Free-running Conditions Research Articles

Phenotyping Circadian Rhythms in Mice.

Circadian rhythms take place with a periodicity of 24 hr, temporally following the rotation of the earth around its axis. Examples of circadian rhythms are the sleep/wake cycle, feeding, and hormone secretion. Light powerfully entrains the mammalian clock and assists in keeping animals synchronized to the 24-hour cycle of the earth by activating specific neurons in the "central pacemaker" of the brain, the suprachiasmatic nucleus. Absolute periodicity of an animal can deviate slightly from 24 hr as manifest when an animal is placed into constant dark or "free-running" conditions. Simple measurements of an organism's activity in free-running conditions reveal its intrinsic circadian period. Mice are a particularly useful model for studying circadian rhythmicity due to the ease of genetic manipulation, thus identifying molecular contributors to rhythmicity. Furthermore, their small size allows for monitoring locomotion or activity in their homecage environment with relative ease. Several tasks commonly used to analyze circadian periodicity and plasticity in mice are presented here including the process of entrainment, determination of tau (period length) in free-running conditions, determination of circadian periodicity in response to light disruption (e.g., jet lag studies), and evaluation of clock plasticity in non-24-hour conditions (T-cycles). Studying the properties of circadian periods such as their phase, amplitude, and length in response to photic perturbation, can be particularly useful in understanding how humans respond to jet lag, night shifts, rotating shifts, or other transient or chronic disruption of environmental surroundings.

Pigment-dispersing factor signaling in the circadian system of Caenorhabditis elegans.

The neuropeptide pigment-dispersing factor (PDF) is important for the generation and entrainment of circadian rhythms in the fruitfly Drosophila melanogaster. Recently two pdf homologs, pdf-1 and pdf-2, and a PDF receptor, pdfr-1, have been found in Caenorhabditis elegans and have been implicated in locomotor activity. In this work, we have studied the role of the PDF neuropeptide in the circadian system of C. elegans and found that both pdf-1 and pdf-2 mutants affect the normal locomotor activity outputs. In particular, loss of pdf-1 induced circadian arrhythmicity under both light-dark (LD) and constant dark (DD) conditions. These defects can be rescued by a genomic copy of the pdf-1 locus. Our results indicate that PDF-1 is involved in rhythm generation and in the synchronization to LD cycles, as rhythmic patterns of activity rapidly disappear when pdf-1 mutants are recorded under both entrained and free-running conditions. The role of PDF-2 and the PDF receptors is probably more complex and involves the interaction between the two pdf paralogues found in the nematode.

Purity of the single frequency mode of a hybrid semiconductor-fiber laser.

The penalty of extending the cavity length of a laser diode when seeking a linewidth reduction is normally revealed by poor side mode suppression, which prevents the laser from operating purely in a single mode of the external cavity. A hybrid laser, based on a C-band semiconductor optical amplifier combined with a long erbium doped fiber external cavity, is carefully engineered to operate with high spectral purity and outstanding stability. For the first time, a side-mode suppression ratio of ≥42 dB, measured at a resolution of 1.16 pm (149 MHz) at all intra-cavity powers above the lasing threshold, is reported. The output power at the peak lasing wavelength is 13.3 dBm. Also, the ability to lock such a hybrid laser to a particular external-cavity mode is realized for the first time. Excluding the effect of mechanical and thermal drifts on the cavity length, the long-term frequency stability is demonstrated to be within ± 11 Hz while the long-term linewidth is 2.26 kHz, measured using the self-beating technique under free running conditions.

Long-term imaging of circadian locomotor rhythms of a freely crawling C. elegans population

BackgroundLocomotor activity is used extensively as a behavioral output to study the underpinnings of circadian rhythms. Recent studies have required a populational approach for the study of circadian rhythmicity in Caenorhabditis elegans locomotion. New methodWe describe an imaging system for long-term automated recording and analysis of locomotion data of multiple free-crawling C. elegans animals on the surface of an agar plate. We devised image analysis tools for measuring specific features related to movement and shape to identify circadian patterns. ResultsWe demonstrate the utility of our system by quantifying circadian locomotor rhythms in wild-type and mutant animals induced by temperature cycles. We show that 13°C:18°C (12:12h) cycles are sufficient to entrain locomotor activity of wild-type animals, which persist but are rapidly damped during 13°C free-running conditions. Animals with mutations in tax-2, a cyclic nucleotide-gated (CNG) ion channel, significantly reduce locomotor activity during entrainment and free-running. Comparison with existing method(s)Current methods for measuring circadian locomotor activity is generally restricted to recording individual swimming animals of C. elegans, which is a distinct form of locomotion from crawling behavior generally observed in the laboratory. Our system works well with up to 20 crawling adult animals, and allows for a detailed analysis of locomotor activity over long periods of time. ConclusionsOur population-based approach provides a powerful tool for quantification of circadian rhythmicity of C. elegans locomotion, and could allow for a screening system of candidate circadian genes in this model organism.

Experimental and Numerical Studies on the Dynamics of X-Band Gunn Oscillator Based Angle Modulator–Demodulator System with Chaotic Modulating Signal

We have studied the performance of a Gunn oscillator (GO) based angle modulator–demodulator system in transmitting chaotic signals in the X-band microwave frequency channel. The principle of bias tuning of a GO is employed to implement the angle modulator used in the transmitter. The said GO is operated in a free running condition and then in a frequency synchronized condition to an external microwave signal, thus generating frequency modulated (FM) and phase modulated (PM) signals, respectively. The demodulator circuit is implemented with a GO phase synchronized to the incoming modulated signal, followed by a microwave mixer multiplying the input and the output signals of the GO and a low pass filter. The response of the system is analytically established, numerically examined and experimentally verified. The obtained results confirm that in the limit of low modulation index and in the linear range of operation of the bias tuned modulator and the phase synchronized detector, a chaos signal can be transmitted and recovered through a microwave channel using the technique of angle modulation.

Melatonin Is Required for the Circadian Regulation of Sleep

Sleep is an evolutionarily conserved behavioral state whose regulation is poorly understood. A classical model posits that sleep is regulated by homeostatic and circadian mechanisms. Several factors have been implicated in mediating the homeostatic regulation of sleep, but molecules underlying the circadian mechanism are unknown. Here we use animals lacking melatonin due to mutation of arylalkylamine N-acetyltransferase 2 (aanat2) to show that melatonin is required for circadian regulation of sleep in zebrafish. Sleep is dramatically reduced at night in aanat2 mutants maintained in light/dark conditions, and the circadian regulation of sleep is abolished in free-running conditions. We find that melatonin promotes sleep downstream of the circadian clock as it is not required to initiate or maintain circadian rhythms. Additionally, we provide evidence that melatonin may induce sleep in part by promoting adenosine signaling, thus potentially linking circadian and homeostatic control of sleep.

Modeling circadian and sleep-homeostatic effects on short-term interval timing.

Short-term interval timing i.e., perception and action relating to durations in the seconds range, has been suggested to display time-of-day as well as wake dependent fluctuations due to circadian and sleep-homeostatic changes to the rate at which an underlying pacemaker emits pulses; pertinent human data being relatively sparse and lacking in consistency however, the phenomenon remains elusive and its mechanism poorly understood. To better characterize the putative circadian and sleep-homeostatic effects on interval timing and to assess the ability of a pacemaker-based mechanism to account for the data, we measured timing performance in eighteen young healthy male subjects across two epochs of sustained wakefulness of 38.67 h each, conducted prior to (under entrained conditions) and following (under free-running conditions) a 28 h sleep-wake schedule, using the methods of duration estimation and duration production on target intervals of 10 and 40 s. Our findings of opposing oscillatory time courses across both epochs of sustained wakefulness that combine with increasing and, respectively, decreasing, saturating exponential change for the tasks of estimation and production are consistent with the hypothesis that a pacemaker emitting pulses at a rate controlled by the circadian oscillator and increasing with time awake determines human short-term interval timing; the duration-specificity of this pattern is interpreted as reflecting challenges to maintaining stable attention to the task that progressively increase with stimulus magnitude and thereby moderate the effects of pacemaker-rate changes on overt behavior.

Optical Negative Feedback for Linewidth Reduction of Semiconductor Lasers

We propose a coherent optical negative feedback method for reducing linewidth of single-mode semiconductor lasers, which requires only an optical filter. Results of numerical analysis indicate that the power spectral density of FM noise can be reduced by more than 40 dB and the subkilohertz spectral linewidth can be obtained with our method when the spectral linewidth is 10 MHz under a free running condition. From a proof-of-concept experiment, we confirmed that the spectral linewidth of a single-mode semiconductor laser is reduced when using our method from 6.4 MHz to 6.5 kHz. This result confirms that the spectral linewidth can be reduced by at least 30 dB, which proves the validity of our method.

Evaluation of thermal comfort in university classrooms through objective approach and subjective preference analysis

Assessing thermal comfort becomes more relevant when the aim is to maximise learning and productivity performances, as typically occurs in offices and schools. However, if, in the offices, the Fanger model well represents the thermal occupant response, then on the contrary, in schools, adaptive mechanisms significantly influence the occupants' thermal preference. In this study, an experimental approach was performed in the Polytechnic University of Bari, during the first days of March, in free running conditions. First, the results of questionnaires were compared according to the application of the Fanger model and the adaptive model; second, using a subjective scale, a complete analysis was performed on thermal preference in terms of acceptability, neutrality and preference, with particular focus on the influence of gender. The user possibility to control the indoor plant system produced a significant impact on the thermal sensation and the acceptability of the thermal environment. Gender was also demonstrated to greatly influence the thermal judgement of the thermal environment when an outdoor cold climate occurs.

P38 MAP kinase regulates circadian rhythms in Drosophila.

The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. We find that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. We also find that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, we show that p38Kb suppresses the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, we find that manipulating p38K signaling in free-running conditions alters the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways.

Injection-locked operation of an optically pumped semiconductor laser.

We demonstrate a single-frequency, continuous-wave, optically pumped semiconductor laser (OPSL) using an injection-locking technique. Parameters such as output coupling, injection power, and injection wavelength are investigated. With the proper parameters, the output power of the injection-locked laser exceeds the output power of its free-running condition. With 1.5 W of injection power from the master laser, over 9 W of output power is achieved at 1015 nm.

Translational regulation of the DOUBLETIME/CKIδ/ε kinase by LARK contributes to circadian period modulation.

The Drosophila homolog of Casein Kinase I δ/ε, DOUBLETIME (DBT), is required for Wnt, Hedgehog, Fat and Hippo signaling as well as circadian clock function. Extensive studies have established a critical role of DBT in circadian period determination. However, how DBT expression is regulated remains largely unexplored. In this study, we show that translation of dbt transcripts are directly regulated by a rhythmic RNA-binding protein (RBP) called LARK (known as RBM4 in mammals). LARK promotes translation of specific alternative dbt transcripts in clock cells, in particular the dbt-RC transcript. Translation of dbt-RC exhibits circadian changes under free-running conditions, indicative of clock regulation. Translation of a newly identified transcript, dbt-RE, is induced by light in a LARK-dependent manner and oscillates under light/dark conditions. Altered LARK abundance affects circadian period length, and this phenotype can be modified by different dbt alleles. Increased LARK delays nuclear degradation of the PERIOD (PER) clock protein at the beginning of subjective day, consistent with the known role of DBT in PER dynamics. Taken together, these data support the idea that LARK influences circadian period and perhaps responses of the clock to light via the regulated translation of DBT. Our study is the first to investigate translational control of the DBT kinase, revealing its regulation by LARK and a novel role of this RBP in Drosophila circadian period modulation.

Characterisation of circadian rhythms of various duckweeds.

The plant circadian clock controls various physiological phenomena that are important for adaptation to natural day-night cycles. Many components of the circadian clock have been identified in Arabidopsis thaliana, the model plant for molecular genetic studies. Recent studies revealed evolutionary conservation of clock components in green plants. Homologues of clock-related genes have been isolated from Lemna gibba and Lemna aequinoctialis, and it has been demonstrated that these homologues function in the clock system in a manner similar to their functioning in Arabidopsis. While clock components are widely conserved, circadian phenomena display diversity even within the Lemna genus. In order to survey the full extent of diversity in circadian rhythms among duckweed plants, we characterised the circadian rhythms of duckweed by employing a semi-transient bioluminescent reporter system. Using a particle bombardment method, circadian bioluminescent reporters were introduced into nine strains representing five duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna gibba, L. aequinoctialis and Wolffia columbiana. We then monitored luciferase (luc+) reporter activities driven by AtCCA1, ZmUBQ1 or CaMV35S promoters under entrainment and free-running conditions. Under entrainment, AtCCA1::luc+ showed similar diurnal rhythms in all strains. This suggests that the mechanism of biological timing under day-night cycles is conserved throughout the evolution of duckweeds. Under free-running conditions, we observed circadian rhythms of AtCCA1::luc+, ZmUBQ1::luc+ and CaMV35S::luc+. These circadian rhythms showed diversity in period length and sustainability, suggesting that circadian clock mechanisms are somewhat diversified among duckweeds.

Peroxiredoxin 2 nuclear levels are regulated by circadian clock synchronization in human keratinocytes

Circadian rhythms are highly conserved time tracking systems regulating important biological processes at both systemic and cellular levels. The present study was aimed to identify proteins and biological functions circadian regulated in human keratinocytes.HaCaT keratinocytes were entrained by temperature cycles, and a proteomic study was performed on cell fractions isolated under free running conditions at constant temperature. Bioinformatics analysis revealed that molecular clock entrainment was associated with changes in molecular components regulating cell proliferation, energy metabolism, transcription, translation and redox balance. Nuclear levels of the antioxidant enzyme Peroxiredoxin 2 (PRDX2) were found to oscillate rhythmically over two entire 24h long cycles. Donwregulation of PRDX2 resulted in upregulation of the mitochondrion-specific Peroxiredoxin 3 (PRDX3), all other members of the Peroxiredoxin family remained unaltered. Furthermore, PRDX2 knockdown increased intracellular levels of reactive oxygen species (ROS) and impaired cell cycle progression and proliferation. HaCaT cells transduced with a scramble shRNA were used as control.Our work is the first to show that nuclear levels of PRDX2 display circadian oscillation participating in the regulation of human keratinocytes redox balance.

Synergic effects of thermal mass and natural ventilation on the thermal behaviour of traditional massive buildings

The energy policies about energy efficiency in buildings currently focus on new buildings and on existing buildings in case of energy retrofit. However, historic and heritage buildings, that are the trademark of numerous European cities, should also deserve attention; nevertheless, their energy efficiency is nowadays not deeply investigated. In this context, this study evaluates the thermal performance of a traditional massive building situated in a Mediterranean city. Dynamic numerical simulations were carried out on a yearly basis through the software DesignBuilder, both in free-running conditions and in the presence of an air-conditioning (AC) system. The results highlight that the massive envelope of traditional residential buildings helps in maintaining small fluctuations of the indoor temperature, thus limiting the need for AC in the mid-season and in summer. This feature is highly emphasised by exploiting natural ventilation at night, which allows reducing the building energy demand for cooling by about 30%.The research also indicates that, for Mediterranean climate, the increase in thermal insulation does not always induce positive effects on the thermal performance in summer, and that it might even produce an increase in the heat loads due to the transmission through the envelope.

The Circadian Clock Gene Csnk1e Regulates Rapid Eye Movement Sleep Amount, and Nonrapid Eye Movement Sleep Architecture in Mice

Efforts to identify the genetic basis of mammalian sleep have included quantitative trait locus (QTL) mapping and gene targeting of known core circadian clock genes. We combined three different genetic approaches to identify and test a positional candidate sleep gene - the circadian gene casein kinase 1 epsilon (Csnk1e), which is located in a QTL we identified for rapid eye movement (REM) sleep on chromosome 15. Using electroencephalographic (EEG) and electromyographic (EMG) recordings, baseline sleep was examined in a 12-h light:12-h dark (LD 12:12) cycle in mice of seven genotypes, including Csnk1e(tau/tau) and Csnk1e(-/-) mutant mice, Csnk1e (B6.D2) and Csnk1e (D2.B6) congenic mice, and their respective wild-type littermate control mice. Additionally, Csnk1e(tau/tau) and wild-type mice were examined in constant darkness (DD). Csnk1e(tau/tau) mutant mice and both Csnk1e (B6.D2) and Csnk1e (D2.B6) congenic mice showed significantly higher proportion of sleep time spent in REM sleep during the dark period than wild-type controls - the original phenotype for which the QTL on chromosome 15 was identified. This phenotype persisted in Csnk1e(tau/tau) mice while under free-running DD conditions. Other sleep phenotypes observed in Csnk1e(tau/tau) mice and congenics included a decreased number of bouts of nonrapid eye movement (NREM) sleep and an increased average NREM sleep bout duration. These results demonstrate a role for Csnk1e in regulating not only the timing of sleep, but also the REM sleep amount and NREM sleep architecture, and support Csnk1e as a causal gene in the sleep QTL on chromosome 15.

Parametric excitation in a magnetic tunnel junction-based spin torque oscillator

Using microwave current injection at room temperature, we demonstrate parametric excitation of a magnetic tunnel junction (MTJ)-based spin-torque oscillator (STO). Parametric excitation is observed for currents below the auto-oscillation threshold, when the microwave current frequency fe is twice the STO free-running frequency f0. Above threshold, the MTJ becomes parametrically synchronized. In the synchronized state, the STO exhibits an integrated power up to 5 times higher and a linewidth reduction of two orders of magnitude, compared to free-running conditions. We also show that the parametric synchronization favors single mode oscillations in the case of multimode excitation.

A Kinetic Study of the Effects of Light on Circadian Rhythmicity of thefrqPromoter ofNeurospora crassa

The role of the frq gene in the Neurospora crassa circadian rhythm has been widely studied, but technical limitations have hindered a thorough analysis of frq circadian expression waveform. Through our experiments, we have shown an improved precision in defining Neurospora's circadian rhythm kinetics using a codon optimized firefly luciferase gene reporter linked to a frq promoter. In vivo examination of this real-time reporter has allowed for a better understanding of the relationship of the light responsive elements of the frq promoter to its circadian feedback components. We provide a detailed phase response curve showing the phase shifts induced by a light pulse applied at different points of the circadian cycle. Using the frq-luc reporter, we have found that a 12-h light:12-h dark cycle (12L:12D) results in a luciferase expression waveform that is more complex and higher in amplitude than that seen in free-running conditions of constant darkness (DD). When using a lighting regime more consistent with solar timing, rather than a square wave pattern, one observes a circadian waveform that is smoother, lower in amplitude, and different in phasing. Using dim light in place of darkness in these experiments also affects the resulting waveform and phasing. Our experiments illustrate Neurospora's circadian kinetics in greater detail than previous methods, providing further insight into the complex underlying biochemical, genetic, and physiological mechanisms underpinning the circadian oscillator.

No Time for Spruce: Rapid Dampening of Circadian Rhythms in Picea abies (L. Karst)

The identification and cloning of full-length homologs of circadian clock genes from Picea abies represent a first step to study the function and evolution of the circadian clock in gymnosperms. Phylogenetic analyses suggest that the sequences of key circadian clock genes are conserved between angiosperms and gymnosperms. though fewer homologous copies were found for most gene families in P. abies. We detected diurnal cycling of circadian clock genes in P. abies using quantitative real-time PCR; however, cycling appeared to be rapidly dampened under free-running conditions. Given the unexpected absence of transcriptional cycling during constant conditions, we employed a complementary method to assay circadian rhythmic outputs and measured delayed fluorescence in seedlings of Norway spruce. Neither of the two approaches to study circadian rhythms in Norway spruce could detect robust ∼24 h cycling behavior under constant conditions. These data suggest gene conservation but fundamental differences in clock function between gymnosperms and other plant taxa.

Circadian Period Integrates Network Information Through Activation of the BMP Signaling Pathway

Author SummaryThe circadian clock controls rhythms in behavior, physiology, and metabolism in all living organisms. The molecular components as well as the neuronal network required to keep this clock running have been identified in several species. In the Drosophila brain this neuronal network is represented by an ensemble of 150 neurons, and among them, those expressing the Pigment Dispersing Factor (PDF) neuropeptide encompass the “central oscillator”—also called master clock as it ensures 24-hour periods—of the fly brain. In this study we show that the widely conserved Bone Morphogenetic Protein (BMP) signaling pathway is present in PDF neurons, and upon adult-specific activation it lengthens the endogenous period of locomotor behavior. We find that period lengthening correlates with delayed accumulation of nuclear PERIOD, a core component of the molecular clock. We also identified a putative DNA binding motif for the BMP pathway nuclear components within the regulatory region of the Clock (Clk) promoter, another core component of the circadian machinery. Interestingly, upon BMP pathway activation endogenous CLK levels are downregulated, thus accounting for the lengthening of the endogenous period. We propose that the endogenous period is a network property commanded by PDF neurons that results from integration of information from both the autonomous molecular clock and the nonautonomous BMP signaling pathway.