveals a Role for the Intergeniculate Leaflet (IGL) in Gene-Targeted Mice

Loss of Dexras1 in gene-targeted mice impairs circadian entrainment to light cycles and produces complex changes to phase-dependent resetting responses (phase shifts) to light. The authors now describe greatly enhanced and phase-specific nonphotic responses induced by arousal in dexras1^?/? mice. In constant conditions, mutant mice exhibited significant arousal-induced phase shifts throughout the subjective day. Unusual phase advances in the late subjective night were also produced when arousal has little effect in mice. Bilateral lesions of the intergeniculate leaflet (IGL) completely eliminated both the nonphotic as well as the light-induced phase shifts of circadian locomotor rhythms during the subjective day, but had no effect on nighttime phase shifts. The expression of FOS-like protein in the suprachiasmatic nucleus (SCN) was not affected by either photic or nonphotic stimulation in the subjective day in either genotype. Therefore, the loss of Dexras1 (1) enhances nonphotic phase shifts in a phase-dependent manner, and (2) demonstrates that the IGL in mice is a primary mediator of circadian phase-resetting responses to both photic and nonphotic events during the subjective day, but plays a different functional role in the subjective night. Furthermore, (3) the change in FOS level does not appear to be a critical step in the entrainment pathways for either light or arousal during the subjective day. The cumulative evidence suggests that Dexras1 regulates multiple photic and nonphotic signal-transduction pathways, thereby playing an essential role modulating species-specific characteristics of circadian entrainment. (Author correspondence: ralph@psych.utoronto.ca)     

PHOTIC INDUCTION OF Fos IN THE SUPRACHIASMATIC NUCLEUS OF AFRICAN MOLE-RATS: RESPONSES TO INCREASING IRRADIANCE

African mole-rats (family Bathyergidae) are strictly subterranean rodent species that are rarely exposed to environmental light. Morphological and physiological adaptations to the underground environment include a severely reduced eye size and regressed visual system. Responses of the circadian system to light, however, appear to be intact, since mole-rats are able to entrain their circadian activity rhythms to the light-dark cycle and light induces Fos expression in the suprachiasmatic nucleus (SCN). Social organization varies from solitary species to highly elaborated eusocial structures, characterized by a distinct division of labor and in which one reproductive female regulates the behavior and reproductive physiology of other individuals in the colony. The authors studied light-induced Fos expression in the SCN to increasing light intensities in four mole-rat species, ranging from strictly solitary to highly social. In the solitary Cape mole-rat, light induces significant Fos expression in the SCN, and the number of Fos-immunopositive cells increases with increasing light intensity. In contrast, Fos induction in the SCN of social species was slightly greater than, but not statistically different from, the dark-control animals as is typical of most rodents. One species showed a trend for an increase in expression with increased light, whereas a second species showed no trend in expression. In the naked mole-rat, Fos expression appeared higher in the dark-controls than in the animals exposed to light, although the differences in Fos expression were not significant. These results suggest a gradient in the sensitivity of the circadian system to light in mole-rats, with a higher percentage of individuals that are unresponsive to light in correlation with the degree of sociality. In highly social species, such as the naked mole-rat that live in a relatively stable subterranean milieu in terms of food availability, temperature, constant darkness, and devoid of 24-h cyclic environmental cues, the temporal coordination of rest-wake activities may be dependent on social interactions and social status rather than on photic regulation of the circadian timing system. (Author correspondence: moosthuizen@zoology.up.ac.za)     

LACK OF CALBINDIN-D28K ALTERS RESPONSE OF THE MURINE CIRCADIAN CLOCK TO LIGHT

A strong stimulus adjusting the circadian clock to the prevailing light-dark cycle is light. However, the circadian clock is reset by light only at specific times of the day. The mechanisms mediating such gating of light input to the CNS are not well understood. There is evidence that Ca²⁺ ions play an important role in intracellular signaling mechanisms, including signaling cascades stimulated by light. Therefore, Ca²⁺ is hypothesized to play a role in the light-mediated resetting of the circadian clock. Calbindin-D28k (CB; gene symbol: Calb1) is a Ca²⁺ binding protein implicated in Ca²⁺ homeostasis and sensing. The absence of this protein influences Ca²⁺ buffering capacity of a cell, alters spatio-temporal aspects of intracellular Ca²⁺ signaling, and hence might alter transmission of light information to the circadian clock in neurons of the suprachiasmatic nuclei (SCN). We tested mice lacking a functional Calb1 gene (Calb1^?/?) and found an increased phase-delay response to light applied at circadian time (CT) 14 in these animals. This is accompanied by elevated induction of Per2 gene expression in the SCN. Period length and circadian rhythmicity were comparable between Calb1^?/? and wild-type animals. Our findings indicate an involvement of CB in the signaling pathway that modulates the behavioral and molecular response to light. (Author correspondence: urs.albrecht@unifr.ch)     

EARLY PROGRAMMING OF ASTROCYTE ORGANIZATION IN THE MOUSE SUPRACHIASMATIC NUCLEI BY LIGHT

The principal pacemaker in mammals, controlling physiology and behavior, is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Early photic experience has long-term effects on the animal's rhythmic behavior, as indicated by alterations in the phase shift induced by a light pulse, and in the expression of the circadian rhythm of locomotor activity under light-dark (LD), constant light (LL), and constant darkness (DD) environments. However, the brain substrates targeted by early light have not yet been identified. Possible candidates are astrocytes, as they develop postnatally in parallel to the circadian system, and are involved in SCN function by modulating intercellular communication and mediating photic input. Here, we reared three groups of mice under different light environments (LD, LL, and DD) during the suckling period. Later on, all mice were entrained to LD, and we determined associated astrocytic modifications by examining the expression of glial fibrillary acidic protein (GFAP) in the SCN. We observed that although LL-reared mice showed lowest GFAP expression in the SCN, as determined by quantification of immunostaining levels, the number of GFAP-positive cells was highest in this group, suggesting structural remodelling of SCN astrocytes by early light experience. These results indicate the postnatal light environment has long-term effects on the astrocytic population of the SCN. We argue that these neurochemical and structural alterations may affect clock function, which may in turn modify animal behavior (Author correspondence: maria.canal@manchester.ac.uk, Jose.Rodriguez-arellano@manchester.ac.uk).     

The cell adhesion molecule EphA4 is involved in circadian clock functions

Circadian (～24 h) rhythms of cellular network plasticity in the central circadian clock, the suprachiasmatic nucleus (SCN), have been described. The neuronal network in the SCN regulates photic resetting of the circadian clock as well as stability of the circadian system during both entrained and constant conditions. EphA4, a cell adhesion molecule regulating synaptic plasticity by controlling connections of neurons and astrocytes, is expressed in the SCN. To address whether EphA4 plays a role in circadian photoreception and influences the neuronal network of the SCN, we have analyzed circadian wheel‐running behavior of EphA4 knockout (EphA4^?/?) mice under different light conditions and upon photic resetting, as well as their light‐induced protein response in the SCN. EphA4^?/? mice exhibited reduced wheel‐running activity, longer endogenous periods under constant darkness and shorter periods under constant light conditions, suggesting an effect of EphA4 on SCN function. Moreover, EphA4^?/? mice exhibited suppressed phase delays of their wheel‐running activity following a light pulse during the beginning of the subjective night (CT15). Accordingly, light‐induced c‐FOS (FBJ murine osteosarcoma viral oncogene homolog) expression was diminished. Our results suggest a circadian role for EphA4 in the SCN neuronal network, affecting the circadian system and contributing to the circadian response to light.     

CONES ARE REQUIRED FOR NORMAL TEMPORAL RESPONSES TO LIGHT OF PHASE SHIFTS AND CLOCK GENE EXPRESSION

In mammals, non-visual responses to light involve intrinsically photosensitive melanopsin-expressing retinal ganglion cells (ipRGCs) that receive synaptic inputs from rod and cone photoreceptors. Several studies have shown that cones also play a role in light entrainment, photic responses of the suprachiasmatic nucleus (SCN), pupil constriction, and sleep induction. These studies suggest that cones are mainly involved in the initial response to light, whereas melanopsin provides a sustained input for non-visual responses during continued light exposure. Based on this idea, we explored the effects of the absence of middle-wavelength (MW)-cones on the temporal responses of circadian behavior and clock gene expression in light. In mice lacking MW-cones, our results show a reduction in behavioral phase shifts in response to light stimulations of short duration at 480 and 530?nm, but no alteration for short-wavelength (360-nm) light exposures. Similarly, induction of the period gene mPer1 and mPer2 mRNAs in the SCN are attenuated in response to light exposures of mid to long wavelengths. Modeling of the photoresponses shows that mice lacking MW-cones have an overall reduction in sensitivity that increases with longer wavelengths. The differences in photic responsiveness are consistent with the idea that cones provide a strong initial phasic input to the circadian system at light-onset and may confer a priming effect on ipRGC responses to sub-threshold light exposures. In summary, the contribution of MW-cones is essential for the normal expression of phase shifts and clock gene induction by light in mammals. (Author correspondence: ouria.benyahya@inserm.fr)     

Behavioral Responses to Combinations of Timed Light,Food Availability,and Ultradian Rhythms in the Common Vole (Microtus arvalis)

Light is the main entraining signal of the central circadian clock, which drives circadian organization of activity. When food is made available during only certain parts of the day, it can entrain the clock in the liver without changing the phase of the central circadian clock. Although a hallmark of food entrainment is a behavioral anticipation of food availability, the extent of behavioral alterations in response to food availability has not been fully characterized. The authors have investigated interactions between light and temporal food availability in the timing of activity in the common vole. Temporally restricted food availability enhanced or attenuated re-entrainment to a phase advance in light entrainment when it was shifted together with the light or remained at the same time of day, respectively. When light-entrained behavior was challenged with temporal food availability cycles with a different period, two distinct activity components were observed. More so, the present data indicate that in the presence of cycles of different period length of food and light, an activity component emerged that appeared to be driven by a free-running (light-entrainable) clock. Because the authors have previously shown that in the common vole altering activity through running-wheel availability can alter the effectiveness of food availability to entrain the clock in the liver, the authors included running-wheel availability as a parameter that alters the circadian/ultradian balance in activity. In the current protocols, running-wheel availability enhanced the entraining potential of both light and food availability in a differential way. The data presented here show that in the vole activity is a complex of individually driven components and that this activity is, itself, an important modulator of the effectiveness of entraining signals such as light and food. (Author correspondence: vanderveen@nd.edu)     

PHASE AND PERIOD RESPONSES OF THE JERBOA JACULUS ORIENTALIS TO SHORT LIGHT PULSES

The phase and period responses to short light pulses were studied in the jerboa, a seasonal, hibernating, nocturnal rodent from the Atlas region in Morocco. The jerboa, which is a saltatory species, showed precise activity onsets and offsets under a light-dark (LD) cycle using infrared captors to record locomotor activity. When released into constant darkness (DD), the majority of animals showed a circadian period (τ) <24?h (mean τ?=?23.89?±?0.13?h) and a lengthening of the activity span, α. Animals were subsequently exposed to up to eight 15-min light pulses, each separated by at least 2 wks, for up to 160 days in DD. During this span, most individuals maintained robust circadian rhythmicity, with clearly defined activity onsets and offsets, similar levels of total activity, duration of α, and percent activity occurring during the subjective night. The phase response curve (PRC) is typical of other nocturnal rodents, with light eliciting delays during late subjective day and early subjective night (CT8–CT19) and advances during late subjective night to early subjective day (CT19–CT2). A dead zone, when light had no effect on phase, is observed during mid-subjective day (CT3–CT8). A few individuals showed large (>9?h) Type 0 phase resetting near the singularity region (CT19) that resulted in a complete phase reversal, but otherwise displayed normal phase-shifting responses at other CT times. The τ response curve showed a decrease in period from early to late subjective night with increases at other times, but these changes were small (maximum <9?min) and highly variable. There was a distinct tendency for animals that had an initial short τ in DD to conserve a short τ during the series of light pulses and, inversely, for animals with long τ to conserve a long τ. This suggests possible constraints on the plasticity of variation of τ in relation to the endogenous period of the animal. (Author correspondence: howard.cooper@inserm.fr)     

The Endogenous Melatonin (MT) Signal Facilitates Reentrainment of the Circadian System to Light-Induced Phase Advances by Acting Upon MT2 Receptors

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors. (Author correspondence: M.Pfeffer@em.uni-frankfurt.de)     

Role of Proinflammatory Cytokines on Lipopolysaccharide-Induced Phase Shifts in Locomotor Activity Circadian Rhythm

We previously reported that early night peripheral bacterial lipopolysaccharide (LPS) injection produces phase delays in the circadian rhythm of locomotor activity in mice. We now assess the effects of proinflammatory cytokines on circadian physiology, including their role in LPS-induced phase shifts. First, we investigated whether differential systemic induction of classic proinflammatory cytokines could explain the time-specific behavioral effects of peripheral LPS. Induction levels for plasma interleukin (IL)-1α, IL-1β, IL-6, or tumor necrosis factor (TNF)-α did not differ between animals receiving a LPS challenge in the early day or early night. We next tested the in vivo effects of central proinflammatory cytokines on circadian physiology. We found that intracerebroventricular (i.c.v.) delivery of TNF-α or interleukin IL-1β induced phase delays on wheel-running activity rhythms. Furthermore, we analyzed if these cytokines mediate the LPS-induced phase shifts and found that i.c.v. administration of soluble TNF-α receptor (but not an IL-1β antagonistic) prior to LPS stimulation inhibited the phase delays. Our work suggests that the suprachiasmatic nucleus (SCN) responds to central proinflammatory cytokines in vivo, producing phase shifts in locomotor activity rhythms. Moreover, we show that the LPS-induced phase delays are mediated through the action of TNF-α at the central level, and that systemic induction of proinflammatory cytokines might be necessary, but not sufficient, for this behavioral outcome. (Author correspondence: dgolombek@unq.edu.ar)     

Signaling in the mammalian circadian clock: the NO/cGMP pathway

Mammalian circadian rhythms are generated by a hypothalamic suprachiasmatic nuclei (SCN) clock. Light pulses synchronize body rhythms by inducing phase delays during the early night and phase advances during the late night. Phosphorylation events are known to be involved in circadian phase shifting, both for delays and advances. Pharmacological inhibition of the cGMP-dependent kinase (cGK) or Ca2+/calmodulin-dependent kinase (CaMK), or of neuronal nitric oxide synthase (nNOS) blocks the circadian responses to light in vivo. Light pulses administered during the subjective night, but not during the day, induce rapid phosphorylation of both p-CAMKII and p-nNOS (specifically phosphorylated by CaMKII). CaMKII inhibitors block light-induced nNOS activity and phosphorylation, suggesting a direct pathway between both enzymes. Furthermore, SCN cGMP exhibits diurnal and circadian rhythms with maximal values during the day or subjective day. This variation of cGMP levels appears to be related to temporal changes in phosphodiesterase (PDE) activity and not to guanylyl cyclase (GC) activity. Light pulses increase SCN cGMP levels at circadian time (CT) 18 (when light causes phase advances of rhythms) but not at CT 14 (the time for light-induced phase delays). cGK II is expressed in the hamster SCN and also exhibits circadian changes in its levels, peaking during the day. Light pulses increase cGK activity at CT 18 but not at CT 14. In addition, cGK and GC inhibition by KT-5823 and ODQ significantly attenuated light-induced phase shifts at CT 18. This inhibition did not change c-Fos expression SCN but affected the expression of the clock gene per in the SCN. These results suggest a signal transduction pathway responsible for light-induced phase advances of the circadian clock which could be summarized as follows: Glu-Ca2+-CaMKII-nNOS-GC-cGMP-cGK-->-->clock genes. This pathway offers a signaling window that allows peering into the circadian clock machinery in order to decipher its temporal cogs and wheels.     

Differential Involvement of the Suprachiasmatic Nucleus in Lipopolysaccharide-Induced Plasma Glucose and Corticosterone Responses

The hypothalamic suprachiasmatic nucleus (SCN) is an essential component of the circadian timing system, and an important determinant of neuroendocrine and metabolic regulation. Recent data indicate a modulatory role for the immune system on the circadian timing system. The authors investigated how the circadian timing system affects the hypothalamo-pituitary-adrenal (HPA) axis and glucose regulatory responses evoked by an immune challenge induced by lipopolysaccharide (LPS). LPS-induced increases in corticosterone were minimal during the trough of the daily corticosterone rhythm; in contrast, LPS effects on glucose, glucagon, and insulin did not vary across time-of-day. Complete ablation of the SCN resulted in increased corticosterone responses but did not affect LPS-induced hyperglycemia. The paraventricular nucleus (PVN) of the hypothalamus is an important neuroendocrine and autonomic output pathway for hypothalamic information, as well as one of the main target areas of the SCN. Silencing the neuronal activity in the PVN did not affect the LPS-induced corticosterone surge and only slightly delayed the LPS-induced plasma glucose and glucagon responses. Finally, surgical interruption of the neuronal connection between hypothalamus and liver did not affect the corticosterone response but slightly delayed the LPS-induced glucose response. Together, these data support the previously proposed circadian modulation of LPS-induced neuroendocrine responses, but they are at variance with the suggested major role for the hypothalamic pacemaker on the autonomic output of the hypothalamus, as reflected by the effects of LPS on glucose homeostasis. The latter effects are more likely due to direct interactions of LPS with peripheral tissues, such as the liver. (Author correspondence: a.kalsbeek@amc.uva.nl)     

Early- and Late-Emerging Drosophila melanogaster Fruit Flies Differ in Their Sensitivity to Light During Morning and Evening

The authors derived early and late populations of fruit flies showing increased incidence of emergence during morning or evening hours by imposing selection for timing of emergence under 12:12?h light/dark (LD) cycles. From previous studies, it was clear that the increased incidence of adult emergence during morning and evening hours in early and late populations was a result of evolution of divergent and characteristic emergence waveforms in these populations. Such characteristic waveforms are henceforth referred to as “evolved emergence waveforms” (EEWs). The early and late populations also evolved different circadian clocks, which is evident from the divergence in their clock period (τ) and photic phase response curve (PRC). Although correlation between emergence waveforms and clock properties suggests functional significance of circadian clocks, τ and PRCs do not satisfactorily explain the early and late emergence phenotypes. In order to understand the functional significance of the PRC for early and late emergence phenotypes, the authors investigated whether circadian clocks of these flies exhibit any difference in photosensitivity under entrained conditions. Such differences would suggest that the light requirement for circadian entrainment of the emergence rhythm in early and late populations is different. To test this, they examined if early and late flies differ in their light utilization behavior, first by assaying their emergence rhythm under complete photoperiod and then in three different skeleton photoperiods. The results showed that early and late populations require different durations of light during the morning and evening to achieve their EEWs, suggesting that for the circadian entrainment of the emergence rhythm, early and late flies utilize light from different parts of the day. (Author correspondence: vsharma@jncasr.ac.in or vksharmas@gmail.com)     

Circadian Modulation of Acute Alcohol Sensitivity But Not Acute Tolerance in Drosophila

An increased understanding of the factors affecting behavioral and neurological responses to alcohol and alcohol physiology is necessary given the tremendous toll alcohol abuse and alcoholism exert on individuals and society. At the behavioral and molecular levels, the response to alcohol appears remarkably conserved from Drosophila to humans, suggesting that investigations across model species can provide insight into the identification of common modulatory factors. We investigated the interaction between the circadian clock and alcohol sensitivity, alcohol tolerance, and alcohol absorbance in Drosophila melanogaster. Using a loss-of-righting reflex (LoRR) assay, we found that flies exhibit a circadian rhythm in the LoRR, with the greatest sensitivity to alcohol occurring from mid to late night, corresponding to the flies' inactive phase. As predicted, a circadian rhythm in the LoRR was absent in circadian mutant flies and under conditions in which the circadian clock was nonfunctional. Circadian modulation of the response to alcohol was not due to circadian regulation of alcohol absorbance. Similar to other animals, Drosophila develop acute and chronic tolerance to alcohol upon repeat exposures. We found that the circadian clock did not modulate the development of acute alcohol tolerance measured as the difference in sensitivity to alcohol between naïve and pre-exposed flies. Thus, the circadian clock modulates some, but not all, of the behavioral responses to alcohol exposure, suggesting that specific mechanisms underlie the observed circadian modulation of LoRR rather than global cellular circadian regulation. This study provides valuable new insights in our understanding of the circadian modulation of alcohol-induced behaviors that ultimately could facilitate preventative measures in combating alcohol abuse and alcoholism. (Author correspondence: lyons@bio.fsu.edu)     

Validating the Use of Wrist-Level Light Monitoring for In-Hospital Circadian Studies

This clinical methods comparison study describes the difference between light levels measured at the wrist (Actiwatch-L) and at the eye (Daysimeter) in a postoperative in-patient population. The mean difference between the two devices was less than 10 lux at light levels less than 5000 lux. Agreement between the devices was found to decrease as eye-level light exposure increased. Measurements at eye level of 5000 lux or more corresponded to a difference between the devices of greater than 100 lux. Agreement between the eye- and wrist-level light measurements also appears to be influenced by time of day. During the day, the measurement differences were on average 50 lux higher at eye level, whereas at night they were on average 50 lux lower. Although the wrist-level monitor was found to underestimate light exposure at higher light levels, it was well tolerated by participants in the clinical setting. In contrast, the eye-level monitor was cumbersome and uncomfortable for the patients to wear. This study provides light-exposure data on patients in real conditions in the clinical environment. The results show that wrist-level monitoring provides an adequate estimate of light exposure for in-hospital circadian studies. (Author correspondence: g.warman@auckland.ac.nz)     

A Honey Bee (Apis mellifera) Light Phase Response Curve

The authors report a phase response curve (PRC) for individual honey bees (Apis mellifera) to single 1-h light pulses (1000 lux) using an Aschoff Type 1 protocol (n?=?134). The bee PRC is a weak (Type 1) PRC with a maximum advance of 1.5?h between circadian time (CT) 18 and 3 and a maximum delay of 1.5?h between CT 12 and 18. This is the first published honey bee light PRC and provides an important resource for chronobiologists and honey bee researchers. It may also have practical applications for what is an economically important species frequently transported across different time zones. (Author correspondence: G.warman@auckland.ac.nz)     

Factors Associated With Shift Work Disorder in Nurses Working With Rapid-Rotation Schedules in Japan: The Nurses’ Sleep Health Project

Workers who meet the criteria for shift work disorder (SWD) have elevated levels of risk for various health and behavioral problems. However, the impact of having SWD on shiftworkers engaged in rapid-rotation schedules is unknown. Moreover, the risk factors for the occurrence of SWD remain unclear. To clarify these issues, we conducted a questionnaire-based, cross-sectional survey on a sample of shiftworking nurses. Responses were obtained from 1202 nurses working at university hospitals in Tokyo, Japan, including 727 two-shift workers and 315 three-shift workers. The questionnaire included items relevant to age, gender, family structure, work environment, health-related quality of life (QOL), diurnal type, depressive symptoms, and SWD. Participants who reported insomnia and/or excessive sleepiness for at least 1 mo that was subjectively relevant to their shiftwork schedules were categorized as having SWD. The prevalence of SWD in the sampled shiftworking nurses was 24.4%; shiftworking nurses with SWD showed lower health-related QOL and more severe depressive symptoms, with greater rates of both actual accidents/errors and near misses, than those without SWD. The results of logistic regression analyses showed that more time spent working at night, frequent missing of nap opportunities during night work, and having an eveningness-oriented chronotype were significantly associated with SWD. The present study indicated that SWD might be associated with reduced health-related QOL and decreased work performance in shiftworking nurses on rapid-rotation schedules. The results also suggested that missing napping opportunities during night work, long nighttime working hours, and the delay of circadian rhythms are associated with the occurrence of SWD among shiftworking nurses on rapid-rotation schedules. (Author correspondence: yuinoue@tokyo-med.ac.jp)