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Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein ( Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;Dbp KI/+ “liver reporter” mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;Dbp(KI/+) “liver reporter” mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

Understanding the health consequences of chronic disruption of circadian rhythms can contribute to improving prevention strategies for shift workers. Chronic circadian disruption in shift work has been linked to a higher risk of stroke. Dysregulated immune responses are also linked to circadian disruption and may be a factor in stroke outcomes in shift workers. In this study, we test the hypotheses that specific schedules of circadian disruption exacerbate inflammatory responses in the brain, causing an increase in infarct size after experimentally induced ischemic stroke. Mice were exposed to 1 of 5 different lighting schedules followed by a 30-min middle cerebral artery occlusion, then reperfusion and 3-day recovery. A history of weekly phase advances resulted in an increased infarct volume versus the control lighting schedule. These effects were shift-direction specific, nonpermanent, and required multiple shifts to occur. In a separate cohort, stereotaxic injections of lipopolysaccharide were given bilaterally after exposure to 1 of 3 different lighting schedules. Ratios of pro- to anti-inflammatory cytokine expression show dysregulated responses after a history of phase advances. We conclude that chronic circadian disruption leads to worsened stroke outcome in a direction- and schedule-specific manner likely because of priming of the inflammatory response in the brain. These pieces of evidence suggest that the health impacts of shift work may be improved by targeting shift work scheduling, inflammatory mediators, or both.

The proinflammatory cytokine interferon (IFN-gamma) is an immunomodulatory molecule released by immune cells. It was originally described as an antiviral agent but can also affect functions in the nervous system including circadian activity of the principal mammalian circadian pacemaker, the suprachiasmatic nucleus. IFN-gamma and the synergistically acting cytokine tumor necrosis factor-alpha acutely decrease spontaneous excitatory postsynaptic activity and alter spiking activity in tissue preparations of the SCN. Because IFN-gamma can be released chronically during infections, the authors studied the long-term effects of IFN-gamma on SCN neurons by treating dispersed rat SCN cultures with IFN-gamma over a 4-week period. They analyzed the effect of the treatment on the spontaneous spiking pattern and rhythmic expression of the "clock gene," Period 1. They found that cytokine-treated cells exhibited a lower average spiking frequency and displayed a more irregular firing pattern when compared with controls. Furthermore, long-term treatment with IFN-gamma in cultures obtained from a transgenic Per1-luciferase rat significantly reduced the Per1-luc rhythm amplitude in individual SCN neurons. These results show that IFN-gamma can alter the electrical properties and circadian clock gene expression in SCN neurons. The authors hypothesize that IFN-gamma can modulate circadian output, which may be associated with sleep and rhythm disturbances observed in certain infections and in aging.

The immune system is regulated by circadian clocks within the brain and immune cells. Environmental circadian disruption (ECD), consisting of a 6-h phase advance of the light:dark cycle once a week for 4 weeks, elevates the inflammatory response to lipopolysaccharide (LPS) both in vivo and in vitro. This indicates that circadian disruption adversely affects immune function; however, it remains unclear how the circadian system regulates this response under ECD conditions. Here, we develop an assay using ex vivo whole-blood LPS challenge to investigate the circadian regulation of immune responses in mice and to determine the effects of ECD on these rhythms. LPS-induced IL-6 release in whole blood was regulated in a circadian manner, peaking during subjective day under both entrained and free-running conditions. This LPS-induced IL-6 release rhythm was associated with daily variation in both white blood cell counts and immune cell responsiveness. ECD increased the overall level of LPS-induced IL-6 release by increasing immune cell responsiveness and not by affecting immune cell number or the circadian regulation of this rhythm. This indicates that ECD produces pathological immune responses by increasing the proinflammatory responses of immune cells. Also, this newly developed whole blood assay can provide a noninvasive longitudinal method to quantify potential health consequences of circadian disruption in humans.

The suprachiasmatic nucleus (SCN) orchestrates synchrony among many peripheral oscillators and is required for circadian rhythms of locomotor activity and many physiological processes. However, the unique effects of methamphetamine (MAP) on circadian behavior suggest the presence of an SCN-independent, methamphetamine-sensitive circadian oscillator (MASCO). Substantial data collected using rat models show that chronic methamphetamine dramatically lengthens circadian period of locomotor activity rhythms and induces rhythms in animals lacking an SCN. However, the anatomical substrate and the molecular components of the MASCO are unknown. The response to MAP is less well studied in mice, a model that would provide the genetic tools to probe the molecular components of this extra-SCN oscillator. The authors tested the effects of chronic MAP on 2 strains of intact and SCN-lesioned mice in constant dark and constant light. Furthermore, they applied various MAP availability schedules to SCN-lesioned mice to confirm the circadian nature of the underlying oscillator. The results indicate that this oscillator has circadian properties. In intact mice, the MASCO interacts with the SCN in a manner that is strain, sex, and dose dependent. In SCN-lesioned mice, it induces robust free-running locomotor rhythmicity, which persists for up to 14 cycles after methamphetamine is withdrawn. In the future, localization of the MASCO and characterization of its underlying molecular mechanism, as well as its interactions with other oscillators in the body, will be essential to a complete understanding of the organization of the mammalian circadian system.

The ability to form an association between the time and the place of food availability, namely time-place learning, is presumably important for survival. The present study was designed toexamine time-place learning and to identify exogenous and endogenous factors that may affect this behavior in rats. In an initial experiment, rats displayed poor time-place behavior and appeared to prefer the feeder that was closer to the center aisle and water supply. When these cueswere minimized in a subsequent experiment, rats consistently displayed the time-place discrimination by exhibiting food anticipatory activity (FAA) at the correct location prior to each mealtime. These rats also showed significant correlations between the level of FAA and the amount of dopamine turnover (the dihydroxyphenylacetic acid/dopamine ratio) in the nucleus accumbens and paraventricular nucleus of the hypothalamus, indicating possible involvement of regional dopaminergic activity in time-place behavior. No correlation was found for norepinephrine, epinephrine, or serotonin. In addition, the correlation between FAA and dopamine turnover was not foundwhen rats were entrained to only one meal per day. Together, the data suggest that rats can learn the time- place discrimination under proper experimental conditions and that dopamine may play a role in the expression of this behavior.

The feeding-entrained circadian oscillator (FEO) organizes locomotor activity and other variables to anticipate daily timed meals. Whether the biological substrate for the FEO is in the central nervous system or in the periphery, there must be communication between the gut and the brain to result in a behavioral output. To investigate potential neural routes of communication, rats with suprachiasmatic lesions were given systemic capsaicin (total dose: 100 mg/kg, ip) to produce visceral deafferentation. Deafferentation was confirmed using the phenyl-p-benzoquinone stretch test and the corneal irritation test. A 3-h meal was made available at the same time each day while wheel running was recorded for several weeks. Results indicated that rats with capsaicin lesions were somewhat more active overall and during nonanticipation times, but the onset time and the amount of anticipatory wheel running did not differ from vehicle-treated controls. In addition, reentrainment following a phase delay of mealtime and the persistence of anticipatory activity during food deprivation were similar between the groups. Since capsaicin deafferentation and subdiaphragmatic vagotomy do not prevent food-anticipatory activity, it is likely that communication between the gut and the brain is accomplished via a humoral route.