Constant Dark Conditions Research Articles

Circadian Rhythm of Activity in Japanese Quail in Constant Darkness: Variability of Clarity and Possibility of Selection

In conditions of constant darkness, interindividual variability in the clarity of circadian rhythmicity was observed in sexually immature young quail, with birds classified as more or less rhythmic or arrhythmic. The relative clarity of this circadian rhythm was observed on the actograms by measurement of the autocorrelation coefficient ratio over 12 cycles. Autocorrelation coefficients were calculated from sequential series of total activity over 12-minute periods. Crosses of selected phenotypes with different clarities of rhythmicity were conducted in order to study the possibility of selection of this characteristic. From a random population (N = 42, twice), pairs of the most rhythmic birds (3 families), and pairs of arrhythmic birds (4 families) were reared. Autocorrelation coefficient ratios of Fl birds from rhythmic families (N = 54) were greater than those of Fl birds (N = 48) from arrhythmic families (t-test, p <. 0001). These ratios in offspring were significantly correlated with that of the mean parent of each clutch of siblings (N= 102, r =.35, p =. 0003). This result was maintained in a second generation (F2) of birds, for which significant differences in expressed rhythmicity were observed. That is, autocorrelation coefficient ratios of F2 birds from two rhythmic families (TV = 30) were greater than those of F2 birds from arrhydimic families (N = 20) (t-test, p =. 039). Comparison of F2 outbred and inbred birds from rhythmic pairs showed greater values of autocorrelation coefficient ratios in the case of inbred birds (N = 16) than for outbred birds (N = 30; t-test, p =. 036). There was no difference between outbred (N = 20) and inbred birds (N = 15) from arrhythmic pairs. Therefore, selection of a rhythmic strain seems possible, whereas crosses between two arrhythmic birds may also give rise to rhythmic birds. Comparisons between rhythmic birds of different families did not show differences in the free-running period of the circadian rhythm, which is true also for rhythmic birds bred from two arrhythmic parents. Therefore, our selection procedure did not seem to be based on the characteristics of the pacemaker itself, but rather on a downstream event. Although the parents were not selected on the basis of quantity of activity per cycle or on the duration of the active phase, significant differences among the offspring of different families were shown. (Chronobiology International, 75(3), 219–230, 1998)

Pituitary adenylate cyclase-activating polypeptide rhythm in the rat pineal gland

Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated to stimulate melatonin synthesis in the rat pineal gland. Circadian rhythms of melatonin concentration are well known. However, it has not been clarified whether PACAP contents in the pineal gland show circadian rhythm. In this study, we measured PACAP contents in the rat pineal gland throughout the day under 12:12 h light-dark cycle or constant dark conditions. A significant fluctuation was observed in the PACAP content under light-dark conditions but not under constant darkness. On the other hand, the pituitary gland showed no significant variation throughout the day under either conditions. These observations suggest that PACAP may participate in the modulation of melatonin synthesis depending on light conditions in the pineal gland.

Induced dormancy and colour polymorphism in seeds of the bull thistleCirsium vulgare(Savi) Ten.

AbstractTo colonize newly disturbed areas, weedy species must disperse their seeds in space and/or in time.Cirsium vulgarehas poor dispersal in space, but can form a persistent seed bank. Fresh seeds are capable of germinating in either light/dark or constant dark conditions under favourable diurnal temperatures of 25°C: 10°C. Despite this lack of dormancy, a large proportion of seeds do not germinate in the autumn after dispersal. Variation in germination requirements according to seed colour was also examined. Fresh seeds were placed under one of two overwintering treatments at 5°C, alternating light/dark or constant darkness. Some seeds of all colours: white, light, intermediate, dark and black germinated under these conditions. Six months later, ungerminated seeds subjected to the light/dark treatment did not require light for germination when placed under optimal temperatures. However, most seeds that had overwintered in constant darkness required light for germination. In the field, this induced dormancy would prevent seeds from germinating if buried or located in deep shade. Such seeds have the potential of forming a persistent seed bank. The proportion of dormant seeds did not appear to be associated with seed colour. Seeds incubated at 10°C following overwintering under light/dark conditions germinated to a higher percentage than seeds overwintered in darkness, regardless of the light conditions for germination. The results of this study help to explain the contradiction between delayed germination in the field and the lack of seed dormancy.

Spatial and Temporal Expression of theperiodandtimelessGenes in the Developing Nervous System ofDrosophila: Newly Identified Pacemaker Candidates and Novel Features of Clock Gene Product Cycling

The circadian timekeeping system of Drosophila functions from the first larval instar (L1) onward but is not known to require the expression of clock genes in larvae. We show that period (per) and timeless (tim) are rhythmically expressed in several groups of neurons in the larval CNS both in light/dark cycles and in constant dark conditions. Among the clock gene-expressing cells there is a subset of the putative pacemaker neurons, the "lateral neurons" (LNs), that have been analyzed mainly in adult flies. Like the adult LNs, the larval ones are also immunoreactive to a peptide called pigment-dispersing hormone. Their putative dendritic trees were found to be in close proximity to the terminals of the larval optic nerve Bolwig's nerve, possibly receiving photic input from the larval eyes. The LNs are the only larval cells that maintain a strong cycling in PER from L1 onward, throughout metamorphosis and into adulthood. Therefore, they are the best candidates for being pacemaker neurons responsible for the larval "time memory" (inferred from previous experiments). In addition to the LNs, a subset of the larval dorsal neurons (DNLs) expresses per and tim. Intriguingly, two neurons of this DNL group cycle in PER and TIM immunoreactivity almost in antiphase to the other DNLs and to the LNs. Thus, the temporal expression of per and tim are regulated differentially in different cells. Furthermore, the light sensitivity associated with levels of the TIM protein is different from that in the heads of adult Drosophila.

A circadian enhancer mediates PER-dependent mRNA cycling in Drosophila melanogaster.

Genes expressed under circadian-clock control are found in organisms ranging from prokaryotes to humans. In Drosophila melanogaster, the period (per) gene, which is required for clock function, is transcribed in a circadian manner. We have identified a circadian transcriptional enhancer within a 69-bp DNA fragment upstream of the per gene. This enhancer drives high-amplitude mRNA cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (PER) dependent. An E-box sequence within this 69-bp fragment is necessary for high-level expression, but not for rhythmic expression, indicating that PER mediates circadian transcription through other sequences in this fragment.

Day-night variation of pituitary adenylate cyclase-activating polypeptide (PACAP) level in the rat suprachiasmatic nucleus

Adenylate cyclase-activating polypeptide (PACAP) is synthesized in the retinal ganglion cells which terminate on vasoactive intestinal polypeptide neurons in the suprachiasmatic nucleus (SCN), the location of circadian clock. To examine whether PACAP exhibits daily variations in the rat SCN, we measured endogenous PACAP contents throughout the day under 12:12 h light-dark or constant dark conditions. PACAP level was low during the light periods, high during the dark periods, and was stable under constant dark conditions. In the periventricular nucleus of the hypothalamus and cerebral cortex, PACAP content did not show any significant variation throughout the day. Our findings suggest that PACAP content in the SCN may be changed by lighting conditions. Thus, PACAP-containing neurons may play certain roles in the entrainment of circadian rhythms.

Development of Vasoactive Intestinal Peptide mRNA Rhythm in the Rat Suprachiasmatic Nucleus

Development of the daily rhythm of vasoactive intestinal peptide (VIP) mRNA in the rat suprachiasmatic nucleus (SCN), a main locus of circadian oscillation, was investigated by in situ hybridization. The phenotypic expression of VIP neurons occurred in two developmental stages in the ventrolateral portion of the SCN (VLSCN): the first was found before birth in the rostral part, and the second occurred in the main part between postnatal day (P) 10 and P20. The latter period coincided with the time that the massive VIP-efferent fibers project to the subparaventricular zone. In the adult and P20, the VIP mRNA signals of the SCN showed a clear diurnal rhythm with a trough in the light phase and a peak in the dark phase under light/dark (LD) conditions, but under constant dark (DD) conditions, no VIP mRNA fluctuations were observed. At P10, however, it was found that SCN VIP mRNA showed a peak at the transition from night to day and a trough at early dark period in LD conditions, in sharp contrast to the night peak in the adult rhythm. In DD conditions, a light-phase peak and a dark-phase trough were also observed at P10, contrasting the arrhythmic feature at adult stage. The present findings suggest that daily VIP rhythm was first generated in the early developed clock-controlled rostral SCN neurons, and later regulated by light-dependent main VLSCN neurons.

Performance of Western Flower Thrips (Thysanoptera: Thripidae) on Cultivars of Miniature Rose

The performance of western flower thrips, Frankliniella occidentalis (Pergande), on cultivars of miniature rose, Rosa chinensis ‘minima’ , was measured via larval development and adult oviposition experiments using leaf and petal disks. The time for larvae to complete development on both leaf and petal disks differed significantly among cultivars, although the differences were much more pronounced on petal tissue. Developmental time was prolonged on petal tissue from cultivars with red and orange flowers and generally shortest on white- and yellow-flowered cultivars. Larvae failed to complete development on petal tissue from one red- and one orange-Howe red cultivar. In a no-choice test during 10 d, female thrips deposited significantly different numbers of eggs in flower petal tissue among cultivars, with red- and orange-flowered cultivars being among the least preferred. In choice tests comparing oviposition during 48 h in petal disks from cultivars with the same flower color, a preferred cultivar was revealed in each flower color group. In choice tests under constant light and constant dark conditions, using pair combinations of petal disks from the preferred cultivars, flower color had no effect on choice of oviposition substrate. When leaf and flower petal disks from the same cultivar were compared in choice tests, leaf tissue was consistently the preferred substrate for oviposition,

Metabolic rate and level of activity determined in tilapia (Oreochromis mossambicus Peters) by direct and indirect calorimetry and videomonitoring

In order to correlate the metabolic rate and locomotor activity level of fish, a calorimetric system (Sétaram GF 108) was supplemented with a video tracking and motion analysis system. The motion analysis system is based on digital image processing. Once every second, two images (with an interval of 120 ms) are digitized and subtracted. The difference between the two images is used as a measure of the animal's movement. The activity of different complex movements, like turning, accelerations, fin movements, and branchial ventilation, were thus quantified. The combination of motion analysis with calorimetry gave some interesting results. Firstly, during extreme hypoxia, tilapia does not become lethargic as has been described for the anoxia-tolerant crucian carp [1]. The locomotor activity level during severe hypoxia corresponded to the locomotor activity level during normoxia in the restricted area of the calorimetric vessel. This implies that the calorimetrically determined reduction of the heat flux by 50% under these conditions can be ascribed to a reduction in the cellular energy metabolism — metabolic depression. Secondly, the metabolic rate under constant light conditions was elevated from 11–18%, and the animals showed strong fluctuations in the heat flux; periods of aerobic metabolism alternated with periods of anaerobic metabolism. This was in contrast to the experiments under constant dark conditions in which the metabolic rate was around the standard metabolic rate (SMR). Under the applied conditions, no correlation was observed between heat production measurements and locomotor activity. This may possibly be ascribed to the limited size of the calorimetric vessel in which the animals' metabolic rates were around SMR. The observed oscillations in metabolic rate under light conditions could be another disrupting factor; oscillations in the circulation and ventilation could be responsible for this phenomenon.

Endogenous swimming rhythms of blue crab, Callinectes sapidus , megalopae: effects of offshore and estuarine cues

Larvae of the blue crab Callinectes sapidus Rathbun develop on the continental shelf. The postlarval stage (megalopa) occurs near the surface and is transported shoreward by wind-driven surface currents. It then uses selective tidal stream transport for migration up an estuary. Endogenous swimming rhythms were measured under constant dark conditions in the laboratory in megalopae collected from the Newport River Estuary (North Carolina), the Delaware Bay, and offshore from the Newport River Estuary. Megalopae from all areas had a similar circadian activity rhythm, in which they swam during the time of the day phase in the field and were inactive at night. This rhythm predicts the presence of a reverse, diel, vertical-migration pattern offshore which would contribute to the location of megalopae near the surface during the day. The rhythm lacks obvious ecological significance in estuaries because it does not contribute to selective tidal stream transport and would increase vulnerability to visual predators during the day. Attempts to entrain a circatidal rhythm in swimming by cyclic and step changes in salinity were unsuccessful, as the circadian rhythm persisted. The rhythm also continued in the presence of the eelgrass Zostera marina, which is a site of settlement and metamorphosis in the field. Thus, megalopae enter estuaries with a solar day rhythm in activity. This rhythm, however, is not expressed, because light inhibits swimming during the day upon exposure to estuarine water. Since this light inhibition is removed in offshore waters, the rhythm would be expressed if, after entering an estuary, megalopae were transported back to offshore areas.

Elevated Nighttime Activity of Chick Pineal / Channels Requires Protein Synthesis

Free-running circadian rhythms in melatonin secretion persist in dissociated chick pineal cells. Calcium and cyclic AMP interact at several levels in the regulation of melatonin biosynthesis and secretion. Extracellular Ca2+ is required for optimal stimulation of melatonin secretion by cAMP analogues and protagonists. Increased Ca2+ influx during the circadian night is thought to play a role in the circadian clock regulation of melatonin secretion. We have recently described a nonselective cationic channel, ILOT, in chick pineal cells that is regulated by the intrinsic circadian oscillator. Active ILOT channels are detected only during the nighttime and may explain the nocturnal increase in Ca2+ influx. The mechanism by which the activity of ILOT is regulated by the circadian oscillator is not known. In the present study, the effect of the translational inhibitor anisomycin (10(-6) M) on the nighttime activity of ILOT channels was examined. The results show that protein synthesis is required for the detection of ILOT channel activity during the nighttime in cells maintained on light-dark cycles or constant dark conditions.

Zebrafish interphotoreceptor retinoid-binding protein: differential circadian expression among cone subtypes.

Retinoid trafficking between the photoreceptors and pigmented epithelium is probably mediated by interphotoreceptor retinoid-binding protein (IRBP), a 124-145 kDa glycolipoprotein in mammals and amphibians. In these animals, IRBP is composed of four homologous regions (modules) 300 amino acids in length. We have determined the primary structure of zebrafish IRBP and its expression pattern by northern analysis, reverse transcriptase-polymerase chain reaction and in situ hybridization under a variety of lighting conditions. Zebrafish IRBP is half the size (66.3 kDa) of mammalian IRBP because it is composed of only two modules, similar to goldfish IRBP. The first half of the zebrafish protein is most similar to the first module of mammalian IRBP and the second half to the fourth module of mammalian IRBP. This suggests that during the evolution of the ray-finned fish (Actinopterygii), the middle two modules were lost. Each of the modules contains conserved hydrophobic domains which may form the ligand-binding pocket. The expression of zebrafish IRBP mRNA is sevenfold higher in the middle of the light period (at mid-light) than in the middle of the dark period (at mid-dark). This rhythm persists for 2 days under conditions of constant light or constant darkness, then dampens to an intermediate level by 8 days of constant conditions. At mid-light, IRBP mRNA is expressed by all cone types and to a lesser extent by the rods. At mid-dark, the mRNA is restricted to the ultraviolet-sensitive short single cones. These data suggest that IRBP expression is regulated by circadian and light-driven mechanisms that act differentially on the various photoreceptor subtypes in the zebrafish retina.

The tau mutation shortens the period of rhythmic photoreceptor outer segment disk shedding in the hamster

The outer segments of vertebrate retinal photoreceptors undergo periodic shedding of membrane from their distal tips. This circadian rhythm of disk shedding persists with a period of about 24 h in the absence of external time cues. A circadian oscillator controlling photoreceptor disk shedding may exist in the eye, but in addition, the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) may also influence ocular rhythms including that of disk shedding. The tau mutation directly affects the SCN, and shortens the period of locomotor activity from 24 h in wild-type hamsters to 20 h in homozygous mutants. Here we show that homozygous tau-mutant hamsters in a 20-h light/dark cycle exhibit a 20-h oscillation in the rate of disk shedding, with peak phagosome numbers in the retinal pigmented epithelium occurring just after light onset. The numbers of phagosomes are significantly elevated from mid-dark levels prior to light onset, indicating that the disk shedding cycle anticipates dawn. Under conditions of constant darkness, the disk shedding rhythm in tau-mutant hamsters persists with a period of approximately 20 h. These results indicate that a rhythm of retinal photoreceptor outer segment disk shedding exists in the hamster eye, and that the period of this rhythm is shortened by the tau mutation.

Effects of photoperiod on the circadian timing of pheromone response in male Trichoplusia ni: Relationship to the modulatory action of octopamine

The pheromone-mediated upwind flight of male cabbage looper moths was measured throughout the scotophase in a series of photoperiods, to determine the phase-relationship between the time of peak response to pheromone during the dark and the photoperiod cues. Male upwind flight and source contact was measured to a low dose of pheromone, one that would best reveal the presence of a temporal peak in male sensitivity. Results failed to support the idea that there is a narrow ‘window’ of maximal sensitivity during the dark that is phase-regulated by either the lights off, or lights on signal. Rather, there was a clear relationship between the lights off signal and the time (2–3h) to reach maximal response levels, regardless of the duration of the dark. Once the point of maximal response was reached, response levels displayed relatively constant levels until the very end of the dark period. Experiments involving shifts in the time of lights off or lights on also supported the idea that the lights off cue is critical for expression and timing of the response rhythm. Other experiments confirmed that a circadian oscillator is involved, evidenced by the rhythmic expression of male behavior mirroring the duration of expected dark periods in two photoperiods when males were tested in constant dark conditions. Also, constant dark and light conditions significantly affected the pattern of change in levels of octopamine observed in the brain in a light:dark regime. Of particular interest, the pattern of decrease in octopamine levels correlated well with the pattern of response to sex pheromone over the dark, supporting an earlier hypothesis that octopamine modulates neural pathways involved in perception of the odor signal, and that this action is triggered by the lights off cue. The results support the conclusion that the lights off photoperiod cue is a critical time in which a number of physiological process are initiated that will affect male response thresholds for a number of behaviors during the dark, including our previously demonstrated modulatory action of octopamine on the sensitivity of male moths to sex pheromone.

A cationic channel regulated by a vertebrate intrinsic circadian oscillator.

Secretory cells of the chicken pineal gland exhibit light-sensitive circadian rhythms in melatonin release that persist in vitro. Melatonin secretion is positively regulated by cyclic AMP and intracellular Ca2+ (refs 8, 10-15). Cyclic AMP analogues are more effective at stimulating melatonin secretion during the circadian night owing in part to increased Ca2+ influx at those times. However, this cannot be attributed to increased activity of L-type Ca2+ channels. Here we describe an unusual 40-pS cationic channel (ILOT) in cultured chicken pineal cells that is permeable to Ca2+ and active in the night but not during the day. ILOT is not voltage- or stretch-activated, it has a characteristically long open time, and its gating persists in excised inside-out patches in the absence of Ca2+ or cyclic nucleotides. Daily rhythms in ILOT gating are also observed in previously entrained chicken pineal cells free-running under constant dark conditions. Nighttime ILOT activity is not suppressed by brief light pulses.

Light- and temperature-entrained circadian regulation of activity and mRNA accumulation of 1-aminocyclopropane-1-carboxylic acid oxidase in Stellaria longipes.

Stem and leaf tissues of Stellaria longipes Goldie (prairie ecotype) exhibit circadian rhythmicity in the activity and mRNA abundance for 1-aminocyclopropane-1-carboxylic acid oxidase (EC 1.4.3). The steady-state mRNA levels and enzymatic activity levels fluctuated with a period of approximately 24 h and reached their maxima by the middle of the light phase and minima by the middle of the dark phase. The oscillations showed damping under constant light, constant dark and constant temperature conditions, indicating that the rhythm is entrained by an external signal. The results indicate that light/dark cycles have greater entraining effects than temperature cycles. A 15-min red light pulse, but not a blue light pulse, could reset rhythm in continuous darkness, suggesting the possible role of a red-light signal transduction pathway in the circadian regulation of 1-aminocyclopropane-1-carboxylic acid oxidase.

A circadian clock regulates rod and cone input to fish retinal cone horizontal cells.

In the vertebrate retina, the light responses of post-receptor neurons depend on the ambient or background illumination. Using intracellular recording, we have found that a circadian clock regulates the light responses of dark-adapted fish cone horizontal cells. Goldfish were maintained on a 12-hr light/12-hr dark cycle. At different times of the day or night, retinas were superfused in darkness for 90 min ("prolonged darkness"), following which horizontal cells were impaled without the aid of any light flashes. In some of the experiments, fish were kept in constant darkness for 3-48 hr prior to surgery. After prolonged darkness during the night, but not during the day, the light responses of L-type cone horizontal cells resembled those of rod horizontal cells with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Following light sensitization during the night and day, the light responses of rod and cone horizontal cells were clearly different with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Under conditions of constant darkness for two full light/dark cycles, average responses of cone horizontal cells to a bright light stimulus during the subjective day were greater than during the subjective night. Prior reversal of the light/dark cycle reversed the 24-hr rhythm of cone horizontal cell responses to bright lights. In addition, following one full cycle of constant darkness, average cone horizontal cell spectral sensitivity during the subjective night closely matched that of rod horizontal cells, whereas average cone horizontal cell spectral sensitivity during the subjective day was similar to that of red (625 nm) cones. These results indicate that the effects of dark adaptation depend on the time of day and are regulated by a circadian clock so that cone input to cone horizontal cells predominates in the day and rod input predominates in the night.

Altered circadian rhythmicity in the Wocko mouse, a hyperactive transgenic mutant.

Induced changes in the level of daily activity can alter the period of the mammalian circadian clock. In this report, we examined the period of the circadian rhythm of wheel-running activity in a transgenic neurological mouse mutant, Wocko. Wocko mice display a dominant behavioral phenotype that consists of hyperactivity, circling and head tossing. The period of the circadian rhythm of wheel-running activity in constant dark conditions was significantly shorter in mice expressing the Wocko mutation than in their normal littermates. Total activity, monitored by the interruption of an array of infrared beams, was significantly elevated in Wocko mice. These findings support the view that spontaneous exercise can modulate the circadian timekeeping system.

Light-responsive and rhythmic gene expression of psbA2 in cyanobacterium Microcystis aeruginosa K-81.

We cloned, sequenced and characterized the psbA2 gene of the cyanobacterium Microcystis aeruginosa K-81 strain. The deduced amino acid sequence of PsbA2 exhibited extensive similarity to the D1 protein, which is known to be a core protein in the reaction center of the photosynthetic Photosystem II in cyanobacteria. The N-terminal amino acid sequence of PsbA2 also showed that this protein is a Form II type of D1 protein, as defined for Synechococcus sp. strain PCC 7942. The psbA2 gene was transcribed as a 1.2kb monocistron, and the potential promoter had an Escherichia coli consensus sequence. The light-responsive psbA2 message exhibited rhythmicity under conditions of constant darkness after prior entrainment to light and dark cycles.

The expression of locomotor circadian rhythm in female German cockroach, Blattella germanica (L.).

Fifteen percent of intact female German cockroaches (n = 13), Blattella germanica (L.) (Dictyoptera: Blattellidae), had weak free-running locomotor rhythmicity under 28 degrees C and constant darkness conditions. However, 86% of ovariectomized females (n = 14) showed a strong free-running rhythm under the same conditions with a circadian period of 23.60 +/- 0.15 h, similar to the male's period of 23.45 +/- 0.03 h. In addition, the locomotory activities occurred mainly during the subjective night under DD conditions as was the case in males. These results indicated that female locomotion was under the control of a circadian oscillator, which was masked by the existence of ovaries. This internal masking effect could be removed by the existence of males, but females had no effect on the locomotor pattern of another female. Since the male failed to entrain female locomotion, its role as a zeitgeber was excluded. That the locomotory pattern of the females still coincided with their reproductive cycle when exposed to male odor suggests that exposure to a male only partially removed the internal masking effects.