Clocks for the city: circadian differences between forest and city songbirds.

To keep pace with progressing urbanization organisms must cope with extensive habitat change. Anthropogenic light and noise have modified differences between day and night, and may thereby interfere with circadian clocks. Urbanized species, such as birds, are known to advance their activity to early morning and night hours. We hypothesized that such modified activity patterns are reflected by properties of the endogenous circadian clock. Using automatic radio-telemetry, we tested this idea by comparing activity patterns of free-living forest and city European blackbirds (Turdus merula). We then recaptured the same individuals and recorded their activity under constant conditions. City birds started their activity earlier and had faster but less robust circadian oscillation of locomotor activity than forest conspecifics. Circadian period length predicted start of activity in the field, and this relationship was mainly explained by fast-paced and early-rising city birds. Although based on only two populations, our findings point to links between city life, chronotype and circadian phenotype in songbirds, and potentially in other organisms that colonize urban habitats, and highlight that urban environments can significantly modify biologically important rhythms in wild organisms.

A new role for cryptochrome in a Drosophila circadian oscillator.

Cryptochromes are flavin/pterin-containing proteins that are involved in circadian clock function in Drosophila and mice. In mice, the cryptochromes Cry1 and Cry2 are integral components of the circadian oscillator within the brain and contribute to circadian photoreception in the retina. In Drosophila, cryptochrome (CRY) acts as a photoreceptor that mediates light input to circadian oscillators in both brain and peripheral tissue. A Drosophila cry mutant, cryb, leaves circadian oscillator function intact in central circadian pacemaker neurons but renders peripheral circadian oscillators largely arrhythmic. Although this arrhythmicity could be caused by a loss of light entrainment, it is also consistent with a role for CRY in the oscillator. A peripheral oscillator drives circadian olfactory responses in Drosophila antennae. Here we show that CRY contributes to oscillator function and physiological output rhythms in the antenna during and after entrainment to light-dark cycles and after photic input is eliminated by entraining flies to temperature cycles. These results demonstrate a photoreceptor-independent role for CRY in the periphery and imply fundamental differences between central and peripheral oscillator mechanisms in Drosophila.

The estimation of the diffusion constant and solubility of O(2)in tissue using kinetics.

The diffusion of a gas through a substance in which it is soluble is analogous to the passage of electric current through a circuit with both capacitance and resistance. We model steady-state diffusion employing this analogy, and extend the model to include a description of the kinetics of systems under circumstances of changing partial pressure, applying two physical constants from electrical circuitry to gas diffusion: capacitance (zeta) and resistance (R). We represent the substrate of the diffusion as a capacitor being charged through a resistor after the rapid imposition of a voltage change. Using the insight derived from this model we have devised an experimental system that allows us to approximate both D, the diffusion coefficient, and alpha, solubility, directly from the kinetic data. We do this by recording the exponential change in P(O(2))on one side of a sheet of material both with and without the addition of a purely resistive barrier of known resistivity. The method was used to estimate D and alpha for distilled water at a number of temperatures, olive oil, and the belly skin of Rana catesbeiana.

Tuning in the transcriptome: basins of attraction in the yeast cell cycle.

Image processing techniques and wavelet analyses have been applied to the yeast cell cycle expression microchip data to reveal large-scale temporally coherent structures and high frequency oscillations in mRNA levels through the cycle. Because transitions in expression frequently occur in phase, they appear as peaks or troughs in colour maps and contour plots of expression levels. Although apparent in the untreated data, these transitions were identified and enhanced by convolution of a Laplacian kernel with the expression arrays of the first 4096 genes. Transitions associated with maximum up- or down-regulation of mRNA levels appear as bands at 30-40 min intervals through two cell cycles. Time-frequency analyses using wavelet transforms support these visualization techniques and lead to the conclusion that, with respect to gene expression, the dominant period is not the cell cycle (90-120 min) but, more commonly, the higher frequency 30-40 minute submultiple of the cycle period.

Morphology of the pupal heart, adult heart, and associated tissues in the fruit fly, Drosophila melanogaster.

The early pupal heart of the fruit fly Drosophila melanogaster has recently been the subject of intense physiological and molecular work, yet it has not been well described, nor has it been compared with the heart of the adult fly. In the work reported here, the hearts of adults and early pupae of D. melanogaster were studied by scanning and transmission electron microscopy and by light microscopy. The hearts of adults and early pupae both consist of a tube of circular striated muscle one cell in thickness. The alary muscles, which suspend the heart, are more delicate in the adult compared to the early pupa. The pericardial cells in both early pupae and adults are connected to the heart by connective tissue radiating from the alary muscles or dorsal diaphragm. We confirm that four major changes occur in the heart during metamorphosis: 1) a conical chamber is formed de novo in the first and second abdominal segments; 2) the adult heart curves to conform to the contour of the abdomen; 3) a layer of longitudinal striated muscle appears on the ventral surface of the heart; 4) a fourth pair of ostia is added to the three already present in the early pupa; and note additionally that 5) the ostia appear as simple openings in the heart of the early pupa but are valve-like in the adult.

A persistent circhoral ultradian rhythm is identified in human core temperature.

There have been inconclusive reports of intermittent rhythmic fluctuations in human core temperature, with the fluctuations having a period of about an hour. However, there has been no definitive demonstration of the phenomenon. This is likely due to the intermittency and seeming instability of the events. They have been assumed to be secondary rather than autonomous phenomena, putatively arising from the oscillation between rapid eye movement (REM) and non-REM (NREM) sleep. In this study, we report identification of a clear, persistent circhoral ultradian rhythm in core temperature with a period for this study sample of 64 +/- 8 minutes. It appeared simultaneously with an intact circadian core temperature rhythm, persisted despite complex perturbations in core temperature brought about by the sequelae of 40 h of sleep deprivation, and could not be attributed to sleep stage alternation or other endogenous or exogenous factors. Analysis of power spectra using the maximum entropy spectral analysis (MESA) method, which can uncover hidden rhythmicities, demonstrated that the apparent intermittency of the rhythm is due to periodic interference of this rhythm by other rhythmic events. The persistence of this oscillation suggests that, in this system as in the endocrine system, circhoral regulation is an integral component of thermoregulatory control. Identifying the source and functional role of this novel rhythm warrants further work.

Courtship bout duration in per circadian period mutants in Drosophila melanogaster.

Mutations on the period locus of Drosophila melanogaster influence circadian periods as well as the rhythm in inter-pulse intervals in male courtship song; perL mutations produce long circadian periods and courtship song rhythms and per(s) mutations produce short circadian periods and courtship song rhythms. Thus, these mutations influence timing mechanisms over both long and short behavioral time horizons. We examined if the mean courtship duration of male Drosophila melanogaster cycles rhythmically, and if mutations at the period locus influence courtship bout duration. We measured the courtship bout durations of the following: (1) wild type Canton-S (per+) males; (2) perL males; and (3)per(s) males. Rhythmicity of courtship bout duration could not be mathematically determined. Mean courtship bout duration did not differ among the three groups; thus, mutations at the period locus did not influence mean courtship duration. There was a nonsignificant trend for per+ males that were successful at mating to have longer mean courtship duration than unsuccessful males.

Quantitative analysis of Drosophila period gene transcription in living animals.

To determine the in vivo regulatory pattern of the clock gene period (per), the authors recently developed transgenic Drosophila carrying a luciferase cDNA fused to the promoter region of per. They have now carried out noninvasive, high time-resolution experiments allowing high-throughput monitoring of circadian bioluminescence rhythms in individual living adults for several days. This immediately solved several problems (resulting directly from individual asynchrony within a population) that have accompanied previous biochemical experiments in which groups of animals were sacrificed at each time point. Furthermore, the authors have developed numerical analysis methods for automatically determining rhythmicity associated with bioluminescence records from single flies. This has revealed some features of per gene transcription that were previously unappreciated and provides a general strategy for the analysis of rhythmic time series in the study of molecular rhythms.

The effects of period mutations and light on the activity rhythms of Drosophila melanogaster.

Strains of Drosophila melanogaster homozygous for alleles of the period gene (perO, perL, perS, and per+) were reared for multiple generations either in light:dark cycles (LD), continuous illumination (LL), or chronic darkness (DD). The locomotor activity of adult flies from these cultures was monitored in either LL or DD. Flies that were reared and tested in DD had a lower proportion of individuals with normal circadian rhythms than flies reared in LD or LL and tested in DD. The activity rhythms of DD-reared DD-tested animals, when present, showed phase coherence within two out of seven populations, while 8 out of 10 LL-reared DD-tested showed phase coherence. Flies tested in LL were largely devoid of circadian rhythms regardless of their rearing environment. Ultradian rhythms were more evident under conditions disruptive to circadian rhythmicity, but were observed in the presence and absence of circadian rhythms. The periods of the ultradian rhythms of LL-reared DD-tested and LD-reared DD-tested flies varied significantly among genotypes, while in other rearing and testing regimes, no relationship was found.

Neurophysiological Correlates of the Behavioral Response to Light in the Sea Anemone Anthopleura elegantissima.

Neurophysiological responses to light in Anthopleura elegantissima do not involve the ectodermal slow system 1 (SS1). Activities in both the endodermal slow system 2 (SS2) and the through conducting nerve net (TCNN) change when the lighting changes, but the response is not consistent. Thus, photoreception in A. elegantissima probably occurs in the endoderm because SS2 and the TCNN are involved and SS1 is not. We hypothesize either that the photoreception occurs in sensory cells in a local nerve net, with the information then being transmitted to the muscles, or that the muscles themselves are light sensitive. In either case, the TCNN and SS2 become involved after the transduction, and as a consequence--rather than the cause--of muscular activation. The conducting systems of zooxanthellate specimens have a higher frequency of activity than those of apozooxanthellate individuals.

Effects of deuterium oxide and temperature on heart rate in Drosophila melanogaster.

A non-intrusive optical technique has been developed to monitor heartbeat in late third-instar Drosophila larvae. Heartbeat in this insect is an oscillation that is not temperature compensated. Deuterium oxide lengthens the period of a number of high and low frequency oscillators and clocks in a variety of organisms. To determine whether deuterium affects heart rate, flies were raised on proteated and deuterated media and their heartbeat was monitored at four temperatures ranging from 18 to 33 degrees C. The rate of heartbeat increased linearly with increasing temperature, and decreased with increasing concentrations of deuterium. There was a significant interaction between temperature and deuterium: the higher the concentration of deuterium oxide the less temperature-sensitive was the heart rate. Raising temperatures also increased the amount of "noise" in the rhythm: signal-to-noise ratio, which characterizes the amount of power in a rhythmic signal, decreased with increasing temperatures. Deuterium oxide had no effect on signal-to-noise ratio.

The development of ultradian and circadian rhythms in premature babies maintained in constant conditions.

Twenty very premature babies, born at 24-29 weeks gestation, have been studied while they were maintained in intensive care with continuous intravenous feeding and constant ambient lighting and temperature. Hourly records of insulated skin temperature and heart rate were made for a continuous period of 6-17 weeks, always starting the recording within 24 h of birth. The development of rhythms within the ultradian, circadian and infradian domains was sought by methods including maximum entropy spectral analysis and autocorrelation. Circadian and ultradian rhythms were present, but not regularly so; rather they appeared and disappeared erratically in successive weeks. As a consequence, the group as a whole did not show an increasing rhythmicity with chronological age. In some cases, babies were later placed in a ward in which the lighting was dimmed at night, and feeding by mouth at regular intervals was instituted. There was some evidence for increases in circadian and ultradian rhythmicity after these changes. These results enable inferences to be drawn as to the origin of fetal rhythms in the third trimester of pregnancy, as well as speculation to be made on the ontogeny of ultradian and circadian rhythms in the neonate.

Mutational analysis of the Drosophila miniature-dusky (m-dy) locus: effects on cell size and circadian rhythms.

A mutational analysis has been performed to explore the function of the Drosophila melanogaster miniature-dusky (m-dy) locus. Mutations at this locus affect wing development, fertility and behavior. The genetic characterization of 13 different mutations suggests that m and dy variants are alleles of a single complex gene. All of these mutations alter wing size, apparently by reducing the volume of individual epidermal cells of the developing wing. In m mutants, epidermal cell boundaries persist in the mature wing, whereas they normally degenerate 1-2 hr after eclosion in wild-type or dy flies. This has permitted the direct visualization of cell size differences among several m mutants. Mutations at the m-dy locus also affect behavioral processes. Three out of nine dy alleles (dyn1, dyn3 and dyn4) lengthen the circadian period of the activity and eclosion rhythms by approximately 1.5 hr. In contrast, m mutants have normal circadian periods, but an abnormally large percentage of individuals express aperiodic bouts of activity. These behavior genetic studies also indicate that an existing "rhythm" mutation known as Andante is an allele of the m-dy locus. The differential effects of certain m-dy mutations on wing and behavioral phenotypes suggest that separable domains of function exist within this locus.

A new mutation at the period locus of Drosophila melanogaster with some novel effects on circadian rhythms.

A new period mutation has been induced and characterized in D. melanogaster. It causes flies to be apparently arrhythmic in tests of locomotor activity and thus is superficially similar to the original per01 mutant. Yet, the new "zero" allele, per04, has some novel properties and effects: Behaviorally, per04 adults often exhibit weak, long-period rhythms of locomotor activity in constant darkness; this low-frequency rhythmicity usually was not obvious in the analog behavioral records but was readily revealed by spectral analyses. These treatments of the data also extracted hidden high-frequency (ultradian) rhythms in many of the behavioral records, of the type associated with per01 and other per-nulls. The wide range of periodicities exhibited by different per04-expressing flies implies the expression of multiple oscillatory modes by this mutant. The new mutation also leads to a tendency for flies to be hyperactive during activity monitoring and is thus dissimilar to the other arrhythmic variants in the per gene but similar to the effects of a deletion of the locus. During light:dark cycling, per04 adults once more behave differently from other per0's and in fact tend to resemble wild-type flies in these conditions. The new mutation is not caused by the same nucleotide substitution that created a stop codon in the original arrhythmic per mutant and, as it turns out, per02 and per03 as well. per04 is also not a null variant at the transcriptional level; but it leads to an anomalous form of per mRNA, which is smaller than the normal 4.5 kb species encoded by this clock gene.

High-resolution analysis of locomotor activity rhythms in disconnected, a visual-system mutant of Drosophila melanogaster.

Free-running locomotor activity and eclosion rhythms of Drosophila melanogaster, mutant at the disconnected (disco) locus, are substantially different from the wild-type phenotype. Initial periodogram analysis revealed little or no rhythmicity (Dushay et al., 1989). We have reanalyzed the locomotor activity data using high-resolution signal analysis (maximum-entropy spectral analysis, or MESA). These analyses, corroborated by autocorrelograms, uncovered significant residual circadian rhythmicity and strong ultradian rhythms in most of the animals tested. In this regard the disco mutants are much like flies expressing mutant alleles of the period gene, as well as wild-type flies reared throughout life in constant darkness. We hypothesize that light normally triggers the coupling of multiple ultradian oscillators into a functional circadian clock and that this process is disrupted in disco flies as a result of the neural lesion.

Further evidence that the circadian clock in Drosophila is a population of coupled ultradian oscillators.

We hypothesize that ultradian oscillators are coupled to yield a composite circadian clock in Drosophila. In such a system, period would be a function of the tightness of coupling of these oscillators, increasing as coupling loosens. Ultradian oscillations would become apparent under weak coupling or in the absence of coupling. A new technique for calculating signal-to-noise ratio (SNR) for biological rhythms to characterize their precision has yielded support for this hypothesis. SNR of rhythms of the allelic series of mutations at the period (per) locus of Drosophila melanogaster were compared. Per(o) was the noisiest, grading through perL, per+, and pers, the least noisy. SNR decreases significantly with increasing period in pers, per+, and perL; per(o) typically has multiple ultradian oscillations and the lowest SNR. At least 70% of perL individuals also exhibit ultradian periodicities.