Constant Dark Conditions Research Articles

After-ripening, Light Conditions, and Cold Stratification Influence Germination of Marula [Sclerocarya birrea (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro] Seeds

Marula [Sclerocarya birrea (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro (Anacardiaceae)] is used in many African countries as a food crop and is also in demand for industrial purposes. The fruit pulp has high vitamin C levels and the nuts have a high protein and oil content. The fruit pulp is commercially used in the production of an alcoholic beverage (Amarula Cream) and the oil is gaining importance in the cosmetic industry. Although attempts are being made to domesticate this high-value indigenous tree, there is very limited information available on aspects of seed germination. Our study investigated the role of light, temperature, cold stratification, and after-ripening on seed germination of S. birrea. Temperatures between 25 and 35 °C favored germination of opercula-removed seeds under continuous dark conditions. White light completely inhibited seed germination with the inhibitory effect being reversed when seeds were transferred to dark conditions. This photoinhibitory effect on opercula-removed seeds was lost after 12 months of seed storage at room temperature in the dark. Cold stratification (5 °C) of intact seeds for 14 days significantly improved germination (65%) as compared with nonstratified seeds (32%). Pregermination treatments (acid scarification, boiling water, dry heat, soaking, and plant growth regulators) of S. birrea seeds did not promote germination. Seeds of S. birrea can be considered orthodox because they tolerated desiccation without significant loss of viability. Both intact and opercula-removed seeds readily imbibe water suggesting physiological rather than physical dormancy. The highest germination percentage was recorded under constant dark conditions at 25 °C for opercula-removed seeds exposed to an after-ripening period of 12 months. This study indicates that after-ripening, light conditions, and cold stratification are critical factors for germination of S. birrea seeds.

Evidences on the transient disruption of Sabella spallanzanii (Polychaeta, Sabellidae) fan activity rhythm in laboratory constant darkness

Circadian and ultradian rhythms were analyzed in Sabella spallanzanii tested in laboratory constant darkness conditions. A video camera took images per 30 s during a week. The number of times animals opened the fan per 30 min was counted by an observer. Resulting time series were subdivided into 24‐h segments in order to assess the change of the activity rhythm over consecutive days. Periodogram and waveform analyses measured the periodicity and the pattern of fluctuation, at each cycle. Results indicate that animals express a major circadian peak in the first day of test, and this progressively spits into subcomponents from days 2 to 4. At days 5 and 6 the circadian peak is reconstituted. The activity phase of a rhythm (α) and its amplitude of oscillation were also computed per 24‐h segments. The values of different time series were averaged at corresponding 24‐h stages. A significant increment of mean α was observed from days 1 to 4, while conversely the amplitude decreased. From days 5 to day 6, an opposite trend in both variables was observed. These results are descriptive but can be interpreted in the context of models accounting for the generation of ultradian rhythms when controlling oscillators became transiently uncoupled.

Period gene expression in the diurnal degu (Octodon degus) differs from the nocturnal laboratory rat (Rattus norvegicus)

Recent data suggest that both nocturnal and diurnal mammals generate circadian rhythms using similarly phased feedback loops involving Period genes in the suprachiasmatic nuclei (SCN) of the hypothalamus. These molecular oscillations also exist in the brain outside of the SCN, but the relationship between SCN and extra-SCN oscillations is unclear. We hypothesized that a comparison of "diurnal" and "nocturnal" central nervous system Per rhythms would uncover differences in the underlying circadian mechanisms between these two chronotypes. Therefore, this study compared the 24-h oscillatory patterns of Per1 and Per2 mRNA in the SCN and putative striatum and cortex of Octodon degus (degu), a diurnal hystricognath rodent, with those of the nocturnal laboratory rat, Rattus norvegicus. The brains of adult male degus and rats were collected at 2-h intervals across 24 h in entrained light-dark and constant darkness conditions, and sections were analyzed via in situ hybridization. In the SCN, degu Per1 and Per2 hybridization signal exhibited 24-h oscillatory patterns similar in phasing to those seen in other rodents, with peaks occurring during the light period and troughs during the dark period. However, Per1 remained elevated for five fewer hours in the degu than in the rat, and Per2 remained elevated for two fewer hours in the degu. In brain areas outside of the SCN, the phase of Per2 hybridization signal rhythms in the degu were 180 degrees out of phase with those found in the rat, and Per1 hybridization signal lacked significant rhythmicity. These results suggest that, while certain basic components of the transcriptional-translational feedback loop generating circadian rhythms are similar in diurnal and nocturnal mammals, there are variations that may reflect adaptations to circadian niche.

Sex‐specific differences in spatial behaviour in the flesh fly Sarcophaga crassipalpis

Abstract Territoriality in the flesh fly Sarcophaga crassipalpis (Diptera: Sarcophagidae) is studied in the laboratory. In rectangular enclosures, male flies exhibit a lower tolerance (occupation of the same physical space) of same‐sex conspecifics than do females. In circular arenas, male flies show significantly higher levels of spatial separation among themselves (as determined from nearest neighbour analyses) than do females: males show a slight tendency towards a uniform distribution, whereas females exhibit a slight tendency towards clustering. The male spatial behaviour occurs during the photophase but not the scotophase, suggesting that visual cues are required for maintenance of inter‐individual spacing. No significant differences in male spacing behaviour occur between subjective day and subjective night in either constant dark or constant light conditions, suggesting that spatial patterning is not driven by a circadian rhythm.

From daily behavior to hormonal and neurotransmitters rhythms: Comparison between diurnal and nocturnal rat species

Mammalian species can be defined as diurnal or nocturnal, depending on the temporal niche during which they are active. Even if general activity occurs during nighttime in nocturnal rodents, there is a patchwork of general activity patterns in diurnal rodents, including frequent bimodality (so-called crepuscular pattern, i.e., dawn and dusk peaks of activity) and a switch to a nocturnal pattern under certain circumstances. This raises the question of whether crepuscular species have a bimodal or diurnal – as opposed to nocturnal – physiology. To this end, we investigated several daily behavioral, hormonal and neurochemical rhythms in the diurnal Sudanian grass rat ( Arvicanthis ansorgei) and the nocturnal Long–Evans rat ( Rattus norvegicus). Daily rhythms of general activity, wheel-running activity and body temperature, with or without blocked wheel, were diurnal and bimodal for A. ansorgei, and nocturnal and unimodal for Long–Evans rats. Moreover, A. ansorgei and Long–Evans rats exposed to light–dark cycles were respectively more and less active, compared to conditions of constant darkness. In contrast to other diurnal rodents, wheel availability in A. ansorgei did not switch their general activity pattern. Daily, unimodal rhythm of plasma leptin was in phase-opposition between the two rodent species. In the hippocampus, a daily, unimodal rhythm of serotonin in A. ansorgei occurred 7 h earlier than that in Long–Evans rats, whereas a daily, unimodal rhythm of dopamine was unexpectedly concomitant in both species. Multiparameter analysis demonstrates that in spite of bimodal rhythms linked with locomotor activity, A. ansorgei have a diurnally oriented physiology.

Phospholipase C beta 4 in mouse hepatocytes: Rhythmic expression and cellular distribution

BackgroundCircadian regulated physiological processes have been well documented in the mammalian liver. Phospholipases are important mediators of both cytoplasmic and nuclear signaling mechanisms in hepatocytes, and despite a potentially critical role for these enzymes in regulating the temporal aspect of hepatic physiology, their involvement in the circadian liver clock has not been the subject of much investigation. The phospholipase C β4 (PLCβ4) enzyme is of particular interest as it has been linked to circadian clock function. In general, there is no knowledge of the role of the PLCβ4 isozyme in mammalian hepatocytes as this is the first report of its expression in the mammalian liver.ResultsWe found that in the liver of mice housed on a light:dark cycle, PLCβ4 protein underwent a significant circadian rhythm with a peak occurring during the early night. In constant darkness, the protein rhythm was more robust and peaked around dusk. We also observed a significant oscillation in plcβ4 gene expression in the livers of mice housed in both photoperiodic and constant dark conditions. The cellular distribution of the protein in hepatocytes varied over the course of the circadian day with PLCβ4 primarily cytoplasmic around dusk and nuclear at dawn.ConclusionOur results indicate that PLCβ4 gene and protein expression is regulated by a circadian clock in the mouse liver and is not dependent on the external photoperiod. A light-independent daily translocation of PLCβ4 implies that it may play a key role in nuclear signaling in hepatocytes and serve as a daily temporal cue for physiological processes in the liver.

Foraging behaviour and habitat selection in pit-building antlion larvae in constant light or dark conditions

Pit-building antlion larvae are small sit-and-wait arthropod predators that dig conical pits in sandy soils. We investigated the effect of exposure to constant light versus constant dark conditions on antlion behaviour. Antlions tended to relocate less often, construct pits more frequently and construct larger pits in constant light. We interpret this as an indication of dichotomous behaviour under light versus dark conditions such that antlion larvae hunt during the day and modify their positions at night. Antlion larvae immediately responded to the complete switch in this illumination treatment and adapted their behaviour to fit the new conditions. Prey capture success did not differ between the constant light and constant dark conditions. When provided with a choice between light and dark conditions, the vast majority of larvae preferred the former. Those that preferred constant dark, however, were on average larger than those that preferred constant light. We thus suggest that since larger antlion larvae possess larger fat reserves, they can afford reductions in foraging activity (possibly to increase safety). An examination of the antlion trade-off between their preference for light and their preference for sand depth (which may affect their ability to evade predators) showed that their response to illumination was much more prominent, irrespective of the sand depth. Finally, combining light with other factors such as temperature and density may further elucidate microhabitat selection in pit-building antlion larvae.

Light‐ and dark‐dependent orexinergic neuronal signals promote neurodegenerative phenomena accounting for distinct behavioral responses in the teleost Thalassoma pavo

The neuropeptides hypocretins/orexins (ORX) are known to control state-dependent activities such as sleep-wakefulness, energy homeostasis, thermoregulation, and maternal behaviors. To date, interests regarding environmental-related ORX-ergic neuronal functions have dealt with mammals; only recently is attention beginning to be directed toward aquatic vertebrates. Here, photoperiod-dependent effects of ORX-A on behavioral, neurodegenerative and transcriptional activities were evaluated in some forebrain areas of a teleost Labridae (ornate wrasse, Thalassoma pavo). The ornate wrasse, when treated intraperitoneally with a high physiological dose (100 ng/g) of ORX-A and exposed to a natural photoperiod (12L:12D), exhibited very high (P < 0.001) locomotion and feeding behaviors. ORX-A in the presence of a constant light photoperiod accounted for numerically even greater (>500%) feeding frequencies. Conversely, constant dark conditions very strongly reduced feeding habits and moderately (P < 0.05) increased resting states. In this case, the same ORX-A and photoperiodic conditions responsible for altered behaviors also induced neurodegenerative processes in the different hypothalamic, mesencephalic, and telencephalic neuronal fields. Interestingly, this ORX-A treatment seemed to be correlated to greater up-regulatory patterns of ORX receptor mRNA in these same brain areas, above all under constant light conditions rather than natural photoperiod. On the other hand, telencephalic sites provided a very active expression capacity during the dark phase. Overall, these results suggest for the first time that at least in the ornate wrasse, light- and dark-dependent ORX-ergic neuronal activities are able to cause short- and long-term abnormal motor behaviors, likely through neurodegenerative and transcriptional events in a brain regional manner.

Differential entrainment of a social rhythm in adolescent mice

Daily routines in animal activities range from sleep–wake cycles, to foraging bouts, to social interactions. Among animals living within groups, it is unclear whether the motivations that underlie social interactions respond to daily light–dark (LD) cycles or endogenous circadian rhythms. Employing two mouse strains (BALB/cJ [BALB] and C57BL/6J [B6]) with genetically based differences in social affect and circadian rhythms, we examined how social investigation (SI) is modulated by social deprivation and circadian factors. We found a genetic influence on SI that was moderated by the preceding duration of social deprivation, requiring 3–6h of social isolation prior to testing. Following 6h of social deprivation, the SI responses of adolescent B6 mice were greater than those of BALB mice only when the isolation period was imposed during the dark phase of the LD cycle. When B6 mice were weaned into conditions of constant darkness, a novel, endogenous social rhythm emerged, which was characterized by two pronounced peaks of social responsiveness (relative to one peak under LD entrainment) that were separated by 12-h intervals. Irrespective of the lighting conditions during social isolation, the SI responses of adolescent BALB mice did not oscillate across the day. Similar strain-dependent patterns of sociability were evident within groups of mice that were left undisturbed in their home cage under LD entrainment or constant darkness. Overall, genetic influences on the social phenotypes of adolescent mice are thus moderated by an interaction between social deprivation and oscillations of an endogenous social rhythm that entrains to the LD cycle.

Photic and circadian regulation of melatonin production in the Mozambique tilapia Oreochromis mossambicus

Diverse circadian systems related to phylogeny and ecological adaptive strategies are proposed in teleosts. Recently, retinal photoreception was reported to be important for the circadian pacemaking activities of the Nile tilapia Oreochromis niloticus. We aimed to confirm the photic and circadian responsiveness of its close relative—the Mozambique tilapia O. mossambicus. Melatonin production in cannulated or ophthalmectomized fish and its secretion from cultured pineal glands were examined under several light regimes. Melatonin production in the cannulated tilapias was measured at 3-h intervals; it fluctuated daily, with a nocturnal increase and a diurnal decrease. Exposing the cannulated fish to several light intensities (1500–0.1 lx) and to natural light (0.1 and 0.3 lx) suppressed melatonin levels within 30 min. Static pineal gland culture under light–dark and reverse light–dark cycles revealed that melatonin synthesis increased during the dark periods. Rhythmic melatonin synthesis disappeared on pineal gland culture under constant dark and light conditions. After ophthalmectomy, plasma melatonin levels did not vary with light–dark cycles. These results suggest that (1) Mozambique tilapias possess strong photic responsiveness, (2) their pineal glands are sensitive to light but lack circadian pacemaker activity, and (3) they require lateral eyes for rhythmic melatonin secretion from the pineal gland.

Direct effect of light on 24-h variation of aqueous humor protein concentration in Sprague–Dawley rats

Sprague–Dawley rats 10–12 weeks of age were entrained to a standard light–dark cycle with lights turned on at 6 am and off at 6 pm. Variations of 24-h aqueous humor protein concentration were determined. Samples were taken every 4 h ( N = 10–14) under the standard light–dark condition at 8 pm, midnight, 4 am, 8 am, noon, and 4 pm. Under an acute constant dark condition, when lights were not turned on at 6 am, samples were collected at 8 am, noon, 4 pm, and 8 pm. Aqueous humor protein concentrations under the standard light–dark condition were found in the range of 0.305 ± 0.115 mg/ml (mean ± SD, N = 10) at midnight to 1.505 ± 0.342 mg/ml ( N = 14) at noon. The 3 light-phase protein concentrations were each higher than the 3 dark-phase concentrations. Aqueous humor protein concentrations at 8 am, noon, and 4 pm under the acute constant dark condition were each higher than the concentrations at 8 pm (after both 2 h and 26 h in the dark), midnight, and 4 am, demonstrating an endogenously driven 24-h pattern. At 8 am, noon, and 4 pm, protein concentrations were 56–147% higher when exposed to light. Intraocular pressure (IOP) was monitored using telemetry in separate groups of light–dark entrained rats under the standard light–dark condition and the acute constant dark condition. The 24-h IOP pattern was inverse to the 24-h pattern of aqueous humor protein concentration under the standard light–dark condition, and this IOP pattern was not altered by the acute constant dark condition. In conclusion, an endogenously driven 24-h variation of aqueous humor protein concentration occurred in Sprague–Dawley rats with higher concentrations during the light-phase than the dark-phase. This endogenous pattern of protein concentration was accentuated by a direct effect of light, which was unrelated to the 24-h pattern of IOP.

DEC1 Modulates the Circadian Phase of Clock Gene Expression

DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E' boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E'-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E' box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1-/- mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.

Circadian leaflet movements of Tylosema esculentum (Burch) A. Schreib, and the abolishment of these diurnal movements by potassium deficiency

Tylosema esculentum (Burch) A. Schreib is an underutilized drought-tolerant legume, native to the arid and semi-arid regions of southern Africa, which produces protein- and oil-rich seed and tubers with potential for use as human food and animal fodder. Laboratory experiments were conducted, in order to investigate the nature of the characteristic leaflet movements of this species and the potential effects of potassium on them. The rhythm of leaflet movements was maintained in constant dark conditions and at several temperature regimes and it is also synchronized with the light cycles. Substantially, this study confirms the endogenous nature of T. esculentum leaflet movements, since they have all the main criteria of circadian rhythms. The crucial role of potassium on these movements and consequently on the total water economy of the species is also indicated, as long as there was an undoubted absence of leaflet movements in the potassium-deficient plants. This effect has to be taken seriously into account for any future attempts of the cultivation of the plant in arid and semi-arid environments.

Cycloheximide Induces Synchronous Swelling of Perialgal Vacuoles Enclosing Symbiotic Chlorella vulgaris and Digestion of the Algae in the Ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.

Dopamine modulates diurnal and circadian rhythms of protein phosphorylation in photoreceptor cells of mouse retina

Many aspects of photoreceptor metabolism are regulated as diurnal or circadian rhythms. The nature of the signals that drive rhythms in mouse photoreceptors is unknown. Dopamine amacrine cells in mouse retina express core circadian clock genes, leading us to test the hypothesis that dopamine regulates rhythms of protein phosphorylation in photoreceptor cells. To this end we investigated the phosphorylation of phosducin, an abundant photoreceptor-specific phosphoprotein. In mice exposed to a daily light-dark cycle, robust daily rhythms of phosducin phosphorylation and retinal dopamine metabolism were observed. Phospho-phosducin levels were low during the daytime and high at night, and correlated negatively with levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid. The effect of light on phospho-phosducin levels was mimicked by pharmacological activation of dopamine D4 receptors. The amplitude of the diurnal rhythm of phospho-phosducin was reduced by > 50% in D4 receptor-knockout mice, due to higher daytime levels of phospho-phosducin. In addition, the daytime level of phospho-phosducin was significantly elevated by L-745,870, a dopamine D4 receptor antagonist. These data indicate that dopamine and other light-dependent processes cooperatively regulate the diurnal rhythm of phosducin phosphorylation. Under conditions of constant darkness a circadian rhythm of phosducin phosphorylation was observed, which correlated negatively with the circadian rhythm of 3,4-dihydroxyphenylacetic acid levels. The circadian fluctuation of phospho-phosducin was completely abolished by constant infusion of L-745,870, indicating that the rhythm of phospho-phosducin level is driven by dopamine. Thus, dopamine release in response to light and circadian clocks drives daily rhythms of protein phosphorylation in photoreceptor cells.

Modeling the Effects of Cell Cycle M-phase Transcriptional Inhibition on Circadian Oscillation

Circadian clocks are endogenous time-keeping systems that temporally organize biological processes. Gating of cell cycle events by a circadian clock is a universal observation that is currently considered a mechanism serving to protect DNA from diurnal exposure to ultraviolet radiation or other mutagens. In this study, we put forward another possibility: that such gating helps to insulate the circadian clock from perturbations induced by transcriptional inhibition during the M phase of the cell cycle. We introduced a periodic pulse of transcriptional inhibition into a previously published mammalian circadian model and simulated the behavior of the modified model under both constant darkness and light–dark cycle conditions. The simulation results under constant darkness indicated that periodic transcriptional inhibition could entrain/lock the circadian clock just as a light–dark cycle does. At equilibrium states, a transcriptional inhibition pulse of certain periods was always locked close to certain circadian phases where inhibition on Per and Bmal1 mRNA synthesis was most balanced. In a light–dark cycle condition, inhibitions imposed at different parts of a circadian period induced different degrees of perturbation to the circadian clock. When imposed at the middle- or late-night phase, the transcriptional inhibition cycle induced the least perturbations to the circadian clock. The late-night time window of least perturbation overlapped with the experimentally observed time window, where mitosis is most frequent. This supports our hypothesis that the circadian clock gates the cell cycle M phase to certain circadian phases to minimize perturbations induced by the latter. This study reveals the hidden effects of the cell division cycle on the circadian clock and, together with the current picture of genome stability maintenance by circadian gating of cell cycle, provides a more comprehensive understanding of the phenomenon of circading gating of cell cycle.

Computational design and evolution of the oscillatory response under light–dark cycles

Circadian clocks are biological systems behaving as oscillators even in constant dark conditions. We propose to use a new strategy based on computational design to provide evidence on the origin and evolution of molecular clocks. We design synthetic molecular clocks having a reduced number of genes and some of them showing architectures found in nature. We analyse the response of our models under diverse forcing light–dark (LD) cycles. Our methodology allows us to evolve networks in silico using various selective pressures, which we apply to the analysis of clocks evolved to be either autonomous or phase locked. Our designed networks either have an oscillatory response with the same period as the forcing LD cycle, or they maintain their free-running period. Our methodology will allow analysing the automatic creation of a free-running period under various LD forcing functions and learning new design principles for circadian clocks.

Gemvid, an open source, modular, automated activity recording system for rats using digital video

Measurement of locomotor activity is a valuable tool for analysing factors influencing behaviour and for investigating brain and sleep functions. Several methods have been described in the literature for measuring the amount animal movement but most are flawed or expensive. Here, we describe an open source, modular, low-cost, user-friendly, highly sensitive, non-invasive system that records all the movements of a rat in its cage. Our activity monitoring system quantifies overall free movements of rodents without any markers, using a commercially available CCTV and a newly designed motion detection software developed on a GNU/Linux-operating computer. The operating principle is that the amount of overall movement of an object can be expressed by the difference in total area occupied by the object in two consecutive picture frames. The application is based on software modules that allow the system to be used in highthroughput work-flow. Documentation, example files, source code and binary files can be freely downloaded on the web. In a series of experiments with objects of pre-defined oscillation frequencies and movements, we documented the sensitivity, reproductibility and stability of our system. We also compared data obtained with our system and data obtained with an Actiwatch device. Finally, to validate the system, results obtained from the automated observation of 6 rats during 7 days in a regular light cycle are presented and are accompanied by a stability test. The validity of this system is further demonstrated through the observation of 2 rats in constant dark conditions that displayed the expected free running of their circadian rhythm. In conclusion, the present study describes a system that relies on video frame differences to automatically quantify overall free movements of a rodent without any markers. It allows the monitoring of rats in their own environment for an extended period of time. By using a low-cost, open source hardware/software solution, laboratories can greatly simplify their data acquisition and analysis pipelines and improve their workload.

Circadian expression of 86- and 84-kDa heat shock proteins in the mouse suprachiasmatic nucleus

Circadian rhythm pervades in many aspects of the biological processes including basic cellular functions. Here we examined the circadian gene expression of two forms of 90 kDa heat shock proteins referred to HSP86 and HSP84 in the mouse suprachiasmatic nucleus, the circadian center. In both light-dark, and constant dark conditions, Hsp86 mRNA showed an overt circadian rhythm showing a peak at (subjective) night and a trough at (subjective) day. Hsp84 mRNA also showed the similar expression profile, but the amplitude was weaker. These results indicate that gene expression of molecular chaperone such as Hsp86 and Hsp84 are regulated by the circadian clock.

Cis-Combination of the Classic perS and perL Mutations Results in Arrhythmic Drosophila with Ectopic Accumulation of Hyperphosphorylated PERIOD Protein

The 1st circadian "clock" gene identified was the X-linked period (per) gene in Drosophila melanogaster. In the pioneering initial report, Konopka and Benzer (1971) characterized 3 alleles of per that shortened (per (S); approximately 19 h), lengthened (per (L); approximately 29 h), or abolished (per (0)) circadian behavioral rhythms. They also showed that transheterozygotes carrying the per (S) and per (L) mutations exhibit robust behavioral rhythms with nearly normal periods of approximately 23 h, highlighting the semidominant nature of many clock mutants. In this study, per (0) flies bearing a doubly mutated per transgene that carries both the per (S) and per (L) alleles (per (0); per (S/L)) were analyzed for behavioral and molecular rhythms. Unlike singly mutated versions, the per (0);per ( S/L) transgenic flies are arrhythmic in constant dark conditions and exhibit little, if any, entrainment to daily light-dark cycles. In a wildtype per (+) background, expression of per ( S/L) abolishes behavioral rhythms, indicating that it functions in a transdominant negative fashion. Biochemical analysis of head extracts revealed that only hyperphosphorylated isoforms of the PERS/L protein are detected throughout a daily cycle, and the levels remain constant. Intriguingly, little if any PERS/L is observed in key pacemaker neurons that control daily activity rhythms, consistent with the notion that hyperphosphorylated isoforms of PER are unstable. Nonetheless, PERS/L is detected in ectopic cells in the brain, in which it exhibits an unusual localization, mainly staining the periphery of the nucleus. These results suggest that posttranslational mechanisms play a key role in limiting the accumulation of PER to specific cells. On a broader scope, our results indicate that the semidominant effects of period-altering alleles observed in trans are not necessarily preserved in the cis-configuration and that novel phenotypes can emerge.