Circadian disruption promotes tumor-immune microenvironment remodeling favoring tumor cell proliferation.

Circadian disruption negatively affects physiology, posing a global health threat that manifests in proliferative, metabolic, and immune diseases, among others. Because outputs of the circadian clock regulate daily fluctuations in the immune response, we determined whether circadian disruption results in tumor-associated immune cell remodeling, facilitating tumor growth. Our findings show that tumor growth rate increased and latency decreased under circadian disruption conditions compared to normal light-dark (LD) schedules in a murine melanoma model. Circadian disruption induced the loss or inversion of daily patterns of M1 (proinflammatory) and M2 (anti-inflammatory) macrophages and cytokine levels in spleen and tumor tissues. Circadian disruption also induced (i) deregulation of rhythmic expression of clock genes and (ii) of cyclin genes in the liver, (iii) increased CcnA2 levels in the tumor, and (iv) dampened expression of the cell cycle inhibitor p21WAF/CIP1 , all of which contribute to a proliferative phenotype.

Factors that influence magnetic orientation in Caenorhabditis elegans.

Magnetoreceptive animals orient to the earth's magnetic field at angles that change depending on temporal, spatial, and environmental factors such as season, climate, and position within the geomagnetic field. How magnetic migratory preference changes in response to internal or external stimuli is not understood. We previously found that Caenorhabditis elegans orients to magnetic fields favoring migrations in one of two opposite directions. Here we present new data from our labs together with replication by an independent lab to test how temporal, spatial, and environmental factors influence the unique spatiotemporal trajectory that worms make during magnetotaxis. We found that worms gradually change their average preferred angle of orientation by ~ 180° to the magnetic field during the course of a 90-min assay. Moreover, we found that the wild-type N2 strain prefers to orient towards the left side of a north-facing up, disc-shaped magnet. Lastly, similar to some other behaviors in C. elegans, we found that magnetic orientation may be more robust in dry conditions (< 50% RH). Our findings help explain why C. elegans accumulates with distinct patterns during different periods and in differently shaped magnetic fields. These results provide a tractable system to investigate the behavioral genetic basis of state-dependent magnetic orientation.

Deficits in temporal processing correlate with clinical progression in Huntington's disease.

OBJECTIVES: Precise temporal performance is crucial for several complex tasks. Time estimation in the second-to-minutes range-known as interval timing-involves the interaction of the basal ganglia and the prefrontal cortex via dopaminergic-glutamatergic pathways. Patients with Huntington's disease (HD) present deficits in cognitive and motor functions that require fine control of temporal processing. The objective of the present work was to assess temporal cognition through a peak-interval time (PI) production task in patients with HD and its potential correlation with the Unified Huntington's Disease Rating Scale (UHDRS). MATERIALS AND METHODS: Patients with molecular diagnosis of HD and controls matched by age, sex and educational level (n=18/group) were tested for interval timing in short- (3 seconds), medium- (6 seconds) and long (12 seconds)-duration stimuli. RESULTS: Significant differences were observed in the PI task, with worse performance in HD compared to controls. Patients underestimated real time (left-shifted Peak location) for 6- and 12-second intervals (P<.05) and presented decreased temporal precision for all the intervals evaluated (P<.01). Importantly, a significant correlation was found between time performance and the UHDRS (P<.01). Patients' responses also deviated from the scalar property. CONCLUSIONS: Our results contribute to support that timing functions are impaired in HD in correlation with clinical deterioration. Recordings of cognitive performance related to timing could be a potential useful tool to measure the neurodegenerative progression of movement disorder-related pathologies.

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

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

Glial and light-dependent glutamate metabolism in the suprachiasmatic nuclei.

The suprachiasmatic nuclei, the main circadian clock in mammals, are entrained by light through glutamate released from retinal cells. Astrocytes are key players in glutamate metabolism but their role in the entrainment process is unknown. We studied the time dependence of glutamate uptake and glutamine synthetase (GS) activity finding diurnal oscillations in glutamate uptake (high levels during the light phase) and daily and circadian fluctuations in GS activity (higher during the light phase and the subjective day). These results show that glutamate-related astroglial processes exhibit diurnal and circadian variations, which could affect photic entrainment of the circadian system.

Circadian rhythms in the vegetative state.

OBJECTIVE: To evaluate whether vegetative state patients maintain circadian rhythms. RESEARCH DESIGN: An observational study of five single cases. METHODS AND PROCEDURES: Five chronic vegetative state patients underwent clinical and neurological evaluations and 2-week continuous temperature measurements. MAIN OUTCOMES AND RESULTS: The two patients with traumatic brain injury showed well-formed circadian temperature rhythms and had more reflexive behaviours and relatively low cortical and sub-cortical atrophy, whereas the three patients from anoxic-hypoxic origin demonstrated no cycles or rhythmic behaviour. CONCLUSIONS: The presence of periods of wakefulness does not imply preserved sleep-wake cycling capacity, nor preserved circadian rhythms and it should not be taken as a distinguishing feature for the definition of the vegetative state.

Participation of transcription factors from the Rel/NF-kappa B family in the circadian system in hamsters.

We have studied the presence and activity of components of the nuclear factor-kappaB (NF-kappaB) transcription factor in the hamster circadian system analyzing wheel-running activity, protein expression and DNA binding activity by electrophoresis mobility shift assays (EMSA). Non-rhythmic specific immunoreactive bands corresponding to a NF-kappaB subunit (p65) were found in hamster suprachiasmatic nuclei (SCN) homogenates. The active form of NF-kappaB evidenced by EMSA was clear and specific in SCN nuclear extracts. The administration of the NF-kappaB inhibitor pyrrolidine-dithiocharbamate (PDTC) blocked the light-induced phase advance at circadian time 18 (vehicle+light pulse: 2.08+/-0.46 h, PDTC+light: 0.36+/-0.35 h). These results demonstrate the presence and activity of Rel/NF-kappaB family proteins in the hamster SCN and suggest that these proteins may be related to the entrainment and regulation of circadian rhythms.

Circadian rhythms in airline pilots submitted to long-haul transmeridian flights.

BACKGROUND: Circadian rhythms shift out of phase after transmeridian flights. Desynchronization between body rhythms and the environment is linked to jet lag, which depends on age, flight direction, and number of time zones crossed. METHODS: To investigate this problem in airline pilots, we performed a multivariate analysis of their circadian systems during Madrid-Mexico-Madrid flights (-7 time zones, n = 12) and Madrid-Tokyo-Madrid flights (+8 time zones, n = 21). Telemetry was used to record pilots' activity, skin temperature, and heart rate, obtaining 6 d of continuous data, including 2 d before the flight, the flights themselves, 2 d at the stopover, and 1 d after the return flight. Time series were analyzed by cosinor, and the resulting parameters of the rhythms were compared by ANOVA and Tukey contrasts in every category formed by the age groups (under and over 50 yr old) and flight direction groups. Subjective time estimation of short, intermediate, and long intervals was recorded. Other psychological variables were measured, including anxiety, tiredness, and performance. RESULTS AND CONCLUSIONS: Activity/rest and heart rate rhythms appeared to be linked to a "weak oscillator." Temperature rhythms manifested a rigid response after the phase shifts of the light/dark cycle, closely related to the biological clock. Subjective time appreciation tended to be overestimated without exhibiting a clear circadian component, but attributable to fatigue and stress. Psychometric evaluation showed that desynchronization affected all the pilots. Some results showed an age-related variability with a more marked influence in younger pilots. No consistent effects regarding flight direction were found.

Rhythmic variation in gamma-aminobutyric acid(A)-receptor subunit composition in the circadian system and median eminence of Syrian hamsters.

Temporal changes in the level of expression of gamma-aminobutyric acid (GABA)(A) receptor subunits alpha2, alpha5, beta1 and beta3 were characterized by Western blot analysis in the hamster suprachiasmatic nuclei, retina and median eminence. A nocturnal maximum in the level of GABA(A) receptor beta1 subunit at midday and midnight (12:00 and 00.00 h) was found in the suprachiasmatic nucleus (SCN), the retina and the median eminence of Syrian hamsters. Alpha2 and beta3 subunit levels peaked during the day in the median eminence. Finally, retinal alpha5 levels were maximal during the night. beta1 temporal changes in the SCN and median eminence, as well as alpha2 variations in the median eminence were maintained under constant dark conditions, suggesting an endogenous control, while the other variations were only observed under light-dark cycle conditions.

Circadian urinary 6-sulphatoxymelatonin, cortisol excretion and locomotor activity in airline pilots during transmeridian flights.

Airline pilots divided into two groups of age (over and under 50 years) were studied before, during and after westbound (Madrid-Mexico City-Madrid, n = 12) and eastbound (Madrid-Tokyo-Madrid, n = 21) flights. A group of 10 age-matched people staying in Madrid were submitted to the same tests and served as a control group. Changes in urinary 6-sulphatoxymelatonin (6-aMTs) and free cortisol excretion (determined in 6-hr intervals) were measured by radioimmunoassay. Using wrist actigraphy, the circadian locomotor activity rhythm (LAR) was also monitored. Maximal baseline excretion of 6-aMTs occurred between 00:00 and 12:00 hr and maximal excretion of cortisol took place between 6:00 and 12:00 hr in the control group. Analysed globally, older pilots exhibited significantly lower values of 6-aMTs than younger ones. In both flight directions, pilots maintained the pattern of excretion of 6-aMTs, corresponding to baseline. The return flight to Madrid from Mexico and Tokyo coincided with a maximum in 6-aMTs excretion. Pilots kept the cortisol pattern found in the control group, with those over 50 years of age exhibiting significantly lower cortisol values than the younger ones. A 7-hr delay in acrophase of LAR after 2 days in Mexico City was found after cosinor analysis, and similar pre-flight values were found after returning to Madrid. An 8-9-hr acrophase advance of LAR was observed after arriving in Tokyo, with acrophase on the post-return flight day still being advanced 3 4 hr as compared to pre-flight values. Decreases in the amplitude of LAR in older pilots were found at Mexico City, as well as at Tokyo stopover and on post-flight day. Data confirm the occurrence of internal desynchronization in airline crewmembers after transmeridian flights.

Rhythmicity of the cGMP-related signal transduction pathway in the mammalian circadian system.

Entrainment of mammalian circadian rhythms requires the activation of specific signal transduction pathways in the suprachiasmatic nuclei (SCN). Pharmacological inhibition of kinases such as cGMP-dependent kinase (PKG) or Ca2+/calmodulin-dependent kinase, but not cAMP-dependent kinase, blocks the circadian responses to light in vivo. Here we show a diurnal and circadian rhythm of cGMP levels and PKG activity in the hamster SCN, with maximal values during the day or subjective day. This rhythm depends on phosphodiesterase but not on guanylyl cyclase activity. Five-minute light pulses increased cGMP levels at the end of the subjective night [circadian time 18 (CT18)], but not at CT13.5. Western blot analysis indicated that the PKG II isoform is the one present in the SCN. Inhibition of PKG or guanylyl cyclase in vivo significantly attenuated light-induced phase shifts at CT18 (after 5-min light pulses) but did not affect c-Fos expression in the SCN. These results suggest that cGMP and PKG are related to SCN responses to light and undergo diurnal and circadian changes.

Delay model of the circadian pacemaker.

We present a simple and realistic model of the circadian pacemaker that can be interpreted in molecular terms. The model, which consists of a single time-delay differential equation, simulates the expression of a generic clock protein that inhibits its own expression through a feedback mechanism. Despite its simplicity, this model fulfils most of the necessary characteristics of a realistic representation of natural circadian clocks: robust and stable oscillations with circadian free-running periods, typical phase response curves and entrainment to environmental zeitgebers. The present model reduces the molecular mechanism necessary to sustain stable oscillations to its bare bones, suggesting that the essential factor is the time-delayed negative feedback of the oscillating protein on its own expression.

Cyclic AMP and protein kinase A rhythmicity in the mammalian suprachiasmatic nuclei.

The levels of cyclic AMP and protein kinase A, as well as the activity of this enzyme, were measured in the hamster suprachiasmatic nuclei at different time points throughout the daily or circadian cycle. Significant diurnal variations for levels of AMPc and the catalytic subunit of protein kinase A and the activity of this enzyme were found. All of these parameters tended to increase throughout the nocturnal phase, reaching higher values at the end of the night and the beginning of the day and minimal values around the time of lights off. This rhythmicity appears to be under exogenous control, since constant darkness abolished fluctuations throughout the circadian cycle. In vitro incubation in the presence of melatonin during the day significantly decreased cyclic AMP levels and basal protein kinase A activity in the SCN, while neither neuropeptide Y nor light pulses affected these parameters. These results suggest a significant diurnal regulation of the cyclic AMP-dependent system in the hamster circadian clock.

Neuroactive steroids alter the circadian system of the Syrian hamster in a phase-dependent manner.

Several steroid compounds affect neuronal function, primarily by modulating the GABAA receptor complex. A circadian variation in the brain concentration of neurosteroids has been reported in rats and humans. We have previously reported that natural occurring or synthetic neuroactive steroids such as androsterone and alphaxalone also have a rhythmic effect on behavior (anesthetic and anticonvulsant activity) and GABAergic activity. In the present work, we have tested the ability of neuroactive steroids to phase shift circadian rhythms in hamsters. The GABA(A) negative modulator dehydroepiandrosterone sulphate (DHEAS) elicited phase advances when administered at CT 6, while the positive modulator androsterone lacked any effect at this time. A complete phase response curve for DHEAS revealed a nonphotic-like effect. DHEAS also blocked the circadian effects of light, while androsterone induced photic-like responses. There is also evidence that neurosteroids may be present and even synthesized in the SCN. Collectively, the results so far indicate that some neuroactive steroids might modulate the activity of the circadian clock.

Conservation of locomotor behavior in the golden hamster: effects of light cycle and a circadian period mutation.

Locomotor activity in rodents is restricted temporally by the animal' s circadian system. The relative stability of both the species-specific pattern and the amount of locomotor activity per cycle suggested that this behavior may be regulated by conservative mechanisms. In these experiments, the wheel-running behavior of golden hamsters carrying the circadian period mutation, tau, was analyzed in animals housed in a 24-h light:dark cycle (LD) and in constant dark (DD) conditions to determine which aspects of this behavior were conserved. In DD, apart from the change in period which defines the mutation, no main effects of allele combination were found in either average amount of activity, activity profile, or length of the activity phase. In LD, wild-type behavior did not differ from that in DD; however, heterozygous mutants exhibited early onsets of activity, significant fragmentation of both activity and rest, an increase in the duration of the active phase, and an overall decrease in the amount of activity. Despite these differences, the total amount of time spent on the wheel in LD or DD was the same for all environment/genotype combinations. The data show that a conservative mechanism that may influence daily patterns of locomotor behavior is related more to a drive to perform the behavior than the quantity or timing of the behavior itself.

Photic control of nitric oxide synthase activity in the hamster suprachiasmatic nuclei.

Circadian rhythms are controlled by an endogenous clock, which in mammals is located in the hypothalamic suprachiasmatic nuclei (SCN). A role for nitric oxide in circadian responses to light has been indicated. To test the role of nitric oxide synthase (NOS) in the SCN and in circadian responses to light, we examined NOS specific activity at different time points and photic conditions. NOS activity was determined by the conversion of 3H-arginine to 3H-citrulline. NOS enzymatic activity in the SCN was significantly higher during the dark phase than during the day, without any changes in the levels of the NOS protein. However, this difference disappeared when animals were placed under constant darkness, and NOS activity was similar at CT 8 and CT 18 (with CT 12 defined as the onset of the subjective night). When 5-min light pulses were administered at these time points (when light would induce no phase shift or a phase advance, respectively), NOS activity was significantly increased almost equally. A spectrophotometric assay was used to determine NO content in the SCN, showing relatively high constitutive levels enhanced by 100 microM glutamate. These results suggest that NOS activity is not controlled by the circadian clock, although it might mediate some of the effects of light on biological rhythms.

Neonatal clomipramine treatment of Syrian hamsters: effect on the circadian system.

The circadian behavior of male Syrian hamsters injected with the serotonin/norepinephrine reuptake inhibitor clomipramine (15 mg/kg from postnatal days 8 to 21) was examined. Clomipramine treatment significantly augmented mean activity values of wheel running rhythm, as well as delayed its acrophase. After a 6-h phase advance of the light-dark cycle, reentrainment of clomipramine-treated hamsters took significantly longer than controls. Clomipramine-treated hamsters exhibited a shorter circadian period than controls in constant light conditions, but no differences were found in constant darkness. Light pulses applied at late subjective night to clomipramine-treated hamsters caused significantly reduced phase advances as compared to controls, while no differences were found in phase delay magnitudes when light pulses were applied during early subjective night. Administration of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) at circadian time 8 significantly advanced the onset of activity to a greater extent in clomipramine-treated hamsters than in controls. The results indicate that neonatal clomipramine treatment of hamsters causes long-lasting changes in the circadian system, by increasing activity levels and by partially inhibiting light-evoked responses. An enhancement of a non-photic, serotonergic-induced response was also unveiled.

The rhythmic GABAergic system.

GABA is the major inhibitory neurotransmitter in the mammalian brain, and has been implicated in the regulation of a variety of behavioral functions, including biological rhythms. The focus of this minireview is the rhythmic variation of the central GABAergic system, comprising fluctuations of GABA levels and turnover, GABA receptor affinity and postsynaptic activity on the chloride ionophore in rodent's brain. Neurochemical rhythms correlated with diurnal and circadian changes in several behaviors associated with the GABA(A) receptor, e.g., anxiolysis-related behavior. GABA is considered to be the principal neurotransmitter of the mammalian circadian system, being present in the suprachiasmatic nuclei and the intergeniculate leaflet. Pharmacological manipulations of GABA(A) receptors phase shift circadian rhythms and alter circadian responses to light. Administration of putative modulators of GABA function, like melatonin or neuroactive steroids, affects the timing of biological rhythms. Therefore, not only does the GABAergic system exhibit strong diurnal and circadian variations, but it also serves as one of the key modulators of the circadian apparatus.

Aging attenuates diurnal variation in hamster locomotion, anxiolysis and GABA turnover.

A decrease in amplitude of wheel running circadian rhythms was found in old (18 month old) Syrian hamsters, as compared with young (3 month old.) animals. In a plus-maze paradigm, amplitude of variation of anxiety-related variables (2400 vs. 1600 h) was significantly impaired in aged hamsters. Cerebral cortex, hypothalamic and pineal gamma-aminobutyric acid (GABA) turnover was higher at night, amplitude of variation being significantly smaller in aged hamsters. The results further support the existence of impaired amplitude of circadian rhythms in aged Syrian hamsters.

Circadian phase shifts to neuropeptide Y In vitro: cellular communication and signal transduction.

Mammalian circadian rhythms originate in the hypothalamic suprachiasmatic nuclei (SCN), from which rhythmic neural activity can be recorded in vitro. Application of neurochemicals can reset this rhythm. Here we determine cellular correlates of the phase-shifting properties of neuropeptide Y (NPY) on the hamster circadian clock in vitro. Drug or control treatments were applied to hypothalamic slices containing the SCN on the first day in vitro. The firing rates of individual cells were sampled on the second day in vitro. Control slices exhibited a peak in firing rate in the middle of the day. Microdrop application of NPY to the SCN phase advanced the time of peak firing rate. This phase-shifting effect of NPY was not altered by block of sodium channels with tetrodotoxin or block of calcium channels with cadmium and nickel, consistent with a direct postsynaptic site of action. Pretreatment with the glutamate receptor antagonists (DL-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium) also did not alter phase shifts to NPY. Blocking GABAA receptors with bicuculline (Bic) had effects only at very high (millimolar) doses of Bic, whereas blocking GABAB receptors did not alter effects of NPY. Phase shifts to NPY were blocked by pretreatment with inhibitors of protein kinase C (PKC), suggesting that PKC activation may be necessary for these effects. Bathing the slice in low Ca2+/high Mg2+ can block phase shifts to NPY, possibly via a depolarizing action. A depolarizing high K+ bath can also block NPY phase shifts. The results are consistent with direct action of NPY on pacemaker neurons, mediated through a signal transduction pathway that depends on activation of PKC.