Constant Dark Conditions Research Articles

Influence of light-dark and lunar cycles on the ocular melatonin rhythms in the seagrass rabbitfish, a lunar-synchronized spawner.

The aim of this study was to investigate the effects of light-dark (LD) cycles and lunar phases on ocular melatonin rhythms in the seagrass rabbitfish, Siganus canaliculatus, a lunar-synchronized spawner. Under a natural 24-hr LD (12.00:12.00) cycle, ocular melatonin levels were low during daylight hours. The levels significantly elevated to peak during the mid-dark phase at 24.00 hr and then declined sharply in the early morning around 06.00 hr. These rhythms disappeared under either constant light (LL) or constant dark (DD) conditions. Melatonin levels remained low in LL compared with those in DD condition. These results suggest that ocular melatonin rhythms in the seagrass rabbitfish are suppressed in the presence of light. When fish were exposed to natural moon phases, ocular melatonin concentrations were higher around the new moon than both the first quarter and full moon phases. Exposure to experimental new moon conditions caused a significant increase in melatonin levels while those of the fish exposed to experimental full moon conditions were decreased. These results suggest that the seagrass rabbitfish perceives moonlight through the eye and that moonlight directly causes melatonin suppression.

Lack of nociceptin receptor alters body temperature during resting period in mice

The role of nociceptin (NOC) receptor on body core temperature (Tcore) control was examined using NOC receptor knockout mice. In homozygote NOC receptor-knockout, wild-type, and control C57BL/6J and 129/SV mice, Tcore was continuously recorded under 12:12 h light:dark (LD) and conditions of constant darkness (DD). The Tcore values during the resting period were higher in the NOC receptor-knockout mice than in both wild-type and control mice under both LD and DD conditions. Spontaneous activity during the resting period and plasma cortisol levels were not different between the NOC receptor-knockout and control mice. The findings herein indicate that the NOC receptor is involved in the control of Tcore during the resting period and is independent of light, physical activity and/or cortisol regulation.

Daily and circadian expression of neuropeptides in the suprachiasmatic nuclei of nocturnal and diurnal rodents

The suprachiasmatic nuclei (SCN) of the hypothalamus are necessary for coordination of major aspects of circadian rhythmicity in mammals. Although the molecular clock mechanism of the SCN has been a field of intense research during the last decade, the role of the neuropeptides in the SCN, including arginine–vasopressin (AVP), vasoactive intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP), in the clock itself or in circadian organization is still largely unknown. Previous studies mainly performed in the rat have examined the profiles of AVP, VIP and GRP mRNA and peptide levels and suggested that the AVP rhythm is controlled by the circadian clock, whereas those of VIP and GRP are directly dependent on lighting conditions. Here, both daily (i.e., under light–dark cycle [LD]) and circadian (i.e., in constant darkness [DD]) profiles of neuropeptide mRNA were investigated in the SCN of the nocturnal mouse Mus musculus and the diurnal rodent Arvicanthis ansorgei to gain insight into a possible role in circadian organization. Our data show that AVP mRNA exhibits a clear circadian rhythm in the SCN peaking by the end of the subjective day in both species. Contrary to what has been observed in rats, oscillations of VIP and GRP mRNA in the SCN are found to be clock-controlled in mice and A. ansorgei, but with different phases for peak expression. While both VIP and GRP mRNA peak during the middle of the subjective night (i.e., with a 6-h lag compared to AVP mRNA) in mice, they peak almost in phase with AVP mRNA in A. ansorgei. Contrary to what has been reported in the rat, mean levels of VIP and GRP peptide mRNA levels tended to be increased by light in the mice. The different circadian organization of SCN neuropeptides mRNA profiles in both light/dark and constant darkness conditions between mice and A. ansorgei could be related with diurnality.

Circadian rhythms of dopamine, glutamate and GABA in the striatum and nucleus accumbens of the awake rat: modulation by light.

Using microdialysis, we investigated the circadian rhythms of the extracellular concentrations of dopamine, glutamate and gamma-aminobutyric acid (GABA) in the striatum and nucleus accumbens of the awake rat. Wistar rats were maintained in a 12 hr dark:12 hr light (12:12) cycle for 2 wk before the experiment began. The neurotransmitter levels were measured every 30 min for 30 hr in control (maintaining the 12:12 cycle) or in experimental conditions under a 24-h light period (continuous light) or under a 24-h dark interval (continuous dark). The dopamine metabolites, DOPAC and HVA, and the main serotonin metabolite, 5-HIAA, were measured along with arginine and glutamine under all conditions. In 12:12 conditions, a circadian rhythm of dopamine, glutamate and GABA was found in both the striatum and nucleus accumbens. Again under 12:12 conditions, DOPAC, HVA, 5-HIAA, and arginine, but not glutamine, fluctuated in a circadian rhythm. In the striatum under constant light conditions, there was a circadian rhythm of dopamine, glutamate, GABA, DOPAC and HVA, but not 5-HIAA. By contrast, when the rats were kept under continuous dark, dopamine and its metabolites, DOPAC and HVA (but not glutamate and GABA), did not fluctuate in a circadian rhythm. In the nucleus accumbens, under both constant light or dark conditions, no changes were found in the circadian rhythm in any of the neurotransmitters and metabolites studied. These findings show that in the striatum, dopamine but not glutamate and GABA, seem to be influenced by light. In the nucleus accumbens, however, the three neurotransmitters had a circadian rhythm, which was independent of light.

Endogenous cardiac activity rhythms of continental slope Nephrops norvegicus (decapoda: nephropidae)

The endogenous cardiac activity rhythm of the Norway lobster Nephrops norvegicus was studied under constant conditions of darkness by means of a computer-aided monitoring system (CAPMON). Time series recordings of the heart rate (beats min−1) were obtained from 47 adult males freshly collected from the continental slope (400–430 m) in the western Mediterranean. Periodogram analysis revealed the occurrence of circadian periodicity (of around 24 h) in most cases. A large percentage of animals showed significant ultradian periods (of around 12 and 18 h). The analysis of the circadian time series revealed the occurrence of peaks of heart rate activity during the expected night phase of the cycle. These results are discussed in relation to the emergence and locomotor activity rhythms of the species.

Circadian expression of clock gene in the optic tectum of Japanese quail

The physiological activity of avian optic tectum (TeO) is known to be regulated by the circadian system. In a previous study, we found clock gene expression in the TeO of Japanese quail. Here we report rhythmic expression of the Per2 gene in the stratum griseum et fibrosum (SGF) of the TeO under a light–dark (LD) cycle, constant darkness (DD), and constant light (LL) conditions. However, light pulse did not affect Per2 expression in the TeO. These results suggest that light stimulus and melatonin rhythm are not essential for rhythmic expression of Per2 in the avian TeO in spite of the localization of melatonin receptors and retinal input.

Locomotor Activity Rhythms of Continental Slope Nephrops Norvegicus (Decapoda: Nephropidae)

The locomotor rhythmicity of the Norway lobster Nephrops norvegicus was studied under constant conditions of darkness in individuals collected on the continental slope |$(400 - 430\;{\rm{m}})$|⁠. Periodogram analysis revealed the occurrence of both circadian (of around 24 h) and ultradian (of around 12 and 18 h) periodicities. Form estimate analysis of the circadian and ultradian time series revealed the occurrence of significant peaks of activity during the expected night phase of the cycle and day-night transitions, respectively. No ultradian locomotor activity rhythms have been reported in previous studies on continental shelf N. norvegicus, suggesting that this phenomenon may be limited to deep-water animals. A discussion is presented to account for the occurrence of the mechanism of ultradian rhythms when there is significant environmental light intensity reduction, as on the continental slope, where the species attains its maximum densities in the western Mediterranean.

Entrainment of the Circatidal Swimming Activity Rhythm in the Cumacean Dimorphostylis asiatica (Crustacea) to 12.5-hour Hydrostatic Pressure Cycles

The cumacean Dimorphostylis asiatica (Crustacea) exhibits a circatidal swimming activity rhythm. The animals were exposed to a 12.5 hr sinusoidal change of hydrostatic pressure of 0.3 atm amplitude in the laboratory. Under constant dark conditions, most of the specimens were entrained to a daily bimodal swimming activity rhythm by the hydrostatic pressure cycle. A small number of individuals exhibited a unimodal daily rhythm, with no apparent entraining from the administered cycles. A marked feature was a flexible phase relationship between the entrained daily bimodal rhythm and the hydrostatic pressure cycles: the swimming activity of most of the specimens occurred around the pressure-decreasing phase, but for a small number of individuals it coincided with the pressure-increasing phase. Such flexibility suggests a weak entraining effect of hydrostatic pressure on the circatidal rhythm of this species. When exposed to 24 hr light-dark cycles and a hydrostatic pressure cycle simultaneously, the specimens exhibited a rhythmic activity entrained by the hydrostatic pressure cycle during the dark period, which closely resembles the temporal activity pattern of this species in the field. The light cycles entrained the swimming activity via direct inhibition and induction of activity (i.e., masking). Under light-dark conditions, the specimens exhibited activity on the pressure-increasing phase more frequently compared with specimens kept in constant darkness.

Diurnal, circadian and photic regulation of calcium/calmodulin-dependent kinase II and neuronal nitric oxide synthase in the hamster suprachiasmatic nuclei

Mammalian circadian rhythms are entrained by light pulses that induce phosphorylation events in the suprachiasmatic nuclei (SCN). Ca 2+-dependent enzymes are known to be involved in circadian phase shifting. In this paper, we show that calcium/calmodulin-dependent kinase II (CaMKII) is rhythmically phosphorylated in the SCN both under entrained and free-running (constant dark) conditions while neuronal nitric oxide synthase (nNOS) is rhythmically phosphorylated in the SCN only under entrained conditions. Both p-CaMKII and p-NOS (specifically phosphorylated by CaMKII) levels peak during the day or subjective day. Light pulses administered during the subjective night, but not during the day, induced rapid phosphorylation of both enzymes. Moreover, we found an inhibitory effect of KN-62 and KN-93, both CaMKII inhibitors, on light-induced nNOS activity and nNOS phosphorylation respectively, suggesting a direct pathway between both enzymes which is at least partially responsible of photic circadian entrainment.

The Biological Clock Nucleus: A Multiphasic Oscillator Network Regulated by Light

The circadian clock nucleus of the mammalian brain is composed of thousands of oscillator neurons, each driven by the cell-autonomous action of a defined set of circadian clock genes. A critical question is how these individual oscillators are organized into an internal clock that times behavior and physiology. We examined the neural organization of the suprachiasmatic nucleus (SCN) through time-lapse imaging of a short-half-life green fluorescent protein (GFP) reporter of the circadian clock gene Period 1 (Per1). Using brain slice preparations, Per1 promoter rhythms were resolved at the level of the SCN, and in individual neurons within the SCN, to determine the temporal patterns of rhythmicity resulting from exposure of mice to light/dark cycle (LD) and constant darkness (DD) conditions. Quantitative imaging and patch-clamp electrophysiology were used to define the relationship of Per1 gene expression to neurophysiological output on an individual neuron basis. We found that in both LD and DD, the overall rhythm of the clock nucleus is composed of individual cellular rhythms that peak in distinct phase groups at 3-4 hr intervals. However, the phase relationships of Per1 oscillations to locomotor activity and the phase relationships among individual neuronal oscillators within the SCN are different in LD and DD. There was a positive, linear correlation of Per1 transcription with neuronal spike frequency output, thus Per1::GFP rhythms are representative of physiological rhythmicity. Our results reveal multiple phase groupings of SCN oscillators and suggest that light regulation of oscillator interactions within the SCN underlies entrainment to the photoperiod.

The eyes suppress a circadian rhythm of FOS expression in the suprachiasmatic nucleus in the absence of light

Photic information transmitted from the eyes to the suprachiasmatic nucleus (SCN) is essential for entrainment of circadian behavioral and physiological rhythms in mammals. Under conditions of constant darkness, these rhythms are maintained by the circadian pacemaker cells of the SCN [Bioessays 22 (2000) 23]. It is possible, however, that even in the absence of light, the eyes, which also contain autonomous circadian pacemakers [Science 272 (1996) 419; Chronobiol Int 16 (1999) 229], modulate circadian rhythms in the SCN. Indeed, it was shown recently that removal of the eyes abolishes an endogenous circadian rhythm within cells of the SCN [Nat Neurosci 6 (2003) 111], a finding that led to the suggestion that specific rhythms of the SCN are driven by input from the eyes. In contrast, we show here that removal of the eyes amplifies a normally dampened endogenous circadian rhythm within the SCN, indicating that the eyes can suppress the expression of specific rhythms within the SCN while promoting others.

Optimizing conditions for growth and sporulation of Pyrenophora semeniperda

The effects of photoperiod, light quality, wounding and sealing of culture plates on in vitro growth and sporulation of Pyrenophora semeniperda were examined to define conditions conducive to the abundant and rapid production of conidia. For maximum growth and sporulation, the leaf‐spotting and seedborne plant pathogen required an alternating light/dark sequence. Fewer conidia were produced under conditions of constant illumination or constant darkness. Growth was enhanced by light of wavelengths longer than 500 nm, while sporulation was enhanced by light of wavelengths shorter than 500 nm. Sporulation was enhanced under conditions of alternating temperature with a diurnal photoperiod and by mycelial wounding. Sealing of Petri plates had an inhibitory effect on sporulation. Significantly more conidia of higher quality formed when unsealed cultures, wounded after 7 days, were exposed to 23°C during the light phase and 19°C during darkness. Under these optimal conditions, conidial numbers were increased by 800% or more compared with unwounded cultures grown at constant 25°C in 12 h alternating cool‐white light.

Transcriptional profiling of the chick pineal gland, a photoreceptive circadian oscillator and pacemaker.

The avian pineal gland contains both circadian oscillators and photoreceptors to produce rhythms in biosynthesis of the hormone melatonin in vivo and in vitro. The molecular mechanisms for melatonin biosynthesis are largely understood, but the mechanisms driving the rhythm itself or the photoreceptive processes that entrain the rhythm are unknown. We have produced cDNA microarrays of pineal gland transcripts under light-dark and constant darkness conditions. Rhythmic transcripts were classified according to function, representing diverse functional groups, including phototransduction pathways, transcription/translation factors, ion channel proteins, cell signaling molecules, and immune function genes. These were also organized relative to time of day mRNA abundance in light-dark and constant darkness. The transcriptional profile of the chick pineal gland reveals a more complex form of gene regulation than one might expect from a gland whose sole apparent function is the rhythmic biosynthesis of melatonin. The mRNAs encoding melatonin biosynthesis are rhythmic as are many orthologs of mammalian "clock genes." However, the oscillation of phototransductive, immune, stress response, hormone binding, and other important processes in the transcriptome of the pineal gland, raises new questions regarding the role of the pineal gland in circadian rhythm generation, organization, and avian physiology.

Sex difference in circadian period of body temperature in Clock mutant mice with Jcl/ICR background

The period of circadian rhythm remains remarkably stable in both sexes under conditions of constant darkness. Several studies have indicated that gonadal hormones such as estradiol shorten the period of circadian rhythms. We observed that the free-running period of core body temperature is longer in male homozygous Clock mutant mice than in females, although this difference is not found in wild mice. Furthermore, a gonadectomy had no effect on the period of core body temperature in either sex. This suggests the existence of a sex-related difference of the mammalian circadian system, and that it is independent of peripheral gonadal regulations.

Functional retinal input stimulates expression of astroglial elements in the suprachiasmatic nucleus of postnatal developing rat

Astrocytes are abundant in the hypothalamic suprachiasmatic nucleus (SCN), particularly in the retinorecipient region. Using glial fibrillary acidic protein (GFAP) immunocytochemistry, we investigated the effect of light on the development of astrocytes in the SCN housing under light–dark (LD) or constant dark (DD) conditions after birth. GFAP immunoreactivity in the DD group showed lower levels than those in the LD group at P50. However, there was no difference in density of retinohypothalamic tract (RHT) terminals in the SCN between the DD and LD groups. After the adult pattern of GFAP immunoreactivity was established at P30, transferring rats to different LD conditions produced changes in GFAP immunoreactivity evident when rats were sacrificed at P50. We next examined, using a primary culture of hypothalamic astrocytes, whether neurotransmitters of RHT such as glutamate and pituitary adenylate cyclase activating polypeptide (PACAP) can stimulate GFAP expression directly. PACAP-38 increased the length and number of astrocytic processes but glutamate did not. These findings indicate that the functional aspects of RHT such as the light stimulated release of neurotransmitters is important for the development of astrocytes in rat SCN. Dynamic plasticity of astroglial elements in the SCN occurs even after GFAP shows an adult pattern.

A recessive mutant of Drosophila Clock reveals a role in circadian rhythm amplitude.

The transcription factor Clock (Clk) plays a critical role in animal circadian rhythms. Genetic studies defining its function have relied on two dominant negative alleles, one in Drosophila and one in mice. Here we describe a novel recessive allele of Drosophila Clock, Clk(ar). Homozygous Clk(ar) flies are viable and behaviorally arrhythmic. The Clk(ar) phenotype is caused by a splice site mutation that severely disrupts splicing and reduces Clk activity. Despite the behavioral arrhythmicity, molecular oscillations are still detectable in Clk(ar) flies. Transcription analysis indicates potent effects of Clk(ar) on levels and amplitude of transcriptional oscillations. Taken together with other data, we propose that Clk makes a major contribution to the strength and amplitude of circadian rhythms.

Circadian expression of hnRNP U, a nuclear multi-potent regulatory protein, in the murine suprachiasmatic nucleus

Heterogeneous ribonuclear protein U (hnRNP U/SAF-A) is a nuclear multi-potent regulatory protein. We investigated whether hnRNP U protein and transcript levels undergo circadian changes by immunoblot and quantitative RT-PCR analyses. In the suprachiasmatic nucleus (SCN), hnRNP U immunoreactivity (ir) changed in a robust circadian pattern as it showed a peak at late nighttime in both light/dark and constant dark conditions. hnRNP U transcript levels in the SCN changed in a similar circadian pattern. In the hippocampus, hnRNP transcript levels also showed a peak at late nighttime but hnRNP U-ir showed an opposite pattern as it peaked at late daytime. These findings suggest that hnRNP U participates in nuclear regulatory events that are involved in mammalian central and peripheral circadian clocks.

The activity-regulated cytoskeleton-associated ( arc) gene is a new light-inducible early gene in the mouse suprachiasmatic nucleus

The mammalian circadian system is entrained to the environmental light/dark cycle by shifting the phase of the master clock in the suprachiasmatic nucleus. Accompanying the light-induced phase-shift, a variety of immediate-early genes appears in suprachiasmatic nucleus clock cells, and here, we report the expression of a new immediate-early gene Arc (activity-regulated cytoskelton-associated gene) in mice. Arc messenger RNAs were strongly induced at 30-120 min after the light exposure at subjective night (CT12-CT20) in neurons of the retinorecipient area of the suprachiasmatic nucleus, although their spontaneous expression was absent in usual light-dark cycles and in constant dark conditions. At protein level, ARC appeared not only in the nucleus but also in the perikarya and their processes of the suprachiasmatic nucleus neurons. These findings indicate that Arc is an activity-regulated cytoskeletal gene possibly involved in the light-induced phase-shift of the circadian rhythm.

Circadian Oxygen Consumption Patterns in Continental Slope Nephrops Norvegicus (Decapoda: Nephropidae) in the Western Mediterranean

Abstract The oxygen consumption of Nephrops norvegicus animals collected from the upper continental slope (400 m depth) in the northwestern Mediterranean was monitored under constant conditions of darkness and temperature. Two experiments were performed starting at the beginning of the expected day and at the beginning of the expected night phases, respectively. Mean oxygen consumption values recorded during the expected night were significantly higher than those recorded during the expected day. The slopes of the time series of oxygen consumption data of the two experiments were recalculated in consecutive 1-h intervals, being then averaged for corresponding 1-h time intervals. The plotting over a 24-h cycle of these mean hourly-values revealed a global nocturnal increase in the oxygen consumption in the laboratory. This result is discussed and compared with previously reported catch patterns accounting for emergence from burrows in the field, and locomotor and cardiac activity rhythms recorded in consta...

Serum Cortisol Levels in Goats Exhibit Seasonal But Not Daily Rhythmicity

The aim of the study was to find out whether there is a daily rhythm in goat serum cortisol concentrations, whether the concentration profiles differ between normal light:dark and constant dark conditions, and whether any seasonal variations might be detected in daily cortisol secretion patterns. Seven Finnish landrace goats were kept at indoor temperature (18–23°C) under artficial lighting that approximately simulated the annual changes of daylength at 60°N. Blood samples were collected for cortisol measurements by radioimmunoassay at 2h intervals during six times of the year: winter (light:dark 6:18h), early spring (10:14h), late spring (14:10h), summer (18:6h), early fall (14:10h), and late fall (10:14h). Cortisol profiles were determined for two consecutive days, first in light:dark (LD) conditions and then in continuous darkness (DD). There was no significant daily rhythm in serum cortisol levels in any time of the year, nor did the profiles in LD and DD conditions show any differences. A significant seasonal variation was, however, detected among the overall cortisol levels. In winter, the concentrations were higher than in any other season, and from early spring to summer they were at their lowest. Under equal photoperiods, the cortisol levels were higher in fall than spring. The difference between winter and summer was confirmed in the following year in LD conditions. There was no correlation between the serum cortisol and progesterone levels. The results suggest that the possible circadian variation of cortisol secretion in goats is completely masked by external factors, and the lighting conditions do not have immediate effects on the daily secretion patterns. The seasonal variation in the overall cortisol levels is most probably related to the changes in photoperiod, because other conditions were relatively constant during the experiment.