Rhythms In Animals Research Articles

Sex differences in the response to circadian disruption in diurnal sand rats

ABSTRACT Most animal model studies on physiological functions and pathologies are conducted in males. However, diseases such as depression, type 2 diabetes (T2DM) and cardiovascular disease, all show different prevalence and characteristics in females and males. Moreover, most mammal studies are conducted in nocturnal mice and rats, while modelling diurnal humans. We therefore used male and female fat sand rats (Psammomys obesus), which are diurnal in the wild, as an animal model for T2DM, to explore the effects of mild circadian disruption on behavior, glucose tolerance, cholesterol and heart weight. We found significant differences between the sexes: on average, in response to short photoperiods (SP) acclimation, males showed higher levels of depression-like behavior, lower glucose tolerance, and increased plasma cholesterol levels compared with females, with no effect on heart/body weight ratio. Females, however did show an increase in heart/body weight ratio in response to SP acclimation. We also found that regardless of sex, arrhythmic animals showed higher blood glucose levels, cholesterol levels, heart/body weight ratio, and depressive-like behavior compared with rhythmic animals. Hence, we suggest that the expression of the Circadian Syndrome could be different between males and females. Additional work with females is required to clearly delineate the specific effects in both sexes, and promote sex-based health care, prevention measures and therapies.

The eIF2α Kinase GCN2 Modulates Period and Rhythmicity of the Circadian Clock by Translational Control of Atf4

The integrated stress response (ISR) is activated in response to diverse stress stimuli to maintain homeostasis in neurons. Central to this process is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Here, we report a critical role for ISR in regulating the mammalian circadian clock. The eIF2α kinase GCN2 rhythmically phosphorylates eIF2α in the suprachiasmatic circadian clock. Increased eIF2α phosphorylation shortens the circadian period in both fibroblasts and mice, whereas reduced eIF2α phosphorylation lengthens the circadian period and impairs circadian rhythmicity in animals. Mechanistically, phosphorylation of eIF2α promotes mRNA translation of Atf4. ATF4 binding motifs are identified in multiple clock genes, including Per2, Per3, Cry1, Cry2, and Clock. ATF4 binds to the TTGCAGCA motif in the Per2 promoter and activates its transcription. Together, these results demonstrate a significant role for ISR in circadian physiology and provide a potential link between dysregulated ISR and circadian dysfunction in brain diseases.

Hypothalamic control systems show differential gene expression during spontaneous daily torpor and fasting-induced torpor in the Djungarian hamster (Phodopus sungorus).

Djungarian hamsters are able to use spontaneous daily torpor (SDT) during the winter season as well as fasting-induced torpor (FIT) at any time of the year to cope with energetically challenging environmental conditions. Torpor is a state of severely reduced metabolism with a pronounced decrease in body temperature, which enables animals to decrease their individual energy requirements. Despite sharing common characteristics, such as reduced body mass before first torpor expression and depressed metabolism and body temperature during the torpid state, FIT and SDT differ in several physiological properties including torpor bout duration, minimal body temperature, fuel utilization and circadian organization. It remains unclear, whether SDT and FIT reflect the same phenomenon or two different physiological states. The hypothalamus has been suggested to play a key role in regulating energy balance and torpor. To uncover differences in molecular control mechanisms of torpor expression, we set out to investigate hypothalamic gene expression profiles of genes related to orexigenic (Agrp/Npy), circadian clock (Bmal1/Per1) and thyroid hormone (Dio2/Mct8) systems of animals undergoing SDT and FIT during different torpor stages. Orexigenic genes were mainly regulated during FIT and remained largely unaffected by SDT. Expression patterns of clock genes showed disturbed circadian clock rhythmicity in animals undergoing FIT, but not in animals undergoing SDT. During both, SDT and FIT, decreased Dio2 expression was detected, indicating reduced hypothalamic T3 availability in both types of torpor. Taken together, our results provide evidence that SDT and FIT also differ in certain central control mechanisms and support the observation that animals undergoing SDT are in energetical balance, whereas animals undergoing FIT display a negative energy balance. This should be carefully taken into account when interpreting data in torpor research, especially from animal models of fasting-induced hypometabolism such as mice.

Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator.

BackgroundTalitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species.MethodsWe harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq.ResultsWe identified a comprehensive suite of circadian clock gene homologues in T. saltator including the ‘core’ clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression.DiscussionWe report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our sequencing data contribute to the growing inventory of described comparative clocks. Expression profiling of the identified clock genes illuminates tantalising targets for experimental manipulation to elucidate the molecular and cellular control of clock-driven phenotypes in this crustacean.

Clocks and cardiovascular function.

Circadian clocks in central and peripheral tissues enable the temporal synchronization and organization of molecular and physiological processes of rhythmic animals, allowing optimum functioning of cells and organisms at the most appropriate time of day. Disruption of circadian rhythms, from external or internal forces, leads to widespread biological disruption and is postulated to underlie many human conditions, such as the incidence and timing of cardiovascular disease. Here, we describe in vivo and in vitro methodology relevant to studying the role of circadian rhythms in cardiovascular function and dysfunction.

Dusk but not dawn burrow emergence rhythms of <i>Nephrops norvegicus</i> (Crustacea: Decapoda)

The Norway lobster, Nephrops norvegicus , can be captured by haul nets only during the emergence from its burrow. In the last few decades, an extensive field research revealed distinct diel (24-h–based) catchability patterns at different depths. Laboratory experiments suggested that burrow emergence (used as a proxy of catchability) is endogenously controlled via a circadian system. Results were usually presented in terms of mean effects without a quantification of inter-individual variability and arrhythmia. Here, we studied the burrow emergence of 52 adult Nephrops by an infrared actograph endowed with an artificial burrow. Animals were exposed to 12-12 h light-darkness cycle, simulating photic condition of the lower shelf. Forty-five animals showed rhythmic emergence (87%), while seven were arrhythmic (13%). Rhythmic animals were clustered according to their timing of emergence: 54% at dusk and 4% at dawn. Moreover, other animals showed fully diurnal or nocturnal emergence (10% and 19%, respectively). The comparison of our results with those derived from temporally scheduled trawling indicates that bimodal catch patterns observed in shelf populations are poorly observed during individual experiments in the laboratory, where the same light conditions are simulated. Nephrops burrow emergence seems to be the result of a mixed endogenous-exogenous control, while arrhythmia could also be present in the wild.

Difference of social motivation in quail selected for divergent circadian activity

Endogenous rhythms are adaptive responses to predictable changes of the environment, like the day/night cycle. Some researches demonstrated that social cycles can influence the circadian rhythm, while no study investigated the effect of endogenous rhythmicity on the sociability in Vertebrates. This study investigated whether differences in the functioning of the circadian system was associated with social motivation in Japanese quail ( Coturnix c. japonica).We compared quail from a line expressing a robust circadian rhythm of feeding activity (R) to quail from a line expressing circadian arrhythmicity of feeding activity (A) under constant darkness. Classic behavioral tests evaluated social motivation of these birds.When socially isolated, the motivation of R quail to re-establish contact with conspecifics appeared stronger than that of A quail. When in the presence of conspecifics, R quail faced a stressful situation (change of environment) more calmly than did A quail. Thus, variation of circadian behavioral rhythmicity is associated with variation in social motivation in Japanese quail. Rhythmic animals appeared to respond more appropriately to environmental challenges than arrhythmic animals.

Kinetics of Doubletime Kinase-dependent Degradation of the Drosophila Period Protein

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.

Rhythm-Promoting Actions of Exercise in Mice with Deficient Neuropeptide Signaling

Daily exercise promotes physical health as well as improvements in mental and neural functions. Studies in intact wild-type (WT) rodents have revealed that the brain's suprachiasmatic nuclei (SCN), site of the main circadian pacemaker, are also responsive to scheduled wheel running. It is unclear, however, if and how animals with a dysfunctional circadian pacemaker respond to exercise. Here, we tested whether scheduled voluntary exercise (SVE) in a running wheel for 6 hours per day could promote neural and behavioral rhythmicity in animals whose circadian competence is compromised through genetically targeted loss of vasoactive intestinal polypeptide (VIP(-/-) mice) or its VPAC(2) receptor (Vipr2(-/-) mice). We report that in constant dark (DD), rhythmic VIP(-/-) and Vipr2(-/-) mice show weak free-running rhythms with a period of <23 hours and all wild-type mice are strongly rhythmic with approximately 23.5-hour periodicity. VIP(-/-) and Vipr2(-/-) mice rapidly (<7 days) synchronize to daily SVE, while WT mice take much longer (>35 days). Following 21 to 50 days of SVE, WT mice show small changes in their rhythms, and most Vipr2(-/-) mice now sustain robust near 24-hour behavioral rhythms, whereas very few VIP(-/-) mice do. This study demonstrates that scheduled daily exercise can markedly improve circadian rhythms in behavioral activity and raises the possibility that this noninvasive approach may be useful as an intervention in clinical etiologies in which there are dysfunctions of circadian time keeping.

An automated tracking system for Caenorhabditis elegans locomotor behavior and circadian studies application

Automation of simple behavioral patterns, such as locomotor activity, is fundamental for pharmacological and genetic screening studies. Recently, circadian behaviors in locomotor activity and stress responses were reported in the nematode Caenorhabditis elegans, a well-known model in genetics and developmental studies. Here we present a new method for long-term recordings of C. elegans (as well as other similar-sized animals) locomotor activity based on an infrared microbeam scattering. Individual nematodes were cultured in a 96-well microtiter plate; we tested L15, CeMM and E. coli liquid cultures in long-term activity tracking experiments, and found CeMM to be the optimal medium. Treatment with 0.2% azide caused an immediate decrease in locomotor activity as recorded with our system. In addition to the validation of the method (including hardware and software details), we report its application in chronobiological studies. Circadian rhythms in animals entrained to light–dark and constant dark conditions ( n = 48 and 96 worms, respectively) at 16 °C, were analyzed by LS periodograms. We obtained a 24.2 ± 0.44 h period (52% of significantly rhythmic animals) in LD, and a 23.1 ± 0.40 h period (37.5% of significantly rhythmic animals) under DD. The system is automateable using microcontrollers, of low-cost construction and highly reproducible.

Seasonality of the endogenous activity rhythm in Talitrus saltator (Montagu) from a sandy beach in northeastern Tunisia

The endogenous activity of the sandhopper Talitrus saltator (Montagu) has been investigated in a population from a relatively exposed beach in northeast Tunisia. The animals were found to exhibit a nocturnal circadian rhythm of locomotor activity, with no evidence of a circatidal component. The rhythm was monitored in individual animals over the four seasons, under constant laboratory conditions and seasonal variation in free-running period; the signal-to-noise ratio and waveform of the rhythm have been investigated, as well as the incidence of rhythmic animals in the population. The variation in these rhythm parameters is considered in relation to changes in environmental conditions prevailing at the site of collection.

Homeostatic regulation of sleep in arrhythmic Siberian hamsters.

Sleep is regulated by independent yet interacting circadian and homeostatic processes. The present study used a novel approach to study sleep homeostasis in the absence of circadian influences by exposing Siberian hamsters to a simple phase delay of the photocycle to make them arrhythmic. Because these hamsters lacked any circadian organization, their sleep homeostasis could be studied in the absence of circadian interactions. Control animals retained circadian rhythmicity after the phase shift and re-entrained to the phase-shifted photocycle. These animals displayed robust daily sleep-wake rhythms with consolidated sleep during the light phase beginning about 1 h after light onset. This marked sleep-wake pattern was circadian in that it persisted in constant darkness. The distribution of sleep in the arrhythmic hamsters over 24 h was similar to that in the light phase of rhythmic animals. Therefore, daily sleep amounts were higher in arrhythmic animals compared with rhythmic ones. During 2- and 6-h sleep deprivations (SD), it was more difficult to keep arrhythmic hamsters awake than it was for rhythmic hamsters. Because the arrhythmic animals obtained more non-rapid eye movement sleep (NREMS) during the SD, they showed a diminished compensatory response in NREMS EEG slow-wave activity during recovery sleep. When amounts of sleep during the SD were taken into account, there were no differences in sleep homeostasis between experimental and control hamsters. Thus loss of circadian control did not alter the homeostatic response to SD. This supports the view that circadian and homeostatic influences on sleep regulation are independent processes.

Rest-activity patterns in children with hypopituitarism.

Physical lesions in the region of the suprachiasmatic nuclei, which are the site of a circadian clock, result in abnormal circadian rhythmicity in animals, yet the extent of biological rhythm problems in individuals with anatomic or functional lesions in the hypothalamic-pituitary region are largely unknown. To address this issue, we examined patterns of rest and activity of children with hypopituitarism. Children who were between the ages of 2 and 18 years and had the diagnosis of panhypopituitarism were evaluated. Twenty children were studied, including children with septo-optic dysplasia (SOD), congenital hypopituitarism, brain tumors, closed head trauma, and head irradiation. For assessing patterns of activity, individuals wore Actiwatches for 3-4 weeks to measure patterns of gross motor activity. Seventeen children had normal patterns of rest and activity, with an average period length of 24.01 +/- 0.01 hours. Three children, including 2 with SOD and 1 with a hypothalamic germinoma, showed abnormal activity patterns in which there was not consolidated rest at night. One patient with an optic glioma had nonentrained circadian phase. A proportion of children with hypopituitarism have abnormal daily rest-activity patterns. Children with anterior hypothalamic tumors and SOD seem to be at risk for circadian system dysfunction.

Bright light during lactation alters the functioning of the circadian system of adult rats.

To examine the role of light in the maturation of the circadian pacemaker, twelve groups of rats were raised in different conditions of exposure to constant bright light (LL) during lactation: both duration and timing of LL were varied. We studied the motor activity rhythm of the rats after weaning, first under LL and then under constant darkness (DD). In DD, two light pulses [at circadian time 15 (CT15) and CT22] were applied to test the response of the pacemaker. Greater exposure to LL days during lactation increased the number of rhythmic animals and the amplitude of their motor activity rhythm in the LL stage and decreased the phase delay due to the light pulse at CT15. The timing of LL during lactation affected these variables too. Because the response of the adult to light depended on both the number and timing of LL days during lactation, the exposure to light at early stages may influence the development of the circadian system by modifying it structurally or functionally.

Circadian Rhythms of Locomotor Activity in Zebrafish

As part of an effort to characterize the circadian system of the zebrafish, we examined the circadian regulation of locomotor activity in adult males and females. Gross locomotor activity was measured using infrared movement detectors. The effects of light, dark, and temperature on the amplitude, phase, and free-running periods of locomotor rhythms were determined. When zebrafish were maintained in a 12-h light:12 h dark cycle at 25°C, 86% of the fish were most active during the light phase of the cycle. The phases of free-running rhythms measured after transfer of fish from light cycles to constant conditions indicate that this diurnal activity profile reflects entrained circadian rhythmicity. When animals were maintained in constant conditions, the proportion that expressed significant circadian rhythmicity depended on ambient temperature. At 21°C, 73% of the animals were rhythmic in constant darkness, and 65% were rhythmic in constant light. Fewer (28–59%) were rhythmic at 18°, 25°, and 28.5°C. The free-running period of rhythmic animals was not affected by temperature within this range. The average period was shorter in constant light (LL; 12 lx) than in constant darkness (DD) in all but one experiment, and the difference was statistically significant for animals held at 21°C. These data indicate that zebrafish locomotor activity is regulated by a circadian clock that is temperature compensated. Because rhythmicity is most robust at 21°C, this would be the optimal temperature for future studies of the physiological basis of zebrafish behavioral rhythms.

Crab larval-release rhythms

The same claim, worded differently, is made on the next page-clearly these authors meant what they said: they think the rhythms of these animals are not controlled by an internal clock. It is difficult to imagine how such a misunderstanding could arise: fiddler crabs and Carcinus are the classic organisms used in marine chronobiology studies. The first description of a clock-controlled tidal rhythm was done on a fiddler crab (Bennett e t al. 1957) and the animal has continued to be studied to the present day. Scores of papers on the rhythmicity of this crab, emanating from several labs, have been published (see the 34 yr summary of this work by Palmer 1991). The first study of the internal-clock control of Carcinus maenas rhythms was performed in 1958 (Naylor), and there are at least 50 more papers that have appeared since then from Naylor's lab and several others. The first study of the tidal rhythm of the fish Lipophrypho11s was done in 1965 (Gibson), and recently a nice series of papers (culminating in 1991) on the animal's rhythms have been published by Northcott, Gibson & Morgan; a review of the L. pholis rhythm, and the rhythmicity of other intertidal fishes, was published by Gibson (1992). There cannot be the slightest doubt in the minds of marine chronobiologists (other than Saigusa & Kawagoye) that the tide-associated rhythms of these 3 intertidal animals (and a great many others, see Palmer 1995) are under the control of internal living clocks. Further on in their paper, Saigusa & Kawagoye state that their results 'suggests [that] a light-response mechanism [is] involved even in the circatidal rhythm of intertidal animals' (p 92-93). This assertion is repeated again on p 94: 'This indicates that the 24-h LD cycle functions as the zeitgeber of the circatidal rhythm of Hemigrapsus sanguineus.' This claim creates a problem in my mind. Certainly (to give a n extreme example) one would not expect that the attendance of church each Sunday morning would set the phase of our second-to-second heart-beat rhythm. It is equally illogical to accept a conclusion that the 24-h day-night cycle would set the phase or entrain a n overt 12.4-h organismic tidal rhythm. If it did, the rhythm would no longer match the tides; it would become 24 h in length and lose the survival value once offered by a rhythm. Those 2 comments out of the way, I will turn directly to the rhythm itself. Larvae release in this species is a one-time event, i.e. each individual sheds all its larvae in one ca 6-h interval. This type of behavior is referred to in chronobiological literature a s a 'gated response', meaning it takes place only when a 'gate' is opened to allow the event. When a batch of crabs is collected and brought into the laboratory, they do not all release their offspring on the same day; instead, individuals release at different times over a n interval of several days. And when the response of an entire population, maintained in either a light-dark cycle or constant dim illumination, is plotted it is seen that individual releases occur roughly 25 h later each day. Taking the data at face value, the most obvious interpretation of the population-as-a-whole display is that it is a circa-

Circadian rhythm and the per ACNGGN repeat in the mole rat, Spalax ehrenbergi.

Individual variability in circadian locomotor activity has recently discovered in the blind mole rat, Spalax ehrenbergi. An interesting association was found between different circadian types and two DNA fragments, 5.6 and 5.9 kb long, that contain the ACNGGN repeat sequence, homologous to a part of the period gene of Drosophila. Nine of 12 arrythmic animals showed the 5.6-kb band, while 13 of 17 circadian rhythmic animals had the 5.9-kb band. This repeat exists also in the brain RNA of the mole rat, apparently in higher quantities during the sleeping phase, suggesting that an unusual protein(s), composed of a poly-Thr-Gly segment, affects in circadian rhythm.

Expression of circadian rhythmicity correlates with the number of arginine-vasopressin-immunoreactive cells in the suprachiasmatic nucleus of common voles, Microtus arvalis

We have studied the number of arginine-vasopressin (AVP)-immunoreactive (IR) cells in the suprachiasmatic nuclei (SCN) in common voles, Microtus arvalis, with a strong ( n = 18), weak ( n = 10) or absent ( n = 9) expression of circadian rhythmicity. Spontaneous expression of rhythmicity was assessed from records of wheel running activity and general activity, measured with passive infra-red detection during 4 weeks of continuous low light (LL) conditions. Subsequently, 20 voles were perfused in LL. After additional exposure to a 12:12 h light-dark (LD) cycle during 14 days, the other 17 voles were perfused in the early morning. AVP-positive neurons were visualized immunocytochemically with a polyclonal IgG antibody. AVP-stained cell somata were present predominantly in the dorsomedial SCN, whereas terminals, contacting both AVP positive and negative neurons, were distributed throughout the SCN. In LL conditions, AVP-staining intensity of neurons was low in all 20 voles. Also the number of AVP-IR cells in the SCN in brain sections obtained in LL conditions was low and not different for the three categories of rhythmicity. In LD conditions, the staining intensity of AVP positive SCN neurons was low in rhythmic animals, high in non-rhythmic animals not expressing, and intermediate in animals weakly expressing circadian rhythmicity. The number of AVP positive cells in LD conditions showed a negative correlation with the propensity of animals to lose circadian rhythmicity under LL conditions. Differences in AVP-IR were found solely in the dorsomedial SCN, and neither in the rostral and caudal aspects of the SCN nor in the area adjacent to the SCN. These results suggest that the AVP-neurons of the SCN from part of a discrete neuronal substrate which functionally correlates with the expression of circadian rhythmicity.