Synchronization properties of multi-oscillator circadian systems

Dissertation presented to obtain the Ph.D degree in Computational Biology

Period determination in the food-entrainable and methamphetamine-sensitive circadian oscillator(s)

Daily rhythmic processes are coordinated by circadian clocks, which are present in numerous central and peripheral tissues. In mammals, two circadian clocks, the food-entrainable oscillator (FEO) and methamphetamine-sensitive circadian oscillator (MASCO), are "black box" mysteries because their anatomical loci are unknown and their outputs are not expressed under normal physiological conditions. In the current study, the investigation of the timekeeping mechanisms of the FEO and MASCO in mice with disruption of all three paralogs of the canonical clock gene, Period, revealed unique and convergent findings. We found that both the MASCO and FEO in Per1(-/-)/Per2(-/-)/Per3(-/-) mice are circadian oscillators with unusually short (similar to 21 h) periods. These data demonstrate that the canonical Period genes are involved in period determination in the FEO and MASCO, and computational modeling supports the hypothesis that the FEO and MASCO use the same timekeeping mechanism or are the same circadian oscillator. Finally, these studies identify Per1(-/-)/Per2(-/-)/Per3(-/-) mice as a unique tool critical to the search for the elusive anatomical location(s) of the FEO and MASCO.

Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression

Little is known about plant circadian oscillators, in spite of how important they are to sessile plants, which require accurate timekeepers that enable the plants to respond to their environment. Previously, we identified a circadian clock-associated (CCA1) gene that encodes an Myb-related protein that is associated with phytochrome control and circadian regulation in plants. To understand the role CCA1 plays in phytochrome and circadian regulation, we have isolated an Arabidopsis line with a T DNA insertion that results in the loss of CCA1 RNA, of CCA1 protein, and of an Lhcb-promoter binding activity. This mutation affects the circadian expression of all four clock-controlled genes that we examined. The results show that, despite their similarity, CCA1 and LHY are only partially redundant. The lack of CCA1 also affects the phytochrome regulation of gene expression, suggesting that CCA1 has an additional role in a signal transduction pathway from light, possibly acting at the point of integration between phytochrome and the clock. Our results indicate that CCA1 is an important clock-associated protein involved in circadian regulation of gene expression.

Two distinct oscillators in the rat suprachiasmatic nucleus in vitro.

In the rat suprachiasmatic nucleus slice culture, circadian rhythms in the release of arginine vasopressin and vasoactive intestinal polypeptide were measured simultaneously and longitudinally. The phase relationship between the two peptide rhythms was relatively constant in the culture without a treatment of antimitotic drugs but became diverse by an introduction of antimitotics, which is generally used to reduce the number of glial cells. By monitoring the two rhythms continuously for 6 days, different periods were detected in culture with the antimitotic treatment. Furthermore, N-methyl-D-aspartate shifted the phase of the two peptide rhythms in the same culture differently. These results indicate that the arginine vasopressin and vasoactive intestinal polypeptide release are under control of different circadian oscillators.

Unconjugated bile acids influence expression of circadian genes: a potential mechanism for microbe-host crosstalk

Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression. Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression.

Correct biological timing in Arabidopsis requires multiple light-signaling pathways.

Circadian oscillators provide rhythmic temporal cues for a range of biological processes in plants and animals, enabling anticipation of the day/night cycle and enhancing fitness-associated traits. We have used engineering models to understand the control principles of a plant's response to seasonal variation. We show that the seasonal changes in the timing of circadian outputs require light regulation via feed-forward loops, combining rapid light-signaling pathways with entrained circadian oscillators. Linear time-invariant models of circadian rhythms were computed for 3,503 circadian-regulated genes and for the concentration of cytosolic-free calcium to quantify the magnitude and timing of regulation by circadian oscillators and light-signaling pathways. Bioinformatic and experimental analysis show that rapid light-induced regulation of circadian outputs is associated with seasonal rephasing of the output rhythm. We identify that external coincidence is required for rephasing of multiple output rhythms, and is therefore important in general phase control in addition to specific photoperiod-dependent processes such as flowering and hypocotyl elongation. Our findings uncover a fundamental design principle of circadian regulation, and identify the importance of rapid light-signaling pathways in temporal control.

Clock Genes, Melanopsins, Melatonin, and Dopamine Key Enzymes and Their Modulation by Light and Glutamate in Chicken Embryonic Retinal Cells

The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 mu M glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 mu M glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli. (Author correspondence: amdlcast@ib.usp.br).

Caracterização do perfil do ciclo sono-vigília em ratos sob dessincronização forçada

The circadian behavior associated with the 24 hours light-dark (LD) cycle (T24) is due to a circadian clock , which in mammals is located in the hypothalamic suprachiasmatic nucleus (SCN). Under experimental conditions in which rats are espoused to a symmetric LD 22h cycle (T22) the two SCN regions, ventrolateral (vl) and dorsomedial (dm), can be functionally isolated, suggesting that each region regulates distinct physiological and behavioral components. The vl region regulates the locomotor activity and slow wave sleep (SWS) rhythms, while the dm region assures the body temperature and paradoxical sleep (PS) rhythms regulation. This research aimed to deepen the knowledge on the functional properties of circadian rhythmicity, specifically about the internal desynchronization process, and its consequences to locomotor activity and body temperature rhythms as well as to the sleep-wake cycle pattern in rats. We applied infrared motion sensors, implanted body temperature sensors and a telemetry system to record electrocorticogram (ECoG) and electromyogram (EMG) in two rat groups. The control group under 24h period LD cycle (T24: 12hL-12hD) to the baseline record and the experimental group under 22h period LD cycle (T22: 11hL- 11hD), in which is known to occur the uncoupling process of the circadian locomotor activity rhythm where the animals show two distinct locomotor activity rhythms: one synchronized to the external LD cycle, and another expressed in free running course, with period greater than 24h. As a result of 22h cycles, characteristic locomotor activity moment appear, that are coincidence moments (T22C) and non coincidence moments (T22NC) which were the main focus or our study. Our results show an increase in locomotor activity, especially in coincidence moments, and the inversion of locomotor activity, body temperature, and sleep-wake cycle patterns in non coincidence moments. We can also observe the increase in SWS and decrease in PS, both in coincidence and non coincidence moments. Probably the increases in locomotor activity as a way to promote the coupling between circadian oscillators generate an increased homeostatic pressure and thus increase SWS, promoting the decreasing in PS

Forced Desynchronization of Activity Rhythms in a Model of Chronic Jet Lag in Mice

We studied locomotor activity rhythms of C57/Bl6 mice under a chronic jet lag (CJL) protocol (ChrA(6/2)), which consisted of 6-hour phase advances of the light-dark schedule (LD) every 2 days. Through periodogram analysis, we found 2 components of the activity rhythm: a short-period component (21.01 +/- 0.04 h) that was entrained by the LD schedule and a long-period component (24.68 +/- 0.26 h). We developed a mathematical model comprising 2 coupled circadian oscillators that was tested experimentally with different CJL schedules. Our simulations suggested that under CJL, the system behaves as if it were under a zeitgeber with a period determined by (24 -[phase shift size/days between shifts]). Desynchronization within the system arises according to whether this effective zeitgeber is inside or outside the range of entrainment of the oscillators. In this sense, ChrA(6/2) is interpreted as a (24 - 6/2 = 21 h) zeitgeber, and simulations predicted the behavior of mice under other CJL schedules with an effective 21-hour zeitgeber. Animals studied under an asymmetric T = 21 h zeitgeber (carried out by a 3-hour shortening of every dark phase) showed 2 activity components as observed under ChrA(6/2): an entrained short-period (21.01 +/- 0.03 h) and a long-period component (23.93 +/- 0.31 h). Internal desynchronization was lost when mice were subjected to 9-hour advances every 3 days, a possibility also contemplated by the simulations. Simulations also predicted that desynchronization should be less prevalent under delaying than under advancing CJL. Indeed, most mice subjected to 6-hour delay shifts every 2 days (an effective 27-hour zeitgeber) displayed a single entrained activity component (26.92 +/- 0.11 h). Our results demonstrate that the disruption provoked by CJL schedules is not dependent on the phase-shift magnitude or the frequency of the shifts separately but on the combination of both, through its ratio and additionally on their absolute values. In this study, we present a novel model of forced desynchronization in mice under a specific CJL schedule; in addition, our model provides theoretical tools for the evaluation of circadian disruption under CJL conditions that are currently used in circadian research.

Caracterização do ritmo circadiano de atividade e repouso em saguis idosos (Callithrix Jacchus)

Advanced age may become a limiting factor for the maintenance of rhythms in organisms, reducing the capacity of generation and synchronization of biological rhythms. In this study, the influence of aging on the expression of endogenous periodicity and synchronization (photic and social) of the circadian activity rhythm (CAR) was evaluated in a diurnal primate, the marmoset (Callithrix jacchus). This study had two approaches: one with longitudinal design, performed with a male marmoset in two different phases: adult (three years) and older (9 y.o.) (study 1) and the second, a transversal approach, with 6 old (♂: 9.7 ± 2.0 y.o.) and 11 adults animals (♂: 4.2 ± 0.8 y.o.) (study 2). The evaluation of the photic synchronization involved two conditions in LD (natural and artificial illuminations). In study 1, the animal was subjected to the following stages: LD (12:12 ~ 350: ~ 2 lx), LL (~ 350 lx) and LD resynchronization. In the second study, the animals were initially evaluated in natural LD, and then the same sequence stages of study 1. During the LL stage in study 2, the vocalizations of conspecifics kept in natural LD on the outside of the colony were considered temporal cue to the social synchronization. The record of the activity was performed automatically at intervals of five minutes through infrared sensor and actimeters, in studies 1 and 2, respectively. In general, the aged showed a more fragmented activity pattern (> IV < H and > PSD, ANOVA, p < 0.05), lower levels of activity (ANOVA, p < 0.05) and shorter duration of active phase (ANOVA, p < 0.05) in LD conditions, when compared to adults. In natural LD, the aged presented phase delay pronounced for onset and offset of active phase (ANOVA, p < 0.05), while the adults had the active phase more adjusted to light phase. Under artificial LD, there was phase advance and greater adjustment of onset and offset of activity in relation to the LD in the aged (ANOVA, p < 0.05). In LL, there was a positive correlation between age and the endogenous period () in the first 20 days (Spearman correlation, p < 0.05), with prolonged  held in two aged animals. In this condition, most adults showed free-running period of the circadian activity rhythm with  < 24 h for the first 30 days and later on relative coordination mediated by auditory cues. In study 2, the cross-correlation analysis between the activity profiles of the animals in LL with control animals kept under natural LD, found that there was less social synchronization in the aged. With the resubmission to the LD, the resynchronization rate was slower in the aged (t-test; p < 0.05) and in just one aged animal there was a loss of resynchronization capability. According to the data set, it is suggested that the aging in marmosets may be related to: 1) lower amplitude and greater fragmentation of the activity, accompanied to phase delay with extension of period, caused by changes in a photic input, in the generation and behavioral expression of the CAR; 2) lower capacity of the circadian activity rhythm to photic synchronization, that can become more robust in artificial lighting conditions, possibly due to the higher light intensities at the beginning of the active phase due to the abrupt transitions between the light and dark phases; and 3) smaller capacity of non-photic synchronization for auditory cues from conspecifics, possibly due to reducing sensory inputs and responsiveness of the circadian oscillators to auditory cues, what can make the aged marmoset most vulnerable, as these social cues may act as an important supporting factor for the photic synchronization.

Caracterização do ritmo circadiano de atividade e repouso em saguis idosos (Callithrix Jacchus)

Advanced age may become a limiting factor for the maintenance of rhythms in organisms, reducing the capacity of generation and synchronization of biological rhythms. In this study, the influence of aging on the expression of endogenous periodicity and synchronization (photic and social) of the circadian activity rhythm (CAR) was evaluated in a diurnal primate, the marmoset (Callithrix jacchus). This study had two approaches: one with longitudinal design, performed with a male marmoset in two different phases: adult (three years) and older (9 y.o.) (study 1) and the second, a transversal approach, with 6 old (♂: 9.7 ± 2.0 y.o.) and 11 adults animals (♂: 4.2 ± 0.8 y.o.) (study 2). The evaluation of the photic synchronization involved two conditions in LD (natural and artificial illuminations). In study 1, the animal was subjected to the following stages: LD (12:12 ~ 350: ~ 2 lx), LL (~ 350 lx) and LD resynchronization. In the second study, the animals were initially evaluated in natural LD, and then the same sequence stages of study 1. During the LL stage in study 2, the vocalizations of conspecifics kept in natural LD on the outside of the colony were considered temporal cue to the social synchronization. The record of the activity was performed automatically at intervals of five minutes through infrared sensor and actimeters, in studies 1 and 2, respectively. In general, the aged showed a more fragmented activity pattern (> IV < H and > PSD, ANOVA, p < 0.05), lower levels of activity (ANOVA, p < 0.05) and shorter duration of active phase (ANOVA, p < 0.05) in LD conditions, when compared to adults. In natural LD, the aged presented phase delay pronounced for onset and offset of active phase (ANOVA, p < 0.05), while the adults had the active phase more adjusted to light phase. Under artificial LD, there was phase advance and greater adjustment of onset and offset of activity in relation to the LD in the aged (ANOVA, p < 0.05). In LL, there was a positive correlation between age and the endogenous period () in the first 20 days (Spearman correlation, p < 0.05), with prolonged  held in two aged animals. In this condition, most adults showed free-running period of the circadian activity rhythm with  < 24 h for the first 30 days and later on relative coordination mediated by auditory cues. In study 2, the cross-correlation analysis between the activity profiles of the animals in LL with control animals kept under natural LD, found that there was less social synchronization in the aged. With the resubmission to the LD, the resynchronization rate was slower in the aged (t-test; p < 0.05) and in just one aged animal there was a loss of resynchronization capability. According to the data set, it is suggested that the aging in marmosets may be related to: 1) lower amplitude and greater fragmentation of the activity, accompanied to phase delay with extension of period, caused by changes in a photic input, in the generation and behavioral expression of the CAR; 2) lower capacity of the circadian activity rhythm to photic synchronization, that can become more robust in artificial lighting conditions, possibly due to the higher light intensities at the beginning of the active phase due to the abrupt transitions between the light and dark phases; and 3) smaller capacity of non-photic synchronization for auditory cues from conspecifics, possibly due to reducing sensory inputs and responsiveness of the circadian oscillators to auditory cues, what can make the aged marmoset most vulnerable, as these social cues may act as an important supporting factor for the photic synchronization.

Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators.

Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ultrasensitivity that is specific for repression. We then use stochastic simulation to show that multiple binding sites increase the coherence of oscillations by mitigating the binary noise. Slow values of DNA unbinding rate are also effective in alleviating molecular noise due to the increased distance from the bifurcation point. Our work demonstrates how the number of DNA binding sites and slow unbinding kinetics, which are often omitted in biophysical models of gene circuits, can have a significant impact on the temporal and stochastic dynamics of genetic oscillators.

Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light-dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN`s subregional organization to both photic input processing and rhythmic output control.

Modeling the Emergence of Circadian Rhythms in a Clock Neuron Network

Circadian rhythms in pacemaker cells persist for weeks in constant darkness, while in other types of cells the molecular oscillations that underlie circadian rhythms damp rapidly under the same conditions. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms leading to damped or self-sustained oscillations remain largely unknown. There exist many mathematical models that reproduce the circadian rhythms in the case of a single cell of the Drosophila fly. However, not much is known about the mechanisms leading to coherent circadian oscillation in clock neuron networks. In this work we have implemented a model for a network of interacting clock neurons to describe the emergence (or damping) of circadian rhythms in Drosophila fly, in the absence of zeitgebers. Our model consists of an array of pacemakers that interact through the modulation of some parameters by a network feedback. The individual pacemakers are described by a well-known biochemical model for circadian oscillation, to which we have added degradation of PER protein by light and multiplicative noise. The network feedback is the PER protein level averaged over the whole network. In particular, we have investigated the effect of modulation of the parameters associated with (i) the control of net entrance of PER into the nucleus and (ii) the non-photic degradation of PER. Our results indicate that the modulation of PER entrance into the nucleus allows the synchronization of clock neurons, leading to coherent circadian oscillations under constant dark condition. On the other hand, the modulation of non-photic degradation cannot reset the phases of individual clocks subjected to intrinsic biochemical noise.

Circadian modulation of dopamine receptor responsiveness in Drosophila melanogaster

We investigated the circadian function of Drosophila dopamine receptors by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. We show that the amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the period gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the period-dependent molecular oscillators in the body in the modulation of amine receptor responsiveness.