Bright light does not immediately stop the circadian clock of gonyaulax

Circadian rhythms in acid-stimulated bioluminescence and cell division are observed for at least 16 days in bright continuous light (4.5 milliwatts per square centimeter or 20,000 lux). The photosynthesis rhythm also fails to stop immediately upon transfer of cell suspensions to bright light. After about 4 weeks under these conditions, all rhythms were observed to damp out. In cells transferred from bright light to continuous darkness, the rhythms were reset to about circadian hour 12 to 14, the phase of the beginning of a normal night.     

Light Cycles Given During Development Affect Freerunning Period of Circadian Locomotor Rhythm of period Mutants in Drosophila melanogaster

We reared wild type (Canton-S) and period mutant flies, i.e., per(S) and per(L), of Drosophila melanogaster in constant darkness, constant light or 24h light dark cycles with various light to dark ratios throughout the development from embryo to early adult. The locomotor activity rhythms of newly eclosed individuals were subsequently monitored in the lighting conditions, in which they had been reared, for several days and then in constant darkness. Circadian rhythms were clearly exhibited in constant darkness even in flies reared in constant light and constant darkness as well as flies reared in light-dark cycles, but the freerunning period differed among groups. The results suggest that the circadian clock is assembled without any cyclical photic information, and that the light influences the developing circadian clock of Drosophila to alter the freerunning period. The effects of light on the rhythm differed in some aspects between per(L) flies and the other two strains. Possible mechanisms through which light affects the developing circadian clock are discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Interseasonal variation in the circadian rhythms of locomotor activity and temperature selection in sleepy lizards, <Emphasis Type="Italic">Tiliqua rugosa</Emphasis>

Few studies in non-mammalian vertebrates have examined how various effectors of the circadian system interact. To determine if the daily locomotor and behavioural thermoregulatory rhythms of Tiliqua rugosa are both controlled by the circadian system in different seasons, lizards were tested in laboratory thermal gradients in four seasons and in constant darkness. Circadian rhythmicity for both rhythms was present in each season, being most pronounced in spring and summer and least evident in autumn. Most lizards displayed a unimodal locomotor activity pattern across all seasons. However, some individuals presented a bimodal locomotor activity pattern in spring and summer. Seasonal variations in the phase relationships of both rhythms to the light:dark (LD) cycle were demonstrated. No seasonal differences in the free-running period lengths of either rhythm were detected, raising the possibility that a single circadian pacemaker drives both rhythms in this species. Our present results demonstrate that both rhythms are similarly controlled by the circadian system in each season. Although seasonal variations in the thermal preferences of reptiles both in the field and laboratory have previously been well documented, this study is the first to demonstrate circadian rhythms of temperature selection in a reptile species in each season.     

Circadian rhythms in the activities of brain and liver aldehyde dehydrogenase isozymes in mice

Circadian variations in the activities of aldehyde dehydrogenase (ALDH) isozymes in the subcellular fractions of the brain and liver were investigated in male and female mice of C57BL/6J strain. The rhythms in high Km-ALDH activities of brain and liver mitochondrial fractions which existed in ordinary light-dark cycle were not observed in animals maintained in the continuous darkness for two weeks. The rhythms in high Km-ALDH activities of hepatic soluble and microsomal fractions existed in both ordinary cycle and total darkness but the rhythmic phases were different. In the low Km-ALDH activity of hepatic mitochondrial fraction, the circadian rhythm was similar in two lighting conditions. There was sex difference in the existence of the circadian rhythm. It seems that the ALDH activity of mice is influenced by light-dark cycle and sex hormones.     

Circadian rhythmicity in dopamine content of mammalian retina: role of the photoreceptors

Dopamine, the predominant retinal catecholamine, is a neurotransmitter and neuromodulator known to regulate light-adaptive retinal processes. Because dopamine influences several rhythmic events in the retina it is also a candidate for a retinal circadian signal. Using high performance liquid chromatography (HPLC), we have tested whether dopamine and its breakdown products are rhythmic in Royal College of Surgeons (RCS) rats with normal and dystrophic retinas. In both normal and mutant animals entrained to a 12-h light/12-h dark cycle, we found robust daily rhythms of dopamine and its two major metabolites. To address circadian rhythmicity of dopamine content, rats were entrained to light/dark cycles and released into constant darkness, using the circadian rhythm of wheel-running activity as a marker of each individual's circadian phase. Circadian rhythms of dopamine and metabolite content persisted in both wild type and retinally degenerate animals held for two weeks in constant darkness. Our results demonstrate for the first time clear circadian rhythms of dopamine content and turnover in a free-running mammal, and suggest that rods and cones are not required for dopamine rhythmicity.     

Analysis of three separate probes suggests the absence of endocytosis in Neurospora crassa hyphae

Reports of the existence of endocytosis in filamentous fungi have been conflicting and inconclusive. For this reason, we have tested three independent markers in Neurospora crassa: the electron opaque marker lanthanum (La) and the fluorescent probes Lucifer yellow (LY) and FM4-64. Both La and LY were endocytosed by Saccharomyces cerevisiae cells, which were used as positive controls for endocytosis, but the probes did not accumulate in N. crassa hyphae. Only FM4-64 became internalized into N. crassa hyphae, but it induced abnormal changes in membrane systems and its internalization could be explained by mechanisms other than endocytosis. Together, our results suggest that endocytosis does not occur in N. crassa hyphae and question whether the styryl dyes do in fact reliably report normal endocytosis in filamentous fungi.     

Circadian Locomotor Activity in Freshwater Decapods: An Ecological Approach

Circadian rhythms of locomotor activity shown by freshwater decapods display different patterns among crayfish, Procambarus, and crabs, when exposed to artificial light-dark cycles. Crayfish are mainly nocturnal while a crepuscular activity is observed in crabs of the genus Pseudothelphusa. In constant darkness, free running rhythms are displayed in unimodal or bimodal patterns by crayfish Procambarus; however, Pseudothelphusa continues to show bimodal rhythms. The many studies using locomotor activity indicate that the rhythm in freshwater crabs is circadian in nature, but that a multioscillatory system may be controlling the overt rhythm. In the present study, the implications of different locomotor activity patterns are analyzed in selected freshwater decapods with regard to the interactions between light and the organisms. Crabs and crayfish are commonly found in similar habitats, often sharing the same environment; however, different patterns of locomotor activity as well as different sensitivities of the bouts of activity with regard to entrainment by light, indicate that distinct temporal niches may exist that result in temporal exclusion or low competition.     

Rhythms of differentiation and diacylglycerol in Neurospora.

Although the fungus Neurospora crassa is a relatively simple lower eukaryote, its circadian system may be more complex than previously thought. In this paper we review evidence suggesting that there may be several output pathways coupled in complex ways to a single oscillator, or that there may be more than one oscillator driving independent output pathways. We have described two new rhythms in Neurospora that are not tightly coupled to the rhythm of conidiation bands that is the standard assay for the state of the Neurospora circadian clock. The first is a rhythm in the timing of differentiation, i.e. the production of aerial hyphae and spores. Large regions of the mycelium differentiate synchronously, as if responding to a spatially widespread signal. This rhythm may be distinct from the timer that sets the determination switch controlling the spatial pattern of conidiation bands. The second new rhythm is an oscillation in the levels of the neutral lipid diacylglycerol (DAG). This rhythm is found in all regions of a colony and is not always in phase with the rhythm of conidiation bands. The DAG rhythm shares some characteristics with the differentiation rhythm and has the potential to act as the signal that induces rhythmic differentiation.     

Mutation of the Cys-9 Gene, Which Encodes Thioredoxin Reductase, Affects the Circadian Conidiation Rhythm in Neurospora Crassa

Ten cysteine auxotrophs of Neurospora crassa were examined with regard to the period lengths of their circadian conidiation rhythms. One of the these cysteine auxotrophs, cys-9, showed dramatic changes in the circadian conidiation rhythm. At 10 μM methionine, the cys-9 mutant had a period length that was 5 hr shorter than that of the wild-type strain during the first 3 days after transfer to continuous darkness. At this concentration of methionine, the period length was unstable after the fourth day and varied widely from 11 to 31 hr. In contrast, other cysteine auxotrophs did not show such instability of the period length at any of the concentrations of methionine tested. Furthermore, only the cys-9 mutant exhibited partial loss of the capacity for temperature compensation of the period length. With regard to cold-induced phase-shifting of the circadian conidiation rhythm, the cys-9 mutant was more sensitive than the wild-type strain to low temperature. The cys-9(+) gene was cloned and was found to encode NADPH-dependent thioredoxin reductase. These results indicate that mutation of the gene for thioredoxin reductase results in abnormal expression of the circadian conidiation rhythm in N. crassa.     

Time-lapse analysis of the circadian rhythms of conidiation and growth rate in neurospora

The filamentous fungus Neurospora crassa has frequently served as a model organism for the study of circadian rhythms through its ability to form conidial spores on a daily basis. This phenomenon leaves a spatial pattern of conidiation bands along a solid surface of agar after several days of growth. Using time-lapse video, the authors have quantified the rate of conidiation. They have found that conidia do not form at a specified lag time after the growth front is laid down, but rather the band region tends to simultaneously develop over a short time frame. This produces a sharp peak when the conidiation rate is plotted against time. In addition, the authors used time-lapse video to assay growth rate with greater accuracy than previously reported. It is usually assumed that Neurospora's rate of growth is constant, and this assumption of linear growth has been used extensively to determine period and phase of the conidiation circadian rhythm. The authors have confirmed an earlier report of nonlinear growth rate and have shown that the growth rate varies by a factor of about 2 with each circadian cycle. They have demonstrated that the errors in calculating times of conidiation peaks are maximally 1 to 2 h if linearity is assumed. The conidiation rate and growth rate rhythms are not apparent under conditions (using mutants or high or low temperatures) where the spatial banding rhythm is not observed. In light/dark entraining conditions, the conidiation rate and growth rate rhythms maintain the same phase relationship in different T-cycles. These data are consistent with the hypothesis that the growth rate rhythm is a consequence of the conidiation rate rhythm.     

Fungal development and the COP9 signalosome

The conserved COP9 signalosome (CSN) multiprotein complex is located at the interface between cellular signaling, protein modification, life span and the development of multicellular organisms. CSN is required for light-controlled responses in filamentous fungi. This includes the circadian rhythm of Neurospora crassa or the repression of sexual development by light in Aspergillus nidulans. In contrast to plants and animals, CSN is not essential for fungal viability. Therefore fungi are suitable models to study CSN composition, activity and cellular functions and its role in light controlled development.     

Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster

Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 clock neurons that are roughly classified into seven groups. In the circuit, a group of neurons expressing pigment-dispersing factor (PDF) play an important role in organizing the pacemaking system. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-positive neurons. To explore its role in the circadian pacemaker, we examined the circadian locomotor rhythms of pdf-Gal4/UAS-TNT transgenic flies in which chemical synaptic transmission in PDF-positive neurons was blocked by expressed tetanus toxin light chain (TNT). In constant darkness (DD), the flies showed a free-running rhythm, which was similar to that of wild-type flies but significantly different from pdf null mutants. Under constant light conditions (LL), however, they often showed complex rhythms with a short period and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-positive and PDF-negative clock neurons in the transgenic flies. These results suggest that both PDF and UNT play important roles in the Drosophila circadian clock, and activation of PDF pathway alone by LL leads to the complex locomotor rhythm through desynchronized oscillation among some of the clock neurons.     

Light pulses entrain the circadian activity rhythm of a diurnal rodent (Ammospermophilus leucurus)

Circadian rhythms of wheel-running activity of the antelope ground squirrel (Ammospermophilus leucurus) were entrained by light-dark cycles (LD: 100 1x vs total darkness) with periods (T) between ca 23.75 and 24.75 hr. Two 1-hr light pulses per cycle ('skeleton photoperiods') with T = 24.25 hr as well as one 1-hr light pulse per cycle with Ts of 23.75 and 24.25 hr were effective in entraining the circadian activity rhythms in at least 50% of the antelope ground squirrels. Phase and period responses to single 1-hr light pulses were measured which depend on the initial phase and period of the rhythm. It is concluded that discrete (phasic) light input contributes to the mechanism of entrainment to LD cycles in diurnal rodents.     

Gravity and light effects on the circadian clock of a desert beetle,Trigonoscelis gigas

Circadian function is affected by exposure to altered ambient force environments. Under non-earth gravitational fields, both basic features of circadian rhythms and the expression of the clock responsible for these rhythms are altered. We examined the activity rhythm of the tenebrionid beetle, Trigonoscelis gigas, in conditions of microgravity (microG; spaceflight), earth's gravity (1 G) and 2 G (centrifugation). Data were recorded under a light-dark cycle (LD), constant light (LL), and constant darkness (DD). Free-running period (tau) was significantly affected by both the gravitational field and ambient light intensity. In DD, tau was longer under 2 G than under either 1 G or microG. In addition, tauLL was significantly different from tauDD under microG and 1 G, but not under 2 G.     

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.     

Relationship of atypical melatonin rhythm with two circadian clock outputs in B6D2F(1) mice

Circadian rhythms in body temperature, locomotor activity, and the circadian changes of plasma and pineal melatonin content were investigated in B6D2F(1) mice synchronized by 12 h of light and 12 h of darkness. During 8 wk continuous recording, activity and temperature displayed a marked stable and reproducible circadian rhythm, with both peaks occurring near the middle of darkness. Both 24- and 12-h rhythmic components were also significantly detected. Mean plasma melatonin concentration rose steadily during the light span and reached a maximum (30.6 +/- 10.0 pg/ml) at 11 h after light onset (HALO), then gradually decreased after the onset of darkness to a nadir (4.7 +/- 0.4 pg/ml) at 20 HALO. Mean pineal content followed a pattern parallel to that of plasma concentration (peak at 11 HALO: 17.7 +/- 1.0 pg/gland; trough at 17 HALO: 4.7 +/- 1.0 pg/gland). In addition, a second sharp peak was observed at 21 HALO (20.2 +/- 3.5 pg/gland). Plasma and pineal contents displayed large and statistically significant circadian changes, with a composite rhythm of period (24 + 12 h). This mouse model has predominant production and secretion of melatonin during the day. This possibly contributes to a similar coupling between chronopharmacology mechanisms and the rest-activity cycle in these mice and in human subjects.