DD Conditions Research Articles

Does Lighting Manipulation During Incubation Affect Hatching Rhythms and Early Development of Sole?

Light plays a key role in the development of biological rhythms in fish. Previous research on Senegal sole has revealed that both spawning rhythms and larval development are strongly influenced by lighting conditions. However, hatching rhythms and the effect of light during incubation are as yet unexplored. Therefore, the aim of this study was to investigate the impact of the light spectrum and photoperiod on Solea senegalensis eggs and larvae until day 7 post hatching (dph). To this end, eggs were collected immediately after spawning during the night and exposed to continuous light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white light (LDW), blue light (LDB; λpeak = 463 nm), or red light (LDR; λpeak = 685 nm). Eggs exposed to LDB had the highest hatching rate (94.5% ± 1.9%), whereas LDR and DD showed the lowest hatching rate (54.4% ± 3.9% and 48.4% ± 4.2%, respectively). Under LD conditions, the hatching rhythm peaked by the end of the dark phase, but was advanced in LDB (zeitgeber time 8 [ZT8]; ZT0 representing the onset of darkness) in relation to LDW and LDR (ZT11). Under DD conditions, the same rhythm persisted, although with lower amplitude, whereas under LL the hatching rhythm split into two peaks (ZT8 and ZT13). From dph 4 onwards, larvae under LDB showed the best growth and quickest development (advanced eye pigmentation, mouth opening, and pectoral fins), whereas larvae under LDR and DD had the poorest performance. These results reveal that developmental rhythms at the egg stage are tightly controlled by light characteristics, underlining the importance of reproducing their natural underwater photoenvironment (LD cycles of blue wavelengths) during incubation and early larvae development of fish. (Author correspondence: borja@um.es)

Variation in glutathione status associated with induction and initiation of diapause in eggs of the bivoltine strain of the silkworm Bombyx mori

For the bivoltine (Dazao) strain of the silkworm Bombyx mori L., diapause expression in progeny is induced by exposure to conditions of 25 °C and continuous illumination (LL) during the maternal generation, whereas an environment of 15 °C and constant darkness (DD) results in nondiapause progeny. Initiation of diapause in progeny can be prevented by treatment of diapause‐programmed eggs with hydrochloric acid (HCl) at approximately 24 h post‐oviposition. To investigate whether glutathione is involved in the regulation of diapause induction and initiation in this species, measurements of total glutathione, reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG ratio, glutathione S‐transferase (GST) and peroxiredoxins (Prdx) are compared in eggs incubated under LL and DD conditions, and between diapause eggs and those treated with HCl. Compared with DD, eggs incubated under LL have higher total glutathione (GSH + 2GSSG), lower GSH, higher GSSG, a lower GSH/GSSG ratio, lower GST activity and higher Prdx activity at stages 20–25 of maternal embryogenesis. The lower ratio of GSH/GSSG is indicative of pro‐oxidative conditions during diapause induction, which may result from the stronger oxidation of GSH. Compared with HCl‐treated eggs, diapause eggs have lower total glutathione, no difference in GSH, lower GSSG, a higher GSH/GSSG ratio, no difference in GST activity and lower Prdx between 36 and 72 h post‐oviposition. The higher ratio GSH/GSSG is indicative of reducing conditions during diapause initiation, which may a result of the weaker oxidation of GSH. Moreover, variations of Prdx and GST suggest that Prdx rather than GST plays an important role in the oxidation of GSH during the induction and initiation of diapause.

Circadian rhythms in metabolic variables in Caenorhabditis elegans

Circadian rhythms govern a wide variety of physiological and metabolic functions in most organisms through neural networks, hormones and gene expression. In this work, we studied the circadian variation in metabolic variables of adult C. elegans such as food consumption, pharyngeal contractions, defecation and oxygen consumption. Feeding behavior was clearly rhythmic under LD conditions, with a non-significant trend under DD conditions. In addition, a daily and circadian variation in muscle contraction of the pharynx was observed. Oxygen consumption also showed a circadian fluctuation with a maximum in the middle of the night (a peak was found around ZT18/CT18). Furthermore, defecation behavior also showed a daily variation in the N2 strain (wild type). This work demonstrates that in the adult nematode C. elegans metabolic variables vary daily. In summary, our results will allow us to take full advantage of this widely used animal model (including research in genetics, ageing and developmental biology) for studies in Chronobiology.

TEMPERATURE CYCLES TRIGGER NOCTURNALISM IN THE DIURNAL HOMEOTHERM OCTODON DEGUS

Body temperature regulation within a physiological range is a critical factor for guaranteeing the survival of living organisms. The avoidance of high ambient temperatures is a behavioral mechanism used by homeothermic animals living in extreme environmental conditions. As the circadian system is involved in these thermoregulatory responses, precise phase shifts and even complete temporal niche inversion have been reported. Octodon degus, a mainly diurnal rodent from Chile, has the ability to switch its phase preference for locomotor activity to coincide with the availability of a running wheel. The aims of this work are twofold: to determine whether ambient temperature cycles, with high values during the day and low values at night (HLTa), can induce nocturnal chronotypes in degus previously characterized as diurnal; and to learn whether HLTa cycles are able to act as a zeitgeber in this dual-phase species. To this end, degus were subjected to 24 h HLTa cycles under both 12:12 LD and DD conditions. Two experimental groups were used, one with previous wheel running experience and another naïve group, to study the influence of the thermal cycles and previous wheel running experience on the degus' dual-phasing behavior. Temperature cycles (31.3 ± 1.5°C during the day and 24.2 ± 1.6°C at night) induced a 100% nocturnalism in previously diurnal individuals. Indeed, both entrainment with nocturnal phase angle to LD and nocturnal rhythmicity induced by masking were observed. Moreover, HLTa cycles acted by masking, confining wheel-running activity to the cooler phase under DD conditions, with the naïve group being more sensitive than the experienced one. (Author correspondence: jamadrid@um.es)

Rhythmic oscillations of α-amylase protein and its enzymatic activity levels in Drosophila melanogaster (Diptera: Drosophilidae)

In this report, we show that α-amylase activity is rhythmic in the wild-type fruit fly Drosophila melanogaster, and that this rhythm exhibits the properties of a clock output. Moreover, the rhythm of amylase activity is accompanied by fluctuations in the Amy protein level under 12L : 12D conditions. A strong sexual dimorphism is evident in the oscillations of Amy protein and enzymatic activity. Under light : dark (LD) conditions on the control diet, CantonS wild-type Drosophila melanogaster exhibit a bimodal rhythm of amylase activity, particularly of the AmyD3 (Amy3) isoform, with morning and evening peaks. Under these conditions, Amy protein levels also oscillate significantly, again more strongly for the Amy3 isoform than Amy1 (Amy1). A robust oscillation of Amy3 and Amy1 activity is also observed under DD conditions for both sexes. In constant light (LL) the rhythms dampen out, particularly in the males. A high level of dietary glucose causes an overall decrease in the amplitudes of the rhythmic oscillations of amylase activity, but the processes are nevertheless rhythmic, with peak activities at Zt8 for the females, and at Zt0 for the males in LD. In constant darkness (DD) the rhythms are maintained. Mutants lacking a functioning oscillator, per01, exhibit a slight photoperiodicity in LD, with a decrease in amylase activity in both males and females during the late night in LD, but no rhythmic oscillations in DD.

Individual behavioural rhythmicity is linked to social motivation in Japanese quail

This experiment explored links between individual social traits and behavioural rhythms by comparing the feeding activity rhythms of HSR (high social reinstatement) Japanese quail that will cover long distances to reach conspecifics, to those of LSR (low social reinstatement) quail that will cover only short distances to reach conspecifics. We evaluated the functioning of their circadian and ultradian clocks under constant darkness (DD), and then under photoperiodic conditions (LD). Our results revealed that, under DD conditions, the circadian periods of HSR quail were closer to 24 h than those of LSR quail. Under LD conditions, the daily rhythm of HSR quail was clearer than that of LSR quail. HSR quail synchronized faster and better to the environmental LD cycle than LSR quail. Our results also showed that, under DD conditions, more LSR than HSR quail expressed ultradian rhythmic phenotypes and that the ultradian rhythm of LSR quail was clearer than that of LSR quail. Under LD conditions, these differences between HSR and LSR quail showed the same tendencies. LSR quail seem to present a more functional ultradian system, and thus could possibly synchronise better on an ultradian environmental cue than HSR quail. Our study demonstrated a link between individual rhythmicity and social traits at an intra-specific level. It revealed complex relationships between ultradian, circadian, and daily rhythms and level of social motivation in Japanese quail.

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

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 (alpha), as well as free-running period (tau) 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.

Synaptic vesicles in motor synapses change size and distribution during the day

The morphology of Drosophila motor terminals changes along the day with a circadian rhythm controlled by the biological clock. Here, we used electron microscopy to investigate the size, number, and distribution of synaptic vesicles, at intervals of 6 h during 2 consecutive days, under light-dark (LD) or the first 2 days in constant darkness (DD). We found changes in the size and distribution of vesicles located either at the active zone or in the reserve pool, indicating a circadian rhythm of synapse reorganization. Vesicles at the active zone were generally smaller than those in the reserve pool in LD and DD conditions. The size of active zones vesicles decreased twice in LD, corresponding with times of more intense locomotion activity, but only once in DD conditions.

Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus

In the unicellular cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock under continuous light (LL) conditions. Here, we used high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild-type, kaiABC-null, and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild-type strains, >30% of transcripts oscillated significantly in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was severely attenuated in kaiABC-null strains. Although it has been proposed that KaiC globally represses gene expression, our analysis revealed that dawn-expressed genes were up-regulated by kaiC-overexpression so that the clock was arrested at subjective dawn. Transfer of cells to DD conditions from LL immediately suppressed expression of most of the genes, while the clock kept even time in the absence of transcriptional feedback. Thus, the Synechococcus genome seems to be primarily regulated by light/dark cycles and is dramatically modified by the protein-based circadian oscillator.

Cloning and developmental expression analysis of prokineticin 2 and its receptor PKR2 in the Syrian hamster surpachiasmatic nucleus

Prokineticin 2 (PK2) and its receptor (PKR2) may play important roles in transmitting circadian rhythm information from the suprachiasmatic nucleus (SCN) to downstream neural targets. In this study, we identified PK2 and PKR2 genes in the Syrian hamster through a homologous cloning approach by performing 5′ and 3′-RACE. Sequence alignments indicate significant homology between the mouse, rat and Syrian hamster sequences. In situ hybridization experiments indicate that PK2 and PKR2 are expressed in the SCN, with the expression of PK2 with a pronounced amplitude change across the daily cycle under DD conditions. Recent studies indicate that the molecular machinery of the central clock in the suprachiasmatic nucleus only mature gradually during development. We thus studied the developmental expression of PK2 and PKR2 in the surpachiasmatic nuclei of the perinatal animals. PKR2 is expressed in the Syrian hamster SCN at all time points examined, while the expression of PK2 remains undetectable at prenatal time points. Expression of PK2 begins to be detectable on postnatal day 3 and onwards on day 5 and day 7. The developmental appearance of the PK2 expression rhythm may reflect the maturation process of the central clock's molecular machinery.

Influence of darkness on embryonic diapause termination in dormant Artemia cysts with no experience of desiccation

One of the environmental cues for Artemia embryonic diapause termination (EDT), the nature of which is barely understood, is the absence of light, although light-dark cycles (photoperiod) have been examined in inducing embryonic diapause. The influence of constant darkness (DD) on EDT was investigated in laboratory-produced cysts of Artemia franciscana. In a previous report (Nambu et al., 2008. J Exp Zool 309A:17-24 ), we described a facilitated effect of pretreatment of DD (5-14 days) on EDT in Artemia cysts exposed to a standard rearing condition. In this article, the influence of pretreatment of DD (1-150 days) was investigated in detail by bioassay of hatching. It was found that all the DD conditions significantly promoted EDT. Either the plateau value of the percent hatch increased in response to each DD condition, or the rearing period to attain the half value of the plateau value became shorter. It was also found that DD (1-100 days) led to cyclic changes of EDT: the logistic functions of hatching were cyclically regulated and 30 days-DD cycles of relative values of their parameters were observed synchronously. The effect of DD-duration was not cumulative, and a couple of optimum conditions for higher EDT levels were obtained. The probability of Artemia's encountering DD and the possible meaning of cyclic regulation of EDT by DD in Artemia are discussed.

Two Steady‐Entrainment Phases and Graded Masking Effects by Light Generate Different Circadian Chronotypes in Octodon degus

Processes involved in the operation of the circadian pacemaker are well characterized; however; little is known about what mechanisms drive the overt diurnal, nocturnal, or crepuscular behavior in a species. In this context, dual‐phasing rodents, such as Octodon degus, emerge as a useful model to decipher these keys. Two main chronotypes, nocturnal and diurnal, have been traditionally described in laboratory‐housed degus based on the percentage of activity displayed by the animals during the scotophase or photophase. However, if one considers also the entrainment phase angle during the first days following a change from LD to DD conditions, a third chronotype (intermediate)—or more properly, a continuous grading of circadian expressions between diurnal and nocturnal chronotype—can be observed. Our experiments suggest the pacemaker of the diurnal animal is entrained to the photophase, and light does not exert a negative masking effect. The pacemaker of the nocturnal degus, on the other hand, is entrained to the scotophase, and light exerts a strong negative masking effect. Finally, the intermediate chronotype is characterized by variable negative masking effect of light overlapping a pacemaker entrained to the photophase. The phase shift inversion from diurnal to nocturnal chronotype is related to the availability of a wheel in the cage, and the effect may be located downstream from the clock. However, body temperature rhythm recordings, less affected by masking effects, point to an involvement of the circadian pacemaker in chronotype differentiation, as transient entrainment cycles, and not an abrupt phase shift, were detected after providing access to the wheel. The diurnality of degus seems to be the result of a variety of mechanisms, which may explain how different processes can lead to similar chronotypes.

CLOCK Is Required for Maintaining the Circadian Rhythms of Opsin mRNA Expression in Photoreceptor Cells

In zebrafish, the expression of long-wavelength cone (LC) opsin mRNA fluctuated rhythmically between the day and night. In a 24-h period, expression was high in the afternoon and low in the early morning. This pattern of fluctuation persisted in zebrafish that were kept in constant darkness, suggesting an involvement of circadian clocks. Functional expression of Clock, a circadian clock gene that contributes to the central circadian pacemaker, was found to play an important role in maintaining the circadian rhythms of LC opsin mRNA expression. In zebrafish embryos, in which the translation of Clock was inhibited by anti-Clock morpholinos, the circadian rhythms of LC opsin mRNA expression diminished. CLOCK may regulate the circadian rhythms of LC opsin mRNA expression via cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In control retinas, the concentration of cAMP was high in the early morning and low in the remainder of the day and night. Inhibition of Clock translation abolished the fluctuation in the concentration of cAMP, thereby diminishing the circadian rhythms of opsin mRNA expression. Transient increase of cAMP concentrations in the early morning (i.e. by treating the embryos with 8-bromo-cAMP) restored the circadian rhythms of LC opsin mRNA expression in morpholino-treated embryos. Together, the data suggest that Clock plays important roles in regulating the circadian rhythms in photoreceptor cells.

Time‐of‐day differences in dopamine clearance in the rat medial prefrontal cortex and nucleus accumbens

Circadian rhythms influence cocaine-seeking behavior in rats, and this behavior may be mediated by variability in the rate of extracellular dopamine clearance across the day:night cycle. We used rotating disk electrode voltammetry to examine dopamine clearance and inhibition of clearance by cocaine in the rat medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). Rats were housed under light:dark conditions (LD, 12 h:12 h) or in constant darkness (DD), the latter given just prior to the day of sacrifice. Tissue was collected at 4-h intervals under LD and DD conditions. Under LD, dopamine clearance in both brain regions was greatest at 4h after lights on. Under DD, there was a blunted but still rhythmic pattern of dopamine clearance across the 24-h cycle. Cocaine-induced inhibition of dopamine clearance in the mPFC was not different across the day:night cycle in rats under LD. Paradoxically, under DD, dopamine clearance in the mPFC was enhanced by cocaine at ZT16, 4 h into the subjective night, and only minimally inhibited at other times. In the NAc, cocaine inhibition of dopamine clearance was lowest at ZT4 under LD, and did not vary under DD. We conclude that dopamine clearance varies both in a diurnal and possibly in a circadian manner in the mPFC, and in a diurnal manner in the NAc. These results indicate that light itself may be used to manipulate molecules implicated in drug addiction.

Circadian Phenotypes of Drosophila Fragile X Mutants in Alternative Genetic Backgrounds

Drosophila FMR1 mutants are models of human fragile X syndrome. They show a loss of locomotor activity rhythm and severe degradation of eclosion timing. We analyzed the circadian behavior of FMR1 mutants (dfmr1B55) in two genetic backgrounds, yellow white (yw) and Canton S (CS). The arrhythmic phenotype of circadian locomotor activity in constant darkness (DD) did not significantly change in either genetic background. Surprisingly, eclosion timing was completely restored by backcrossing dfmr1B55 with yw or CS flies. Morphological analysis of the small ventrally located lateral neurons of FMR1 mutants revealed that the dorsal-projection area was significantly larger in arrhythmic than rhythmic flies. In addition, dfmr1B55 mutants in both genetic backgrounds had a significantly lower evening peak in the light-dark (LD) cycle. These results indicate that lack of FMR1 does not affect eclosion timing, but alters locomotor activity patterns in both LD and DD conditions by affecting the arborization of small ventrally located lateral neurons. Thus, the FMR1 gene may regulate the circadian-related locomotor activity of Drosophila.

Widespread Receptivity to Neuropeptide PDF throughout the Neuronal Circadian Clock Network of Drosophila Revealed by Real-Time Cyclic AMP Imaging

The neuropeptide PDF is released by sixteen clock neurons in Drosophila and helps maintain circadian activity rhythms by coordinating a network of approximately 150 neuronal clocks. Whether PDF acts directly on elements of this neural network remains unknown. We address this question by adapting Epac1-camps, a genetically encoded cAMP FRET sensor, for use in the living brain. We find that a subset of the PDF-expressing neurons respond to PDF with long-lasting cAMP increases and confirm that such responses require the PDF receptor. In contrast, an unrelated Drosophila neuropeptide, DH31, stimulates large cAMP increases in all PDF-expressing clock neurons. Thus, the network of approximately 150 clock neurons displays widespread, though not uniform, PDF receptivity. This work introduces a sensitive means of measuring cAMP changes in a living brain with subcellular resolution. Specifically, it experimentally confirms the longstanding hypothesis that PDF is a direct modulator of most neurons in the Drosophila clock network.

Daily variation of melatonin content in the optic lobes of the crab Neohelice granulata

Melatonin is a biogenic amine, known from almost all phyla of living organisms. In vertebrates melatonin is produced rhythmically in the pinealocytes of the pineal gland, relaying information of the environmental light/dark cycle to the organism. With regard to crustaceans only a handful of studies exist that has attempted to identify the presence and possible daily variation of this substance. We set out to investigate whether in the crab Neohelice granulata melatonin was produced in the optic lobes of these animals and underwent rhythmic fluctuations related to the daily light/dark cycle. Our experimental animals were divided into three groups exposed to different photoperiods: normal photoperiod (12L:12D), constant dark (DD), and constant light (LL). The optic lobes were collected every 4 hours over a 24-h period for melatonin quantification by radioimmunoassay (RIA). N. granulata kept under 12 L:12D and DD conditions, showed daily melatonin variations with two peaks of abundance ( p < 0.05), one during the day and another, more extensive one, at night. Under LL-conditions no significant daily variations were noticeable ( p > 0.05). These results demonstrate the presence of a daily biphasic fall and rise of melatonin in the eyestalk of N. granulata and suggest that continuous exposure to light inhibits the production of melatonin synthesis.

A Model-Based Analysis of Chemical and Temporal Patterns of Cuticular Hydrocarbons in Male Drosophila melanogaster

Drosophila Cuticular Hydrocarbons (CH) influence courtship behaviour, mating, aggregation, oviposition, and resistance to desiccation. We measured levels of 24 different CH compounds of individual male D. melanogaster hourly under a variety of environmental (LD/DD) conditions. Using a model-based analysis of CH variation, we developed an improved normalization method for CH data, and show that CH compounds have reproducible cyclic within-day temporal patterns of expression which differ between LD and DD conditions. Multivariate clustering of expression patterns identified 5 clusters of co-expressed compounds with common chemical characteristics. Turnover rate estimates suggest CH production may be a significant metabolic cost. Male cuticular hydrocarbon expression is a dynamic trait influenced by light and time of day; since abundant hydrocarbons affect male sexual behavior, males may present different pheromonal profiles at different times and under different conditions.

Nocturnal Male Sex Drive in Drosophila

Many behaviors and physiological processes including locomotor activity, feeding, sleep, mating, and migration are dependent on daily or seasonally reoccurring, external stimuli. In D. melanogaster, one of the best-studied circadian behaviors is locomotion. The fruit fly is considered a diurnal (day active/night inactive) insect, based on locomotor-activity recordings of single, socially naive flies. We developed a new circadian paradigm that can simultaneously monitor two flies in simple social contexts. We find that heterosexual couples exhibit a drastically different locomotor-activity pattern than individual males, females, or homosexual couples. Specifically, male-female couples exhibit a brief rest phase around dusk but are highly active throughout the night and early morning. This distinct locomotor-activity rhythm is dependent on the clock genes and synchronized with close-proximity encounters, which reflect courtship, between the male and female. The close-proximity rhythm is dependent on the male and not the female and requires circadian oscillators in the brain and the antenna. Taken together, our data show that constant exposure to stimuli emanating from the female and received by the male olfactory and other sensory systems is responsible for the significant shift in intrinsic locomotor output of socially interacting flies.

Daily Oscillation and Photoresponses of Clock Gene, Clock, and Clock‐Associated Gene, Arylalkylamine N‐acetyltransferase Gene Transcriptions in the Rat Pineal Gland

This study was conducted to investigate the circadian rhythms and light responses of Clock and arylalkylamine N‐acetyltransferase (NAT) gene expressions in the rat pineal gland under the environmental conditions of a 12 h light (05:00–17:00 h): 12 h‐dark (17:00–05:00 h) cycle (LD) and constant darkness (DD). The pineal gland of Sprague‐Dawley rats housed under a LD regime (n=42) for four weeks and of a regime (n=42) for eight weeks were sampled at six different times, every 4 h (n=7 animals per time point), during a 24 h period. Total RNA was extracted from each sample, and the semiquantitative reverse transcription polymerase chain reaction (RT‐PCR) was used to determine temporal changes in mRNA levels of Clock and NAT genes during different circadian or zeitgeber times. The data and parameters were analyzed by the cosine function software, Clock Lab software, and the amplitude F test was used to reveal the circadian rhythm. In the DD or LD condition, both the Clock and NAT mRNA levels in the pineal gland showed robust circadian oscillation (p<0.05) with the peak at the subjective night or at nighttime. In comparison with the DD regime, the amplitudes and mRNA levels at the peaks of Clock and NAT expressions in LD in the pineal gland were significantly reduced (p<0.05). In the DD or LD condition, the circadian expressions of NAT were similar in pattern to those of Clock in the pineal gland (p>0.05). These findings indicate that the transcriptions of Clock and NAT genes in the pineal gland not only show remarkably synchronous endogenous circadian rhythmic changes, but also respond to the ambient light signal in a reduced manner.