Phase Of Circadian Clock Research Articles

VIVID interacts with the WHITE COLLAR complex and FREQUENCY-interacting RNA helicase to alter light and clock responses in Neurospora

The photoreceptor and PAS/LOV protein VIVID (VVD) modulates blue-light signaling and influences light and temperature responses of the circadian clock in Neurospora crassa. One of the main actions of VVD on the circadian clock is to influence circadian clock phase by regulating levels of the transcripts encoded by the central clock gene frequency (frq). How this regulation is achieved is unknown. Here we show that VVD interacts with complexes central for circadian clock and blue-light signaling, namely the WHITE-COLLAR complex (WCC) and FREQUENCY-interacting RNA helicase (FRH), a component that complexes with FRQ to mediate negative feedback control in Neurospora. VVD interacts with FRH in the absence of WCC and FRQ but does not seem to control the exosome-mediated negative feedback loop. Instead, VVD acts to modulate the transcriptional activity of the WCC.

The Mammalian Circadian Clock Exhibits Acute Tolerance to Ethanol

Tolerance to ethanol is observed over a variety of time courses, from minutes to days. Acute tolerance, which develops over 5 to 60 minutes, has been observed for both behavioral and neurophysiological variables and may involve changes in signaling through NMDA, GABA, or other receptors. Previous work has shown that both acute and chronic ethanol treatments modulate photic and nonphotic phase resetting of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Although not specifically tested, the data thus far do not point to the development of chronic tolerance to the modulatory effects of ethanol. Here we investigated whether acute tolerance the ethanol occurs with respect to in vitro phase modulation of the SCN clock. Mouse brain slices containing the SCN were pretreated with ethanol for varying lengths of time, followed by treatment concurrent with either glutamate or the serotonin agonist, 8-hydroxy-DPAT (DPAT). The phase of the SCN circadian clock was assessed the following day through extracellular recordings of SCN neuronal activity. SCN neuronal activity normally peaks during mid-day, and this rhythm can be shifted by treatment with either glutamate or DPAT. While concurrent treatment of SCN-containing brain slices with ethanol and glutamate blocks glutamate-induced phase delays of the SCN clock, pretreating the slices with ethanol for > or =15 minutes prevents this inhibition. Likewise, while concurrent treatment with ethanol and DPAT enhances DPAT-induced phase advances of the SCN clock, pretreating the slices with ethanol for > or =30 minutes prevents this enhancement. Both the inhibiting and enhancing effects of ethanol on in vitro SCN clock phase resetting show acute tolerance. Additional experiments are needed to determine whether more slowly developing forms of tolerance also occur with respect to the SCN circadian clock.

Ethanol modulates mammalian circadian clock phase resetting through extrasynaptic gaba receptor activation

Ethanol modulates the actions of multiple neurotransmitter systems, including GABA. However, its enhancing effects on GABA signaling typically are seen only at high concentrations. In contrast, although GABA is a prominent neurotransmitter in the circadian clock of the suprachiasmatic nucleus (SCN), we see ethanol modulation of clock phase resetting at low concentrations (<50 mM). A possible explanation is that ethanol enhances GABAergic signaling in the SCN through activating GABA A receptors that contain the δ subunit (GABA Aδ receptors), which are sensitive to low ethanol concentrations. Therefore, we investigated whether ethanol acts on GABA Aδ receptors in the SCN. Here we show that acute application of the GABA Aδ receptor antagonist, RO15-4513, to mouse hypothalamic slices containing the SCN prevents ethanol inhibition of nighttime glutamate-induced (photic-like) phase delays of the circadian clock. Diazepam, which enhances activity of GABA A receptors containing the γ subunit (GABA Aγ receptors), does not modulate these phase shifts. Moreover, we find that RO15-4513 prevents ethanol enhancement of daytime serotonergic (non-photic) phase advances of the circadian clock. Furthermore, diazepam phase-advances the SCN circadian clock when applied alone in the daytime, while ethanol has no effect by itself at that time. These data support the hypothesis that ethanol acts on GABA Aδ receptors in the SCN to modulate photic and non-photic circadian clock phase resetting. They also reveal distinct modulatory roles of different GABA A receptor subtypes in circadian clock phase regulation.

Chronic ethanol attenuates circadian photic phase resetting and alters nocturnal activity patterns in the hamster

Acute ethanol (EtOH) administration impairs circadian clock phase resetting, suggesting a mode for the disruptive effect of alcohol abuse on human circadian rhythms. Here, we extend this research by characterizing the chronobiological effects of chronic alcohol consumption. First, daily profiles of EtOH were measured in the suprachiasmatic nucleus (SCN) and subcutaneously using microdialysis in hamsters drinking EtOH. In both cases, EtOH peaked near lights-off and declined throughout the dark-phase to low day-time levels. Drinking bouts preceded EtOH peaks by approximately 20 min. Second, hamsters chronically drinking EtOH received a light pulse during the late dark phase [Zeitgeber time (ZT) 18.5] to induce photic phase advances. Water controls had shifts of 1.2 +/- 0.2 h, whereas those drinking 10% and 20% EtOH had much reduced shifts (0.5 +/- 0.1 and 0.3 +/- 0.1 h, respectively; P < 0.001 vs. controls). Third, incremental decreases in light intensity (270 lux to 0.5 lux) were used to explore chronic EtOH effects on photic entrainment and rhythm stability. Activity onset was unaffected by 20% EtOH at all light intensities. Conversely, the 24-h pattern of activity bouts was disrupted by EtOH under all light intensities. Finally, replacement of chronic EtOH with water was used to examine withdrawal effects. Water controls had photic phase advances of 1.1 +/- 0.3 h, while hamsters deprived of EtOH for 2-3 days showed enhanced shifts (2.1 +/- 0.3 h; P < 0.05 vs. controls). Thus, in chronically drinking hamsters, brain EtOH levels are sufficient to inhibit photic phase resetting and disrupt circadian activity. Chronic EtOH did not impair photic entrainment; however, its replacement with water potentiated photic phase resetting.

Neural correlates of arousal‐induced circadian clock resetting: hypocretin/orexin and the intergeniculate leaflet

In Syrian hamsters, some procedures for stimulating behavioural arousal (e.g. running in a novel wheel and sleep deprivation by gentle handling with minimal activity) markedly phase-advance circadian rhythms when applied during the middle of the daily rest period, while other arousal procedures do not (e.g. physical restraint, caffeine and modafinil). The neural basis for this differential effect of arousal procedures on clock resetting is unknown. We used c-fos expression as a marker for neuronal activation to determine whether these arousal procedures differentially activate two nonphotic inputs to the circadian system, the thalamic intergeniculate leaflet (IGL; a proposed nonphotic gateway to the circadian clock) and the hypothalamic hypocretin system (which depolarizes arousal-related cell groups throughout the brain and innervates both the IGL and the peri-suprachiasmatic nucleus region). c-FOS in hypocretin-1-immunoreactive neurons, in hypothalamic nonhypocretin neurons and in the IGL was significantly increased by novel wheel running, gentle handling and physical restraint, but only weakly by systemic injections of modafinil (300 mg/kg) or caffeine (75 mg/kg), at doses that are strongly alerting. Spatial analysis revealed few regional differences in the percentage of cells double-labelled for hypocretin-1 and c-FOS following each treatment. These results suggest that activation of hypocretin neurons (as in the restraint condition) is not sufficient to induce phase shifts, and that gating of arousal effects on circadian clock phase may be downstream from the hypocretin system and from IGL neurons activated by these procedures.

Acute ethanol modulates glutamatergic and serotonergic phase shifts of the mouse circadian clock in vitro

Alcohol abuse is associated with sleep problems, which are often linked to circadian rhythm disturbances. However, there is no information on the direct effects of ethanol on the mammalian circadian clock. Acute ethanol inhibits glutamate signaling, which is the primary mechanism through which light resets the mammalian clock in the suprachiasmatic nucleus (SCN). Glutamate and light also inhibit circadian clock resetting induced by nonphotic signals, including 5-HT. Thus, we investigated the effects of acute ethanol on both glutamatergic and serotoninergic resetting of the mouse SCN clock in vitro. We show that ethanol dose-dependently inhibits glutamate-induced phase shifts and enhances serotonergic phase shifts. The inhibition of glutamate-induced phase shifts is not affected by excess glutamate, glycine or d-serine, but is prevented by excess brain-derived neurotrophic factor (BDNF). BDNF is known to augment glutamate signaling in the SCN and to be necessary for glutamate/light-induced phase shifts. Thus, ethanol may inhibit glutamate-induced clock resetting at least in part by blocking BDNF enhancement of glutamate signaling. Ethanol enhancement of serotonergic phase shifts is mimicked by treatments that suppress glutamate signaling in the SCN, including antagonists of glutamate receptors, BDNF signaling and nitric oxide synthase. The combined effect of ethanol with these treatments is not additive, suggesting they act through a common pathway. Our data indicate further that the interaction between 5-HT and glutamate in the SCN may occur downstream from nitric oxide synthase activation. Thus, acute ethanol disrupts normal circadian clock phase regulation, which could contribute to the physiological and psychological problems associated with alcohol abuse.

The PAS/LOV protein VIVID controls temperature compensation of circadian clock phase and development in Neurospora crassa

Circadian clocks are cellular timekeepers that regulate aspects of temporal organization on daily and seasonal time scales. To allow accurate time measurement, the period lengths of clocks are conserved in a range of temperatures--a phenomenon known as temperature compensation. Temperature compensation of circadian clock period aids in maintaining a stable "target time" or phase of clock-controlled events. Here we show that the Neurospora protein VIVID (VVD) buffers the circadian system against temperature fluctuations. In vvd-null mutants, the circadian period of clock-controlled events such as asexual sporulation (conidiation) is temperature compensated, but the phase of this clock time marker is not. Consistent with delayed conidiation at lower temperatures in vvd(KO) strains, the levels of vvd gene products in the wild type increase with decreasing temperatures. Moreover, vvd(C108A) mutants that lack the light function of VVD maintain a dark activity that transiently influences the phase of conidiation, indicating that VVD influences the time of conidiation downstream from the clock. FREQUENCY (FRQ) phosphorylation is altered in a vvd(KO) strain, suggesting a mechanism by which VVD can influence the timing of clock-controlled processes in the dark. Thus, temperature compensation of clock-controlled output is a key factor in maintaining temperature compensation of the entire circadian system.

Modulation of environmental responses of plants by circadian clocks

Circadian clocks are signalling networks that enhance an organism's relationship with the rhythmic environment. The plant circadian clock modulates a wide range of physiological and biochemical events, such as stomatal and organ movements, photosynthesis and induction of flowering. Environmental signals regulate the phase and period of the plant circadian clock, which results in an approximate synchronization of clock outputs with external events. One of the consequences of circadian control is that stimuli of the same strength applied at different times of the day can result in responses of different intensities. This is known as 'gating'. Gating of a signal may allow plants to better process and react to the wide range and intensities of environmental signals to which they are constantly subjected. Light signalling, stomatal movements and low-temperature responses are examples of signalling pathways that are gated by the circadian clock. In this review, we describe the many levels at which the circadian clock interacts with responses to the environment. We discuss how environmental rhythms of temperature and light intensity entrain the circadian clock, how photoperiodism may be regulated by the relationship between environmental rhythms and the phasing of clock outputs, and how gating modulates the sensitivity of the clock and other responses to environmental and physiological signals. Finally, we describe evidence that the circadian clock can increase plant fitness.

Regulating a Circadian Clock's Period, Phase and Amplitude by Phosphorylation: Insights from Drosophila

Much progress has been made in understanding the molecular underpinnings governing circadian ( approximately 24 h) rhythms. Despite the increased complexity in metazoans whereby inter-cellular networks form the basis for driving overt rhythms, such as wake-sleep cycles in animals, single isolated cells can exhibit all the formal properties of a circadian pacemaker. How do these cell-autonomous rhythm generators operate? Breakthrough studies in Drosophila melanogaster led to the realization that the molecular logic underlying circadian clocks are highly shared. Most notably, interconnected transcriptional-translational feedback loops produce coordinated rhythms in "clock" RNAs and proteins that are required for the daily progression of clocks, synchronization to local time and transducing temporal signals to downstream effector pathways. More recent findings indicate prominent roles for reversible phosphorylation of clock proteins in the core oscillatory mechanism. In this review we focus on findings in Drosophila to explore the multiple levels that reversible phosphorylation plays in clock function. Specific clock proteins in this system are subjected to different phosphorylation programs, which affect three key properties of a circadian oscillator, its period, amplitude and phase. The role of phosphorylation in clocks is of clear relevance to human health because mutations that affect the PERIOD (PER) phosphorylation program are associated with familial sleep disorders. In addition, the central role of phosphorylation in the assembly of a circadian oscillator was dramatically shown recently by the ability to reconstitute a circadian phosphorylation/dephosphorylation cycle in vitro, suggesting that the dynamics of clock protein phosphorylation are at the "heart" of circadian time-keeping.

Glutamatergic activity modulates the phase‐shifting effects of gastrin‐releasing peptide and light

Previous studies have established that microinjection of gastrin-releasing peptide (GRP) into the suprachiasmatic nucleus (SCN) region or third ventricle causes circadian phase shifts similar to those produced by light pulses. Activation of N-methyl-d-aspartate (NMDA) receptors in the SCN region also produces light-like phase shifts. This study was designed to test the effects of (+/-)-2-amino-5-phosphonopentanoic acid (AP5), an NMDA antagonist, and l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a glutamate reuptake inhibitor, on GRP-induced phase shifts. Adult male Syrian hamsters equipped with a surgically implanted guide cannula aimed at the third ventricle were housed in constant darkness until stable free-running rhythms of wheel-running activity were apparent. Microinjection of GRP into the third ventricle at circadian time (CT)13 induced large phase delays. These GRP-induced phase delays were completely blocked by co-administration of AP5, suggesting that GRP-induced phase delays require concurrent activation of NMDA receptors. Microinjection of AP5 alone did not induce significant phase shifts. A second set of experiments was designed to test whether GRP-induced phase shifts would be enhanced by PDC. Co-administration of PDC and GRP elicited significantly larger phase delays at CT13 than GRP alone. However, administration of PDC alone did not induce a significant phase shift. Finally, when administered just prior to a light pulse, PDC elicited significantly larger phase delays than light pulse plus vehicle controls. These data suggest that the effects of GRP on the circadian clock phase are highly dependent on the level of excitation provided by activated NMDA receptors.

Promoting adjustment of the sleep–wake cycle by chronobiotics

Chronobiotics are substances that adjust the timing of internal biological rhythms. Many classes of drugs have been claimed to possess such properties and arouse growing interest as the circumstances for their use in sleep disturbances caused by circadian rhythms alterations (delayed or advanced sleep-phase syndromes, non-24-h sleep–wake disorders, jet lag, shift work sleep disorders and so on) have become progressively more frequent. Amongst the substances potentially presenting chronobiotic properties, a consensus seems to be reached on the possible use of melatonin or its agonists to shift the phase of the human circadian clock, but optimizing the dose, formulation and especially the time of administration require further studies.

The relationship between melatonin and cortisol rhythms: clinical implications of melatonin therapy

AbstractDisturbances in circadian rhythm have been linked to chronic diseases such as insomnia, hypertension, diabetes, and depression. Here we review recent studies on the age‐related changes in cortisol and melatonin rhythms and then present descriptive statistics on our preliminary findings on the rectification of the cortisol rhythms by melatonin therapy in elderly patients with insomnia. In adults, the melatonin onset typically occurs during low cortisol secretion. Administration of exogenous melatonin around dusk will shift the phase of the human circadian clock to earlier hours (advance phase shift) leading to phase advances in circadian rhythms (e.g., sleep, endogenous melatonin, cortisol). With aging, the production of melatonin declines and is shifted to later hours while the production of cortisol increases and its peak occurs earlier in the night. In a randomized placebo‐controlled crossover study with 8 patients with insomnia aged 55 years and older, a group characterized by low and delayed melatonin production, administration of prolonged‐release melatonin in the evening was able to rectify the early onset cortisol production. This delay in nocturnal cortisol onset may explain in part the improvement in sleep quality in elderly patients with insomnia, in schizophrenics, and in depressed patients. Support of circadian pacemaker function by melatonin may provide a new strategy in the treatment of disorders related to impairments in the internal temporal order. The clinical benefit from a decrease in cortisol during the early part of the night may lie beyond the improvement of sleep into a better control of blood pressure, metabolism, and mood. Drug Dev. Res. 65:119–125, 2005. © 2005 Wiley‐Liss, Inc.

Melatonin as a chronobiotic

Melatonin, hormone of the pineal gland, is concerned with biological timing. It is secreted at night in all species and in ourselves is thereby associated with sleep, lowered core body temperature, and other night time events. The period of melatonin secretion has been described as 'biological night'. Its main function in mammals is to 'transduce' information about the length of the night, for the organisation of daylength dependent changes, such as reproductive competence. Exogenous melatonin has acute sleepiness-inducing and temperature-lowering effects during 'biological daytime', and when suitably timed (it is most effective around dusk and dawn) it will shift the phase of the human circadian clock (sleep, endogenous melatonin, core body temperature, cortisol) to earlier (advance phase shift) or later (delay phase shift) times. The shifts induced are sufficient to synchronise to 24 h most blind subjects suffering from non-24 h sleep-wake disorder, with consequent benefits for sleep. Successful use of melatonin's chronobiotic properties has been reported in other sleep disorders associated with abnormal timing of the circadian system: jetlag, shiftwork, delayed sleep phase syndrome, some sleep problems of the elderly. No long-term safety data exist, and the optimum dose and formulation for any application remains to be clarified.

Social influences on mammalian circadian rhythms: animal and human studies.

While light is considered the dominant stimulus for entraining (synchronizing) mammalian circadian rhythms to local environmental time, social stimuli are also widely cited as 'zeitgebers' (time-cues). This review critically assesses the evidence for social influences on mammalian circadian rhythms, and possible mechanisms of action. Social stimuli may affect circadian behavioural programmes by regulating the phase and period of circadian clocks (i.e. a zeitgeber action, either direct or by conditioning to photic zeitgebers), by influencing daily patterns of light exposure or modulating light input to the clock, or by associative learning processes that utilize circadian time as a discriminative or conditioned stimulus. There is good evidence that social stimuli can act as zeitgebers. In several species maternal signals are the primary zeitgeber in utero and prior to weaning. Adults of some species can also be phase shifted or entrained by single or periodic social interactions, but these effects are often weak, and appear to be mediated by social stimulation of arousal. There is no strong evidence yet for sensory-specific nonphotic inputs to the clock. The circadian phase-dependence of clock resetting to social stimuli or arousal (the 'nonphotic' phase response curve, PRC), where known, is distinct from that to light and similar in diurnal and nocturnal animals. There is some evidence that induction of arousal can modulate light input to the clock, but no studies yet of whether social stimuli can shift the clock by conditioning to photic cues, or be incorporated into the circadian programme by associative learning. In humans, social zeitgebers appear weak by comparison with light. In temporal isolation or under weak light-dark cycles, humans may ignore social cues and free-run independently, although cases of mutual synchrony among two or more group-housed individuals have been reported. Social cues may affect circadian timing by controlling sleep-wake states, but the phase of entrainment observed to fixed sleep-wake schedules in dim light is consistent with photic mediation (scheduled variations in behavioural state necessarily create daily light-dark cycles unless subjects are housed in constant dark or have no eyes). By contrast, discrete exercise sessions can induce phase shifts consistent with the nonphotic PRC observed in animal studies. The best evidence for social entrainment in humans is from a few totally blind subjects who synchronize to the 24 h day, or to near-24 h sleep-wake schedules under laboratory conditions. However, the critical entraining stimuli have not yet been identified, and there are no reported cases yet of social entrainment in bilaterally enucleated blind subjects. The role of social zeitgebers in mammalian behavioural ecology, their mechanisms of action, and their utility for manipulating circadian rhythms in humans, remains to be more fully elaborated.

Dynamic regulation of polysialylated neural cell adhesion molecule in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) prominently expresses polysialic acid (PSA), a carbohydrate polymer that is attached to neural cell adhesion molecule (NCAM) and promotes changes in cell interactions. Previous studies have shown that expression of PSA is important for circadian rhythm stability under constant darkness, and for photic entrainment of the SCN circadian clock. In the present study, immunoblot analyses of the Syrian hamster SCN revealed marked diurnal fluctuations in PSA under a 24-h light/dark cycle. PSA levels were reduced by >90% during the mid-to-late dark phase, and were elevated to maximal daytime levels ∼1 h after lights-on. A similar pattern of PSA fluctuation persisted under constant darkness. Exposure of animals under a 24-h light/dark cycle to a 30-min light pulse during the late dark phase dramatically increased SCN contents of PSA within 60 min, and these values returned to basal levels 1–2 h later. There was no effect of light-on expression of PSA in the hippocampus. Parallel studies revealed changes in the NCAM-180 isoform that carries PSA in the brain, suggesting that regulation of PSA may include protein as well as carbohydrate-associated mechanisms. Immunohistological analysis revealed light-induced enhancement of PSA in the SCN subregion containing calbindin D 28K cells. PSA staining was also closely associated with the majority of SCN cells expressing light-inducible Fos protein. This rhythmic, light-inducible expression of PSA within the SCN suggests that dynamic cell interactions are important for the photic regulation of circadian clock phase.

Serotonin phase-shifts the mouse suprachiasmatic circadian clock in vitro

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) receives multiple afferent signals that could potentially modulate its phase. One input, the serotonin (5-HT) projection from the raphe nuclei, has been extensively investigated in rats and hamsters, yet its role(s) in modulating circadian clock phase remains controversial. To expand our investigation of 5-HT modulation of the SCN clock, we investigated the phase-shifting effects of 5-HT and its agonist, (+)8-hydroxy-2-(di- n-propylamino)tetralin (DPAT), when applied to mouse SCN brain slices. 5-HT induced 2–3 h phase advances when applied during subjective day, while non-significant phase shifts were seen after 5-HT application at other times. These phase shifts were completely blocked by the 5-HT antagonist, metergoline. DPAT also induced phase shifts when applied during mid-subjective day, and this effect appeared dose-dependent. Together, these results demonstrate that the mouse SCN, like that of the rat, is directly sensitive to in vitro phase-resetting by 5-HT.

Cirrhosis does not shift the circadian phase of plasma fibrinolysis

OBJECTIVES: The aim of the present investigation was to study the endogenous circadian clock phase rhythm in cirrhotic patients. METHODS: The study population comprised 13 patients with cirrhosis (seven in Child-Pugh class A and six in classes B/C) and nine healthy controls. Plasma melatonin, tissue plasminogen activator antigen, and plasminogen activator inhibitor 1 antigen were measured at 4-h intervals over a 24-h period. Multiple-components rhythmometry using population mean cosinor methods were employed to analyze the findings. RESULTS: All three variables were characterized in both patients and controls by a statistically significant circadian rhythm, with similar profiles. The peak times of tissue plasminogen activator and plasminogen activator inhibitor 1 antigens were practically identical in controls and cirrhotic patients, irrespective of Child-Pugh class (calculated peak at times 6:52, 6:56, and 7:20 for the inhibitor in controls and Child-Pugh class A and classes B/C patients, respectively; p = ns), whereas the peak of melatonin was delayed in classes B/C patients (at times 2:08, 1:56, and 4:00, respectively; p < 0.05). CONCLUSION: The similar circadian phases of plasminogen activator inhibitor antigen in controls and cirrhotic patients in the present investigation indicates that the output rhythm of the internal timekeeping system is not shifted in this pathological condition.

Cirrhosis does not shift the circadian phase of plasma fibrinolysis

The aim of the present investigation was to study the endogenous circadian clock phase rhythm in cirrhotic patients. The study population comprised 13 patients with cirrhosis (seven in Child-Pugh class A and six in classes B/C) and nine healthy controls. Plasma melatonin, tissue plasminogen activator antigen, and plasminogen activator inhibitor 1 antigen were measured at 4-h intervals over a 24-h period. Multiple-components rhythmometry using population mean cosinor methods were employed to analyze the findings. All three variables were characterized in both patients and controls by a statistically significant circadian rhythm, with similar profiles. The peak times of tissue plasminogen activator and plasminogen activator inhibitor 1 antigens were practically identical in controls and cirrhotic patients, irrespective of Child-Pugh class (calculated peak at times 6:52, 6:56, and 7:20 for the inhibitor in controls and Child-Pugh class A and classes B/C patients, respectively; p = ns), whereas the peak of melatonin was delayed in classes B/C patients (at times 2:08, 1:56, and 4:00, respectively; p < 0.05). The similar circadian phases of plasminogen activator inhibitor antigen in controls and cirrhotic patients in the present investigation indicates that the output rhythm of the internal timekeeping system is not shifted in this pathological condition.

In rats, odor-induced Fos in the olfactory pathways depends on the phase of the circadian clock

We used immunostaining for Fos to study the effect of circadian clock phase on odor-induced neuronal activation in the olfactory system in rats. Brief presentation of cedar odor to rats housed in constant darkness stimulated Fos expression in the main olfactory bulb, anterior olfactory nucleus, piriform cortex, and several other odor-responsive structures, both in the subjective day and subjective night phases of the cycle. Fos expression in response to odor, but not basal expression, was greatly enhanced in the subjective night in all structures examined. These findings are consistent with the idea that odor-induced neuronal activation in the olfactory pathways is modulated by the phase of the circadian clock.

Inhibition of protein kinase A phase delays the mammalian circadian clock

The suprachiasmatic nuclei (SCN) contain the mammalian circadian clock whose rhythm of firing rate can be recorded in vitro for several days. Application of a protein kinase A (PKA) inhibitor onto the SCN at Zeitgeber time (ZT) 10 on the first day in vitro phase delayed the rhythm of firing rate expressed by SCN neurons on the subsequent day in vitro. Application of the inhibitor (Rp-cAMPS) at other circadian phases did not phase shift the rhythm. These results suggest that during approximately 1 h in the late subjective day the presence and activity of PKA plays a role in setting the phase of the mammalian circadian clock.