Rhythm In Darkness Research Articles

Defining the role of Drosophila lateral neurons in the control of circadian rhythms in motor activity and eclosion by targeted genetic ablation and PERIOD protein overexpression.

The ventral lateral neurons (LNvs) of the Drosophila brain that express the period (per) and pigment dispersing factor (pdf) genes play a major role in the control of circadian activity rhythms. A new P-gal4 enhancer trap line is described that is mostly expressed in the LNvs This P-gal4 line was used to ablate the LNvs by using the pro-apoptosis gene bax, to stop PER protein oscillations by overexpressing per and to block synaptic transmission with the tetanus toxin light chain (TeTxLC). Genetic ablation of these clock cells leads to the loss of robust 24-h activity rhythms and reveals a phase advance in light-dark conditions as well as a weak short-period rhythm in constant darkness. This behavioural phenotype is similar to that described for disconnected1 (disco1) mutants, in which we show that the majority of the individuals have a reduced number of dorsally projecting lateral neurons which, however, fail to express PER. In both LNv-ablated and disco1 flies, PER cycles in the so-called dorsal neurons (DNs) of the superior protocerebrum, suggesting that the weak short-period rhythm could stem from these PDF-negative cells. The overexpression of per in LNs suppresses PER protein oscillations and leads to the disruption of both activity and eclosion rhythms, indicating that PER cycling in these cells is required for both of these rhythmic behaviours. Interestingly, flies overexpressing PER in the LNs do not show any weak short-period rhythms, although PER cycles in at least a fraction of the DNs, suggesting a dominant role of the LNs on the behavioural rhythms. Expression of TeTxLC in the LNvs does not impair activity rhythms, which indicates that the PDF-expressing neurons do not use synaptobrevin-dependent transmission to control these rhythms.

Effects of intergeniculate leaflet lesions on circadian rhythms in Octodon degus

The intergeniculate leaflet (IGL) modulates photic and nonphotic entrainment of circadian rhythms in nocturnal species, but nothing is known about its role in diurnal species. We investigated the significance of the IGL for circadian rhythm function in the diurnal rodent, Octodon degus, by determining the effects of bilateral electrolytic IGL lesions (IGL X) on: (i) photic entrainment; (ii) reentrainment rates to photic cues following a 6-h phase advance of the light–dark (LD) cycle; (iii) reentrainment rates to nonphotic social and photic cues following a 6-h phase advance of the LD cycle; and (iv) the circadian period ( τ) of the activity rhythm in constant darkness (DD). IGL X significantly lengthened the duration ( α) of the entrained activity rhythm and produced a significantly earlier phase of activity onset under entrained (LD 12:12) conditions, but did not change phase of activity offset, rhythm amplitude or mean daily activity levels. IGL X failed to modify τ of free-running activity rhythms in DD or alter reentrainment rates of circadian rhythms to nonphotic social and photic cues or photic cues alone. Thus, the IGL modulates two parameters of photic entrainment, but is not necessary for reentrainment to either nonphotic social or photic cues. Our results contribute to the growing comparative database on the neural mechanisms controlling circadian rhythms and indicate that the role of the IGL varies across species with no apparent relationship between diurnality–nocturnality and circadian function.

PHOTIC ENTRAINMENT OF CIRCADIAN ACTIVITY PATTERNS IN THE TROPICAL LABRID FISH HALICHOERES CHRYSUS

Yellow wrasses (Halichoeres chrysus) show clear daily activity patterns. The fish hide in the substrate at (subjective) night, during the distinct rest phase. Initial entrainment in a 12h:12h light-dark (12:12 LD) cycle (mean period 24.02h, SD 0.27h, n = 16 was followed by a free run (mean period 24.42h, SD 1.33h) after transition into constant dim light conditions. Light pulses of a comparable intensity as used in the light part of the LD cycles did not result in significant phase shifts of the free-running rhythm in constant darkness. Application of much brighter 3h light pulses resulted in a phase-response curve (PRC) for a fish species, with pronounced phase advances during late subjective night. The PRCs differed from those mainly obtained in other vertebrate taxa by the absence of significant phase delays in the early subjective night. At that circadian phase, significant tonic effects of the light pulses caused a shortening of the circadian period length. Entrainment to skeleton photoperiods of 1:11 LD was observed in five of six wrasses exposed, also after a 3h phase advance of this LD cycle. Subsequently, a 1:11.25 LD cycle resulted in entrainment in four of the six fish. It is suggested that the expression of the circadian system in fish can be interpreted as a functional response to a weak natural zeitgeber, as present in the marine environment. This response allows photic entrainment as described here in the yellow wrasse. (Chronobiology International, 17(5), 613–622, 2000)

Milankovitch-scale palaeoclimate changes in pale–dark bedding rhythms from the Early Cretaceous (Hauterivian and Barremian) of eastern England and northern Germany

Early Cretaceous (Valanginian–Albian) successions in northwestern Europe occupy an ideal palaeoceanographic location in which to study the interplay between the Arctic–Boreal and Tethyan seaways or realms. Marine Early Cretaceous sediments of the North Sea borderlands (north Germany, eastern England) are dominated by fine-grained siliciclastic deposition with carbonates. Occasionally this succession is characterised by visually striking bedding rhythms, comprising calcareous and less calcareous clays or pale–dark alternations on a scale of 0.5–1 m. Such rhythmic sedimentation becomes most dominant in the early to late Hauterivian, parts of the early Barremian and in the Aptian. This study concentrates on late Hauterivian sections in England and Germany, which have remained relatively unaffected by deep burial, tectonism or diagenesis. Changes in geochemistry are related to floral and faunal alternations and thus directly to palaeoenvironment. The wide range of palaeontological and geochemical analyses show conclusively how bedding rhythms, developed in the Milankovitch band, are the result of palaeoclimate change. Pale layers were `Tethyan influenced' in their microflora and fauna, indicating warm surface waters that were poor in nutrients and developed during warm, seasonally arid hinterland climates. Dark layers were `Boreal influenced', displaying features consistent with cooler waters, rich in nutrients. Such dark layers reflect cooler, more humid hinterland conditions. The coincidence of Tethyan taxa with bundles of pale layers displaying high gamma-ray emission and elevated radioactive element contents suggests transgressive or condensed beds. The bundling of pale–dark rhythms coincides with the long-term Milankovitch eccentricity cycle. This paper documents direct links between the reaction of hinterland weathering, marine floras and floras to Milankovitch-scale changes in bed lithology and thickness.

Latitudinal variation in locomotor activity rhythm in adult Drosophila ananassae

The parameters of the circadian rhythm of adult locomotor activity in strains of Drosophila ananassae originating between 6° and 34°N were variable and latitude-dependent. Two representative southern strains became active before sunrise, but one representative northern strain began activity after sunrise in nature. During entrainment to a 12 h light (L) : 12 h dark (D) cycle in the laboratory, the southern strains showed two peaks of activity, at the beginning and end of photophase, whereas the northern strains showed a single uninterrupted activity peak starting about 3 h after the lights-on. Among the strains, the phase angle difference (Ψ) during entrainment to 12 h L : 12 h D varied by about 5 h, the period of free-running rhythm(τ) in constant darkness by 3 h, the duration of the activity phase (α) by 7 h, and the duration of the resting phase (ρ) by 10 h. Lower latitude was correlated with an early Ψ (r = 0.977), a short τ (r = 0.975), a prolonged α (r = -0.995), a short ρ (r = 0.995) and a large α/ρ ratio (r = -0.963).

Organization of rat circadian rhythms during daily infusion of melatonin or S20098, a melatonin agonist.

Daily administration of melatonin or S20098, a melatonin agonist, is known to entrain the free-running circadian rhythms of rats. The effects of the duration of administration on entrainment were studied. The animals demonstrated free-running circadian rhythms (running-wheel activity, body temperature, general activity) in constant darkness. Daily infusions of melatonin or S20098 for 1, 8, or 16 h entrained the circadian rhythms to 24 h. Two daily infusions of 1 h (separated by 8 h) entrained the activity peak within the shorter time interval. The entraining properties of melatonin and S20098 were similar and were affected neither by pinealectomy nor by infusion of 1- or 8-h duration. However, with 16-h infusion, less than half of the animals became entrained. Once entrained, the phase angle between the onset of infusion and the rhythms (onset of activity or acrophase of body temperature) increased with the duration of infusion. Before entrainment, the free-running period increased with the duration of infusion, an effect that was not predictable from the phase response curve.

Circannual variations of lizard circadian activity rhythms in constant darkness

The daily pattern of locomotor activity of Podarcis sicula in the field changes from unimodal in spring to bimodal in summer, becoming unimodal again in autumn. Short-term experiments in which P. sicula collected in different months were tested under constant conditions immediately after capture showed that the activity pattern typical of each season is retained in the lizard circadian locomotor rhythm. In constant conditions, the bimodal pattern is associated with a short free-running period (τ) of the circadian locomotor rhythm and a long circadian activity (α), while the unimodal pattern is associated with a long τ and short α. To test whether seasonal changes in circadian locomotor rhythms are driven by a circannual clock, we recorded locomotor activity of lizards over 12–15 months in constant temperature and darkness. The present results demonstrate, for the first time in a vertebrate, the existence of circannual changes in constant darkness of both τ and α. In most lizards, the longest τ along its circannual cycle is associated with a short α, and the shortest τ in the same cycle with a long α, so that the pattern of mutual association between τ and α is the same as in short-term experiments. Most lizards, however, stayed unimodal all the time. This shows that changes in activity pattern from unimodal to bimodal (and vice versa) are induced by seasonal changes in environmental factors, instead of being incorporated into a circannual rhythm. Circannual changes in τ and α of locomotor rhythms may adaptively predispose the circadian system of P. sicula to a change in activity pattern as soon as seasonal changes in the environment demand it.

No evidence for extraocular photoreceptors in the circadian system of the Syrian hamster.

Campbell and Murphy reported recently that 3 h of bright light (13,000 lux) exposure to the area behind the knee caused phase shifts of the circadian rhythms of both body temperature and saliva melatonin in humans. The authors tested the hypothesis that extraocular photoreception is also involved in the circadian system of the Syrian hamster. Hamsters were bilaterally enucleated (eyes removed), and their backs were shaved. Hamsters with stable free-running rhythms in constant darkness were exposed to direct sunlight for 1 or 3 hours during their subjective night. Intact (control) animals showed phase shifts as expected, but the locomotor activity of enucleated animals was unaffected by the exposure to sunlight. The authors also measured the pineal melatonin content after exposure to sunlight. Pineal melatonin content in intact animals declined markedly as expected, but no decline was observed in the enucleated hamsters. The authors conclude that extraocular phototransduction is not capable of shifting the phase of the hamster's locomotor activity rhythm or of suppressing pineal melatonin synthesis.

Day/night differences in the stimulation of adenylate cyclase activity by calcium/calmodulin in chick pineal cell cultures: evidence for circadian regulation of cyclic AMP.

In chick pineal cell culture, stimulation of adenylate cyclase with the diterpene forskolin was greater during the subjective night than during the subjective day. This rhythm of cyclic AMP (cAMP) stimulation mimicked the rhythm of unstimulated cAMP measured previously during LD cycles from flow-through culture. Direct measurement of adenylate cyclase activity in permeabilized cells revealed an adenylate cyclase activity activated by Ca2+/calmodulin during the night but not during the day. However, this difference in adenylate cyclase activity at two times of the circadian cycle is apparent only when permeabilized cells were prewashed with buffer containing GTE When cAMP was measured from flow-through cultures maintained in continuous darkness to determine whether a circadian clock may regulate cAMP, a low-amplitude rhythm was measured. The circadian rhythm of cAMP was similar to the cAMP rhythm previously measured on LD cycles except that the rhythm in darkness had a lower amplitude. Similar to the suppression of melatonin, cAMP was suppressed by light presented during the middle of the night. LD differences in nocturnal cAMP levels were abolished with dipyridamole, an inhibitor of cyclic GMP (cGMP) phosphodiesterase. These results suggest that the rhythm of cAMP in chick pineal cells involves the stimulation of adenylate cyclase by Ca2+/calmodulin during the night and a GTP-dependent suppression of adenylate cyclase activity during the day. The photic suppression of cAMP at night involves the activation of a dipyridamole-sensitive, cGMP phosphodiesterase.

The role of melatonin and the pineal gland on development and smoltification of Atlantic salmon ( Salmo salar) parr

The present work used measurements of plasma melatonin throughout the year, pinealectomy, and/or intramuscular melatonin implants to investigate the role of melatonin and the pineal gland as intermediaries in the transfer of photic information on daily and calendar time in the control of the timing of the parr–smolt transformation in the Atlantic salmon. An initial experiment confirmed the presence of a seasonally changing light–dark rhythm of melatonin in potential S2 Atlantic salmon smolts, the profile of which accurately reflected the dark phase throughout the annual photoperiodic cycle. In the second experiment, potential S1 smolts were implanted and/or pinealectomised at the winter solstice. Although no variations in growth were observed between groups, smoltification in the pinealectomised plus implanted group was advanced by 3 weeks while the onset of smoltification in the pinealectomised fish was delayed by 3 weeks. In the final experiment, Atlantic salmon parr (3.5–7.0 g) which received melatonin-implants in June were significantly larger than the controls 1 month after implantation; this resulted in all the implanted fish entering the upper modal group by the time of smoltification whereas the control groups exhibited clear bimodal distributions. These results suggest that melatonin implantation and/or pinealectomy can alter the timing of seawater adaptation. Additionally, melatonin was shown to significantly alter the population structure of sibling Atlantic salmon parr resulting in 92% of individuals becoming S1 smolts. However, the mechanism(s) by which the pineal may influence development remains unclear.

Circadian Locomotor Activity Rhythms in the African Clawed Frog, Xenopus laevis: The Role of the Eye and the Hypothalamus

The effects of hypothalamic lesioning and removal of the eyes on locomotor activity rhythms of African clawed frog, Xenopus laevis were examined under light-dark cycles (LD12:12) and constant conditions. Frogs were kept individually and the activity rhythms at the bottom layer of water tank were recorded by means of the infrared photocells. Intact frogs displayed clear entrained nocturnal activity and expressed freerunning activity rhythms in constant darkness (DD), while some frogs did not freerun under co nstant dim light (dimLL) and constant light (LL). Freerunning periods in intact frogs were significantly shorter in dimLL than in DD. Although freerunning periods were shortened after blinding in same individuals, no significant changes in the freerunning periods were observed after blinding under dimLL and LL. When electrolytic lesions to the hypothalamus were performed, all frogs with more than 70% damage of the SCN abolished freerunning rhythms and in frogs with less than 70% damage, 57% of the animals became arrhythmic. In conclusion, (1) There is a circadian pacemaker somewhere outside the eyes, and it is probably situated in the hypothalamusincluding the SCN. (2) Both the eyes and the SCN are involved in the circadian system of the frogs.

A gene pleiotropically controlling developmental and circadian periods in the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae)

A gene causing an 8 h variation in the period of circadian rhythm in the adult locomotor activity was found in the melon fly Bactrocera Cucurbitae by artificial selection for development time from egg to adult eclosion at 25±2°C. The periods of free-running rhythm (mean±SD) in constant darkness were 22.6±0.6 and 22.5±0.4 h, respectively, in two replicate lines selected for short development time, and 30.9±0.9 and 26.5±3.6h, respectively, in two replicate lines selected for long development time. Thus, the development time and the period of circadian clock were correlated. The period locus was autosomal.

A gene pleiotropically controlling developmental and circadian periods in the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae)

A gene causing an 8 h variation in the period of circadian rhythm in the adult locomotor activity was found in the melon fly Bactrocera Cucurbitae by artificial selection for development time from egg to adult eclosion at 25±2°C. The periods of free-running rhythm (mean±SD) in constant darkness were 22.6±0.6 and 22.5±0.4 h, respectively, in two replicate lines selected for short development time, and 30.9±0.9 and 26.5±3.6h, respectively, in two replicate lines selected for long development time. Thus, the development time and the period of circadian clock were correlated. The period locus was autosomal.

Circadian rhythm of the cyanobacterium Synechocystis sp. strain PCC 6803 in the dark.

The cyanobacterium Synechocystis sp. strain PCC 6803 exhibited circadian rhythms in complete darkness. To monitor a circadian rhythm of the Synechocystis cells in darkness, we introduced a PdnaK1::luxAB gene fusion (S. Aoki, T. Kondo, and M. Ishiura, J. Bacteriol. 177:5606-5611, 1995), which was composed of a promoter region of the Synechocystis dnaK1 gene and a promoterless bacterial luciferase luxAB gene set, as a reporter into the chromosome of a dark-adapted Synechocystis strain. The resulting dnaK1-reporting strain showed bioluminescence rhythms with a period of 25 h (on agar medium supplemented with 5 mM glucose) for at least 7 days in darkness. The rhythms were reset by 12-h-light-12-h-dark cycles, and the period of the rhythms was temperature compensated for between 24 and 31 degrees C. These results indicate that light is not necessary for the oscillation of the circadian clock in Synechocystis.

Olfactory bulbectomy impedes social but not photic reentrainment of circadian rhythms in female Octodon degus.

Recent studies demonstrated that nonphotic (social) cues markedly accelerate reentrainment to large phase shifts of the light-dark (LD) cycles in female Octodon degus and that such changes are likely effected by chemosensory stimuli. This experiment investigated the effects of olfactory bulbectomies on (1) socially facilitated reentrainment rates of circadian rhythms following a 6-h phase advance of the LD cycle, (2) photic reentrainment rates of circadian rhythms following a 6-h advance of the LD cycle, (3) photic entrainment, and (4) the circadian period (tau) of activity rhythms in constant darkness (DD). olfactory bulbectomies (BX) blocked socially facilitated reentrainment rates but did not alter reentrainment rates of circadian rhythms to photic cues alone. In addition, BX lowered mean daily locomotor activity levels and decreased the amplitude of the activity rhythm in degus housed in entrained (LD 12:12) conditions but did not alter the phase of activity onset or offset, duration (alpha) of activity, or mean daily core body temperature. Bulbectomies also failed to modify tau of free-running activity rhythms. This experiment confirms that the olfactory bulbs and chemosensory cues are necessary for socially facilitated reentrainment. In contrast to their effects in nocturnal rodents, BX do not produce significant circadian photic changes in diurnal degus. This is the first experiment to determine that chemosensory stimuli modulate the circadian system in a diurnal rodent.

Regulation of thevgf gene in the golden hamster suprachiasmatic nucleus by light and by the circadian clock

By using in situ hybridization in the golden hamster brain, we have found that vgf mRNA levels are induced as a response to light stimulation in the suprachiasmatic nuclei (SCN), the site of the mammalian circadian pacemaker. The induction exhibits delayed kinetics relative to known light-induced immediate early genes: induction of vgf mRNA occurs over a period of 3 to 9 hours after light exposure. Photic induction of vgf expression does not occur in the paraventricular nucleus (PVN) of the hypothalamus, though this nucleus expresses vgf at the mRNA and protein levels. Photic induction of vgf in the SCN occurs only at circadian times when light also causes a phase shift of the circadian system. The irradiance threshold of vgf induction in the SCN closely matches that of the behavioral phase shifting response. In addition, basal expression of vgf in the SCN, but not in the PVN, exhibits a circadian rhythm in constant darkness. The photic induction and circadian rhythm of vgf expression are anatomically separated in the caudal and rostral portions of the SCN, respectively. These results represent the first example of a delayed response to light relative to light-induced immediate early genes at the mRNA level in the SCN.

Rhythmicity of swimming activity in an axenic population of Paramecium multimicronucleatum

A unicellular organism, Paramecium, exhibits circadian rhythm activities in many physiological phenomena, i.e., mating reactivity, photoaccumulation in Paramecium bursaria and mating type reversals in Paramecium multimicronucleatum. In this study, we used an image-processing system to analyze swimming activity in a population of Paramecium multimicronucleatum cultured axenically under 12 h-light/12 h-dark cycles (LD 12∶12). Swimming behavior was recorded both under LD 12∶12 and constant darkness and images tracing the tracks of Paramecium were produced every 4 min. Swimming activity was represented by the occupied area by the tracks relative to the total observed area. It is high during daytime and low at night and exhibits a freerunning rhythm in constant darkness. Furthermore, criteria for two major components of swimming behavior, straight and circle swimming, were established and analyzed. The results indicate that swimming behavior alters depending on the time of day: straight swimming increased during the day and circling was dominant around dusk both under LD 12∶12 and constant darkness.

Twilight transitions promote circadian entrainment to lengthening light-dark cycles.

The upper limits of entrainment of the circadian activity rhythm were compared in hamsters initially exposed to daily light-dark (LD) cycles with either abrupt (LD-rectangular) or simulated twilight (LD-twilight) transitions. Daytime illuminance (10 lx) and the total amount of light emitted per day were the same under the two LD cycles. One-half of the animals in each condition had access to dark nest boxes. The period of the LD cycles was then increased from 24 to 26 h, by 5 min per day. All animals in LD-twilight remained entrained to the lengthening cycle, whereas 60% of those in LD-rectangular began to free run well before the period of the cycle reached 26 h. These effects were independent of nest box availability. The lengthening LD cycles exerted clear aftereffects on the period of the rhythms in constant darkness, the magnitude of which was related to the efficacy of prior entrainment. The results indicate that twilight transitions raise the upper limit of entrainment to LD cycles, suggesting that their inclusion increases the strength of the LD zeitgeber.

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.

Antimanic drugs stabilize hamster circadian rhythms

The circadian wheel-running rhythm of golden hamsters was monitored during chronic oral treatment with four mood-stabilizing drugs in doses relevant for treatment of manic-depressive disorder. Carbamazepine and verapamil shortened the duration of locomotor activity and improved the stability of the pattern of running activity. At comparable doses, valproate had no clear effect on any rhythm variable tested. None of these drugs consistently altered the phase of light-synchronized running rhythms or the period of the rhythm in constant darkness. The results are compared to data showing that lithium, which delays entrained phase and lengthens circadian period in hamsters, also shortens activity duration and increases the stability of wheel-running rhythms. Stabilization of circadian rhythms may be a key action of clinically effective mood-stabilizing drugs.