Accidents in children do not happen at random: Predictable time-of-day incidence of childhood trauma

In a prospective study, 15,110 childhood traumas were recorded by the Pediatric Surgery Service (CHUV, Lausanne) between January 1, 1990 and December 31, 1997. The exact clock hour when the injury occurred and other germane data were obtained. Time series thus obtained were analyzed by several statistical (ANOVA, cosinor, χ², Table Curve, etc.) methods. High statistically significant circadian patterns were detected with a trough at night—almost no traumas/hour (t/h), and a peak in the afternoon (～16:00h)—9.3±0.4 (SD) t/h. Such 24h variation was validated for the whole sample for the entire 8yr study span as well as the data of each year. Neither gender- nor age-related differences in the 24h pattern were detected between children under 5 yr of age, who have not yet attended school and children from 5 to 16 yr of age, who attend school. Small but statistically significant differences in the 24h patterns were observed when categorized by the type of activity associated with the trauma and the place of trauma occurrence. The great stability of the 24h pattern in childhood trauma over the 8yr study span suggests an endogenous origin in addition to the role presumably played by environmental factors. Periods of 12 and 8 h were also detected in the time series. The afternoon peak time of childhood traumas differs from that of adults, which is located ～04:00h in rotating shift workers and automobile drivers and 06:00–08:00h in adult day-workers. The validation of a circadian pattern in childhood traumas with an afternoon peak should be taken into account in the design of children's preventative injury programs.     

An Example of Circular Statistics in Chronobiological Studies: Analysis of Polymorphism in the Emergence Rhythms of Schistosoma Mansoni Cercariae

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals. (Chronobiology International, 17(4), 433–451, 2000)     

Calcium and Photoentrainment in Chick Pineal Cells Revisited: Effects of Caffeine, Thapsigargin, EGTA, and Light on the Melatonin Rhythm

Abstract: Chick pineal cells in dispersed cell culture display a persistent, photosensitive, circadian rhythm of melatonin production and release. Light pulses have at least two distinguishable effects on these cells, i.e., acute suppression of melatonin output and phase shifts (entrainment) of the underlying circadian pacemaker. Previous results linked calcium influx through voltage-sensitive calcium channels in the plasma membrane to acute regulation of melatonin synthesis but denied a role for such influx in entrainment. Those experiments did not, however, address the role of intracellular calcium metabolism. Here we describe the effects of pulses of caffeine, thapsigargin, and EGTA on the melatonin rhythm, and their interactions with the effects of light pulses. Caffeine had two distinguishable effects on these cells, acute enhancement of melatonin output (attributable to phosphodiesterase inhibition) and phase shifts of the circadian pacemaker with a light-like pattern (attributable to effects on intracellular calcium). Phase shifts induced by light and caffeine were not additive. Thapsigargin (which specifically blocks the pump that replenishes intracellular calcium stores, thereby increasing cytoplasmic calcium and depleting intracellular stores) had no phase-shifting effects by itself but reduced the size of the phase advances induced by caffeine or light. Low calcium solution acutely suppressed melatonin output without inducing phase shifts or affecting those induced by caffeine or light. However, addition of EGTA (which specifically chelates calcium, thereby lowering cytoplasmic calcium and depleting intracellular stores) did reduce the size of phase advances induced by caffeine or light, in normal medium or in low calcium solution, without inducing a phase shift by itself at that phase. Taken together, these results point toward a role for intracellular calcium fluxes in entrainment of the circadian pacemaker.     

Interindividual Differences in Chronopharmacologic Effects of Drugs: A Background for Individualization of Chronotherapy

In order to optimize chronotherapeutic schedules (designs), we examined the interindividual differences in chronopharmacologic effects of drugs with consideration of the following three factors: (a) inherited factors of direct relevance to chronopharmacology (genetic variability, gender-related differences) as well as age-related differences; (b) interindividual difference in chronoeffective-ness related to disease (e.g., various types and stages of cancer, affective disorders, etc.) as well as to drug-dependent alteration (phase shifts, distortion) of biological rhythms; and (c) means to solve problems resulting from the need of individualization in chronotherapy. These involve the use of circadian marker rhythms (MR) whose characteristics (peak or trough time, amplitude, etc.) can be precisely quantified and thus are applicable as a reference system for physiologic, pathologic, pharmacologic, and therapeutic uses. The MR has to be specific and pertinent and must be easily monitored and documented. This approach can be further advanced by the use of a battery of MRs rather than a single MR. Other suggested means relate to the fact that chronobiotics (agents capable of influencing parameters of a set of biological rhythms) should be considered (e.g., corticoids and adrenocorticotropic hormone) and/or to the subject's synchronization should be enforced by “conventional” zeitgebers (e.g., bright light, physical activity).     

Interindividual Differences in Chronopharmacologic Effects of Drugs: A Background for Individualization of Chronotherapy

In order to optimize chronotherapeutic schedules (designs), we examined the interindividual differences in chronopharmacologic effects of drugs with consideration of the following three factors: (a) inherited factors of direct relevance to chronopharmacology (genetic variability, gender-related differences) as well as age-related differences; (b) interindividual difference in chronoeffective-ness related to disease (e.g., various types and stages of cancer, affective disorders, etc.) as well as to drug-dependent alteration (phase shifts, distortion) of biological rhythms; and (c) means to solve problems resulting from the need of individualization in chronotherapy. These involve the use of circadian marker rhythms (MR) whose characteristics (peak or trough time, amplitude, etc.) can be precisely quantified and thus are applicable as a reference system for physiologic, pathologic, pharmacologic, and therapeutic uses. The MR has to be specific and pertinent and must be easily monitored and documented. This approach can be further advanced by the use of a battery of MRs rather than a single MR. Other suggested means relate to the fact that chronobiotics (agents capable of influencing parameters of a set of biological rhythms) should be considered (e.g., corticoids and adrenocorticotropic hormone) and/or to the subject's synchronization should be enforced by “conventional” zeitgebers (e.g., bright light, physical activity).     

An Investigation of Natural Genetic Variation in the Circadian System of Drosophila melanogaster: Rhythm Characteristics and Methods of Quantification

Variation in four characteristics of the circadian locomotor activity rhythm was investigated in 24 true-breeding strains of Drosophila melanogaster with a view to establishing methods of phenotypic measurement sufficiently robust to allow subsequent biometric analysis. Between them, these strains formed a representative sample of the genetic variability of a natural population. Period, phase, definition (the degree to which a rhythmic signal was obscured by noise), and rhythm waveform were all found to vary continuously among the strains, although within each strain the rhythm phenotype was remarkably consistent. Each characteristic was found to be sufficiently robust to permit objective measurement using several different methods of quantification, which were then compared.     

An Investigation of Natural Genetic Variation in the Circadian System of Drosophila melanogaster: Rhythm Characteristics and Methods of Quantification

Variation in four characteristics of the circadian locomotor activity rhythm was investigated in 24 true-breeding strains of Drosophila melanogaster with a view to establishing methods of phenotypic measurement sufficiently robust to allow subsequent biometric analysis. Between them, these strains formed a representative sample of the genetic variability of a natural population. Period, phase, definition (the degree to which a rhythmic signal was obscured by noise), and rhythm waveform were all found to vary continuously among the strains, although within each strain the rhythm phenotype was remarkably consistent. Each characteristic was found to be sufficiently robust to permit objective measurement using several different methods of quantification, which were then compared.     

Sleep and circadian rhythms: key components in the regulation of energy metabolism

In this review, we present evidence from human and animal studies to evaluate the hypothesis that sleep and circadian rhythms have direct impacts on energy metabolism, and represent important mechanisms underlying the major health epidemics of obesity and diabetes. The first part of this review will focus on studies that support the idea that sleep loss and obesity are "interacting epidemics." The second part will discuss recent evidence that the circadian clock system plays a fundamental role in energy metabolism at both the behavioral and molecular levels. These lines of research must be seen as in their infancy, but nevertheless, have provided a conceptual and experimental framework that potentially has great importance for understanding metabolic health and disease.     

Mitotic activity of the pars intermedia in the female mouse: age-associated variations in proliferation rate and circadian periodicity

We previously reported daily variations in the mitotic activity of the endocrine cells in the pars intermedia of 21- and 28-day-old male mice. Since cellular proliferation might be affected by factors such as sex and age, we undertook the present experiments to study the mitotic activity of the pars intermedia from 14-, 28-, and 150-day-old female mice. Inbred C3H/S mice, grouped according to age, were housed under standard conditions (12h each of light and dark [LD 12:12]) for periodicity analysis and were killed in lots of 5-11 animals every 4h over a single 24h cycle, with each mouse receiving 2 μg/g of colchicine 4h before decapitation. Pituitaries were excised, extracted, fixed in buffered formaldehyde, embedded in celloidin-paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. We counted the total number of nuclei to estimate the total number of cells monitored and then calculated the mitotic index (metaphases/1000 nuclei). Differences were analyzed for statistical significance by the Student t test. While the 14-day-old animals manifested no significant changes in mitotic activity during the 24h cycle, the 28- and 150-day-old mice showed higher mitotic indices during the period of darkness. The average mitotic activity over the entire cycle, however, was higher in the two groups of younger animals than in the 150-day-old mice. Moreover, the averages for the 28-day-old females were higher than the corresponding values previously reported by us for male mice of the same age. (Chronobiology International, 17(6), 751-756, 2000)     

The precision of circadian clocks: assessment and analysis in Syrian hamsters

Locomotor activity recordings of Syrian hamsters were systematically analyzed to estimate the precision of the overt circadian activity rhythm in constant darkness. Phase variation, i.e., the standard deviation of phase markers around the regression line, varied with the definition of phase. Smallest phase variation was found in the onset of wheel running activity defined by 1h running means of the raw data. Both lower and higher degrees of smoothing lead to decreased precision measured in the overt rhythm. With passive infrared recordings, the midpoint of activity defined by 3h running means was the least variable. This demonstrates that the choice of phase marker should vary between recording methods. Phase variation decreased with increasing activity and was larger in females than in males. By calculating the average cycle variation and serial covariance of consecutive cycles, we estimated the contribution of 'clock' and 'non-clock' related processes to the overt rhythm variability. Variance in precision between phase markers could be shown to be attributable mainly to nonclock processes. Variance in pacemaker cycle length appeared reduced in wheel running activity records compared with passive infrared sensing records, suggesting feedback from running activity onto pacemaker function.     

Norepinephrine Turnover in the Hypothalamus of Adult Male Rats: Alteration of Circadian Patterns by Semistarvation

Norepinephrine (NE) turnover, as estimated by 3-methoxy-4-hydroxyphenylethyleneglycol concentration, was studied in the mediobasal hypothalamus of control and semistarved adult male rats at eight time points of a 24-h period. The marked circadian periodicity of NE turnover with a peak in the dark phase in control rats is completely suppressed in semistarved rats. The average 24-h concentration of the NE precursor tyrosine in brain and of tyrosine flow into brain (calculated from plasma amino acid concentrations) is reduced in semistarved rats, but both brain tyrosine and tyrosine flow show continuing circadian fluctuations.     

Development of Melatonin Synthesis in Chicken Retina: Regulation of Serotonin N-Acetyltransferase Activity by Light, Circadian Oscillators, and Cyclic AMP

In chicken retinas, melatonin levels and the activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme of melatonin biosynthesis, are expressed as circadian rhythms with peaks of levels and activity occurring at night. In the present study, NAT activity was examined in retinas of embryonic and posthatch chicks to assess the ontogenic development of regulation of the enzyme by light, circadian oscillators, and the second messenger cyclic AMP. During embryonic development, NAT activity was consistently detectable by embryonic day 6 (E6). Significant light-dark differences were first observed on E20, and increased to a maximum amplitude of sixfold by posthatch day 3 (PH3). Circadian rhythmicity of NAT activity appears to develop at or prior to hatching, as evidenced by day-night differences of activity in constant darkness observed in PH1 chicks that had been exposed to a light-dark cycle in ovo only. NAT activity is regulated by a cyclic AMP-dependent mechanism. Activity was significantly increased by incubating retinas with forskolin or dibutyryl cyclic AMP as early as E7, and seven- to ninefold increases were observed following treatment with these agents on E14. Thus, development of the cyclic AMP-dependent mechanism for increasing NAT activity significantly precedes that of rhythmicity, suggesting that the onset of rhythmicity may be related to the onset of photoreception or development of the circadian oscillator in chick retina.     

Changes in circadian rhythms of thermoregulation and motor activity in rats as a function of aging: effects of d-amphetamine and alpha-MSH

Thermoregulatory and motor activity circadian cycles are age-dependent. While the level of thermoregulation and motor activity remained almost at the same level during the first 1-15 months during the light portion of the 24-hr cycle, a significant decrease in the level of both rhythms was observed during the dark period. Therefore, older rats exhibited reversed cycles compared with younger rats. Treatments with d-amphetamine resulted in the enhancement of reversal of the cycles. Rats treated with alpha-MSH failed to exhibit a reversal of the cycles. While the effects of d-amphetamine are mediated by the brain DA mesolimbic pathway, it seems that alpha-MSH acts on the dopaminergic system at different sites of action.     

Localization and Characterization of Nitric Oxide Synthase in the Rat Suprachiasmatic Nucleus: Evidence for a Nitrergic Plexus in the Biological Clock

Abstract: Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH-diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of l -[³H]arginine to l -[³H]citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca²⁺/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.     

Zeitgeber Hierarchy in Humans: Resetting the Circadian Phase Positions of Blind People Using Melatonin

Four blind individuals who were thought to be entrained at an abnormal circadian phase position were reset to a more normal phase using exogenous melatonin administration. In one instance, circadian phase was shifted later. A fifth subject who was thought to be entrained was monitored over four years and eventually was shown to have a circadian period different from 24 h. These findings have implications for treating circadian phase abnormalities in the blind, for distinguishing between abnormally entrained and free‐running blind individuals, and for informing the debate over zeitgeber hierarchy in humans.