Search

Environmental cues such as light and timing of food intake influence molecular clocks that produce circadian rhythmicity of many biological functions. The master circadian clock is entrained by light input and synchronizes to peripheral clocks in every organ. Careers that require rotating shift work schedules predispose workers to a constant desynchronization of biological clocks and are associated with increased risk of cardiovascular disease. We utilized a stroke-prone spontaneously hypertensive rat model exposed to a known biological desynchronizer, chronic environmental circadian disruption (ECD), and hypothesized that it would accelerate time to stroke onset. We investigated whether time-restricted feeding could delay stroke onset and evaluated its usefulness as a countermeasure when combined with the disruption of the light cycle. We found that a phase advancing schedule accelerated stroke onset. Restricting food access time to 5 hours/day regardless of lighting delayed stroke onset in both standard 12:12 light:dark or ECD lighting conditions compared with ad-lib feeding, but acceleration by ECD versus control lighting conditions was still observed. Since hypertension is a precursor to stroke in this model, we assessed blood pressure in a small cohort longitudinally using telemetry. Daily systolic and diastolic blood pressure was increased in both control and ECD conditions, thus hypertension was not accelerated to cause earlier strokes. We observed intermittent dampening of rhythms after each shift of the light cycle reminiscent of a relapsing-remitting non-dipping state. Our results suggest that constant ECD rhythms may be associated with increased risk of cardiovascular complications in the presence of cardiovascular risk factors.

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.

The suprachiasmatic nucleus (SCN) is composed of functionally distinct subpopulations of GABAergic neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons, and which aspects result from neuronal interaction within a network. Here, we utilize in vivo miniaturized microscopy to measure fluorescent GCaMP-reported calcium dynamics in arginine vasopressin (AVP)-expressing neurons in the intact SCN of awake, behaving mice. We report that SCN AVP neurons exhibit periodic, slow calcium waves which we demonstrate, using in vivo electrical recordings, likely reflect burst firing. Further, we observe substantial heterogeneity of function in that AVP neurons exhibit unstable rhythms, and relatively weak rhythmicity at the population level. Network analysis reveals that correlated cellular behavior, or coherence, among neuron pairs also exhibited stochastic rhythms with about 33% of pairs rhythmic at any time. Unlike single-cell variables, coherence exhibited a strong rhythm at the population level with time of maximal coherence among AVP neuronal pairs at CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity for the SCN as a whole. These results demonstrate robust circadian variation in the coordination between stochastically rhythmic neurons and that interactions between AVP neurons in the SCN may be more influential than single-cell activity in the regulation of circadian rhythms. Furthermore, they demonstrate that cells in this circuit, like those in many other circuits, exhibit profound heterogenicity of function over time and space.

Nontraditional work schedules, such as shift work, have been associated with numerous health issues, including cardiovascular and metabolic disease. These work schedules can chronically misalign environmental timing cues with internal circadian clock systems in the brain and in peripheral organs, leading to dysfunction of those systems and their associated biological processes. Environmental circadian disruption in the kidney may be an important factor in the increased incidence of hypertension and adverse health outcomes in human shift workers. The relationship between renal rhythmicity and injury resilience is not well understood, especially in the context of environmental, rather than genetic, manipulations of the circadian system. We conducted a longitudinal study to determine whether chronic shifting of the light cycle that mimics shift work schedules would disrupt output rhythms of the kidney and accelerate kidney injury in salt-loaded male spontaneously hypertensive, stroke-prone rats. We observed that chronic shifting of the light-dark (LD) cycle misaligned and decreased the amplitude of urinary volume rhythms as the kidney phase-shifted to match each new lighting cycle. This schedule also accelerated glomerular and tubular injury marker excretion, as quantified by nephrin and KIM-1 compared with rats kept in a static LD cycle. These data suggest that disrupted rhythms in the kidney may decrease resilience and contribute to disease development in systems dependent on renal and cardiovascular functions.

Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.

The Suprachiasmatic Nucleus (SCN) is composed of functionally distinct sub-populations of GABAergic neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons, and which aspects result from neuronal interaction within a network. Here, we utilize in vivo miniaturized microscopy to measure fluorescent GCaMP-reported calcium dynamics in AVP-expressing neurons in the intact SCN of awake, behaving mice. We report that SCN AVP neurons exhibit periodic, slow calcium waves which we demonstrate, using in vivo electrical recordings, likely reflect burst-firing. Further, we observe substantial heterogeneity of function in that AVP neurons exhibit unstable rhythms, and relatively weak rhythmicity at the population level. Network analysis reveals that correlated cellular behavior, or coherence, among neuron pairs also exhibited stochastic rhythms with about 33% of pairs rhythmic at any time. Unlike single-cell variables, coherence exhibited a strong rhythm at the population level with time of maximal coherence among AVP neuronal pairs at CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity for the SCN as a whole. These results demonstrate robust circadian variation in the coordination between stochastically rhythmic neurons and that interactions between AVP neurons in the SCN may be more influential than single-cell activity in the regulation of circadian rhythms. Furthermore, they demonstrate that cells in this circuit, like those in many other circuits imaged in vivo, exhibit profound heterogenicity of function over time and space.Significance StatementThis work is the first to employ two novel in vivo recording techniques, miniaturized calcium microscopy and optogentically-targeted single unit activity recording, to examine the rhythmic behavior of AVP expressing neurons both at the individual neuronal and network level. These results suggest that while AVP neurons are important for organismal rhythmicity, individual cellular rhythms are unstable and diverse. However, we observed correlated activity among these neurons which appears more reliably rhythmic, suggesting that emergent network properties of the SCN may be more relevant for organismal rhythmicity than individual neuronal characteristics.

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;DbpKI/+ “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 in cellular function can be monitored in real time with bioluminescence imaging. In this approach, bioluminescence is produced by an enzymatic reaction, which can be used to report dynamic changes in gene or protein expression in living cells. Bioluminescence imaging in circadian experiments typically uses an ex vivo slice preparation, with the most commonly studied structure being the master clock in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Here we describe procedures for dissecting and collecting SCN slices for bioluminescence imaging experiments.

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 disruption of inflammatory responses is a potential mechanism behind the harmful effects of shift work and is associated with increased risk of hypertension, stroke, obesity, diabetes, and cancer. These responses are linked to the proliferation of leukocytes in shift workers, suggesting a systemic signal as a potential mediator. The purpose of this study was to assess the relationship between systemic inflammation, leukocyte counts, and systemic endotoxemia in samples from a diverse cohort of day workers and shift workers. Participants (normothermic and normotensive) were healthy volunteers, non-smoking, and drug- and medication-free. The following outcomes were measured: C-reactive protein, TNF-α, IL-6, IL-1β, IL-10, leukocyte counts (monocytes, lymphocytes, and neutrophils), and lipopolysaccharide-binding protein (LBP). Risk factors that increase systemic inflammation, such as blood pressure, sleep loss, and cortisol, were also assessed. The results indicated that shift workers slept significantly less than day workers and had significantly increased concentrations of all of the cytokines measured as well as plasma cortisol. Regression models found that after controlling for covariates, shift-work exposure predicted the significant increase observed in IL-10, leukocyte counts, and LBP. Our results suggest that acute increases in low-grade systemic endotoxemia are unresolved during chronic shift-work exposure. This ongoing immune challenge may underlie the disrupted inflammatory responses characteristic of shift-work-related pathologies. Systemic endotoxemia may represent a novel target to investigate the early effects of exposure to shift-work schedules.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A circadian clock governs most aspects of mammalian behavior. Although its properties are in part genetically determined, altered light-dark environment can change circadian period length through a mechanism requiring de novo DNA methylation. We show here that this mechanism is mediated not via cell-autonomous clock properties, but rather through altered networking within the suprachiasmatic nuclei (SCN), the circadian "master clock," which is DNA methylated in region-specific manner. DNA methylation is necessary to temporally reorganize circadian phasing among SCN neurons, which in turn changes the period length of the network as a whole. Interruption of neural communication by inhibiting neuronal firing or by physical cutting suppresses both SCN reorganization and period changes. Mathematical modeling suggests, and experiments confirm, that this SCN reorganization depends upon GABAergic signaling. Our results therefore show that basic circadian clock properties are governed by dynamic interactions among SCN neurons, with neuroadaptations in network function driven by the environment.

Environmental circadian disruption (ECD), characterized by repeated or long-term disruption in environmental timing cues which require the internal circadian clock to change its phase to resynchronize with the environment, is associated with numerous serious health issues in humans. While animal and isolated cell models exist to study the effects of destabilizing the relationship between the circadian system and the environment, neither approach provides an ideal solution. Here, we developed an in vitro model which incorporates both elements of a reductionist cellular model and disruption of the clock/environment relationship using temperature as an environmental cue, as occurs in vivo. Using this approach, we have demonstrated that some effects of in vivo ECD can be reproduced using only isolated peripheral oscillators. Specifically, we report exaggerated inflammatory responses to endotoxin following repeated environmental circadian disruption in explanted spleens. This effect requires a functional circadian clock but not the master brain clock, the suprachiasmatic nucleus (SCN). Further, we report that this is a result of cumulative, rather than acute, circadian disruption as has been previously observed in vivo. Finally, such effects appear to be tissue specific as it does not occur in lung, which is less sensitive to the temperature cycles employed to induce ECD. Taken together, the present study suggests that this model could be a valuable tool for dissecting the causes and effects of circadian disruption both in isolated components of physiological systems as well as the aggregated interactions of these systems that occur in vivo.

Shift work is associated with a higher risk of stroke, but the mechanisms of this relationship are unknown. Photic cues to the suprachiasmatic nucleus (SCN) are the primary source of environmental regulation for circadian rhythms, but non-photic cues such as food intake can synchronize peripheral clocks involved in metabolic functions. Abrupt changes in the timing of such environmental time cues cause environmental circadian disruption (ECD) that may be responsible for chronic and acute diseases in shift workers such as hypertension and stroke. In this study, we used a spontaneously hypertensive stroke-prone rat model to determine whether ECD accelerates stroke onset. We also test the hypothesis that scheduled mealtimes can rescue such effects of ECD. Adult male rats were exposed to either a fixed or shifted (6hr weekly advance shifted) light-dark (LD) 12:12 cycle. Rats were given a high salt diet and 1% NaCl drinking water to increase hypertension onset and establish the conditions for spontaneous hemorrhagic stroke. A second cohort exposed to the same two lighting conditions received 5h scheduled daily mealtimes. Food and water intake were measured to estimate salt load. Neurological assessment scoring was used to determine stroke onset. Circadian activity rhythms stably entrained to the fixed LD cycle but were disrupted in shifted LD rats, causing chronic misalignment as occurs in shift workers. In contrast to the stroke onset in fixed LD conditions, the survival rate was shortened in shifted LD rats with a median survival time of 46.5 days versus 58.5 days, respectively (Log-rank Mantel-Cox test p

Significance The mammalian/mechanistic target of rapamycin (mTOR) kinase resides at the crux of an intracellular signaling network that controls fundamental biological processes. Dysregulation of mTOR signaling is linked to neurological and psychiatric diseases. However, the physiological functions of mTOR signaling in the adult brain are not fully understood. In the current study, we discovered that mTOR in vasoactive intestinal peptide (VIP) neurons plays a key role in regulating neurophysiology in the brain circadian clock and the olfactory system. The conditional mTOR knockout mouse will be a useful model for future investigations of mTOR and/or VIP., Mammalian/mechanistic target of rapamycin (mTOR) signaling controls cell growth, proliferation, and metabolism in dividing cells. Less is known regarding its function in postmitotic neurons in the adult brain. Here we created a conditional mTOR knockout mouse model to address this question. Using the Cre-LoxP system, the mTOR gene was specifically knocked out in cells expressing Vip (vasoactive intestinal peptide), which represent a major population of interneurons widely distributed in the neocortex, suprachiasmatic nucleus (SCN), olfactory bulb (OB), and other brain regions. Using a combination of biochemical, behavioral, and imaging approaches, we found that mice lacking mTOR in VIP neurons displayed erratic circadian behavior and weakened synchronization among cells in the SCN, the master circadian pacemaker in mammals. Furthermore, we have discovered a critical role for mTOR signaling in mediating olfaction. Odor stimulated mTOR activation in the OB, anterior olfactory nucleus, as well as piriform cortex. Odor-evoked c-Fos responses along the olfactory pathway were abolished in mice lacking mTOR in VIP neurons, which is consistent with reduced olfactory sensitivity in these animals. Together, these results demonstrate that mTOR is a key regulator of SCN circadian clock synchrony and olfaction.

Aging produces a decline in the amplitude and precision of 24 h behavioral, endocrine, and metabolic rhythms, which are regulated in mammals by a central circadian pacemaker within the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. Disruption of the circadian system, as experienced during transmeridian travel, can lead to adverse health consequences, particularly in the elderly. To test the hypothesis that age-related changes in the response to simulated jet lag will reflect altered circadian function, we examined re-entrainment of central and peripheral oscillators from young and old PER2::luciferase mice. As in previous studies, locomotor activity rhythms in older mice required more days to re-entrain following a shift than younger mice. At the tissue level, effects of age on baseline entrainment were evident, with older mice displaying earlier phases for the majority of peripheral oscillators studied and later phases for cells within most SCN subregions. Following a 6 h advance of the light:dark cycle, old mice displayed slower rates of re-entrainment for peripheral tissues but a larger, more rapid SCN response compared to younger mice. Thus, aging alters the circadian timing system in a manner that differentially affects the re-entrainment responses of central and peripheral circadian clocks. This pattern of results suggests that a major consequence of aging is a decrease in pacemaker amplitude, which would slow re-entrainment of peripheral oscillators and reduce SCN resistance to external perturbation.

BACKGROUND The mechanism of epithelial to mesenchymal transition (EMT) could be adopted by tumor cells for migration and invasion. We have reported that ARCaPE human prostate cancer cells undergo EMT-like changes during xenograft growth in athymic mice. METHODS In this report, we assessed the extent of EMT by tracking changes in cloned ARCaPE cells expressing red fluorescence protein during successive orthotopic prostate tumor formation. Cancer cells with stromal-like morphology were isolated and examined for EMT-like changes. RESULTS EMT-like morphologic and expression changes were detected after one round of in vivo tumor formation. Importantly, when recovered tumor cells were used in second round xenograft tumor formation, a large fraction of ARCaPE cells showed drastic EMT-like changes, with markedly enlarged cell size and divergent cell shapes similar to those of mesenchymal stromal cells. The morphologic change was accompanied by increased growth and metastasis, as tumor incidence increased while red fluorescent tumor cells could be detected from circulating blood, bone marrow, peritoneal ascites, and lung of the tumor-bearing mice. Recovered clones from these samples had lost epithelial markers but many showed activated stromal marker vimentin expression. The EMT appeared permanent since the newly acquired morphology was sustained after continuous passages. CONCLUSIONS Results from this study demonstrate that through interaction with the host tumor microenvironment, cancer cells acquire cellular plasticity. During xenograft tumor formation and metastasis, a single clone of cancer cells could yield a heterogeneous population, with a substantial number of tumor cells adopting mesenchymal stroma-like phenotypes. Prostate 70: 518–528, 2010. © 2009 Wiley-Liss, Inc.

The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins—called plus-end tracking proteins (+TIPs)—bind to and control the plus-end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus-end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end-binding protein 1 (EB1) in vitro and co-localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus-end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere-associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus-end localization of MCAK. Importantly, aurora B-mediated phosphorylation disrupts the TIP150–MCAK association in vitro. We reason that TIP150 facilitates the EB1-dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.

Circadian rhythms regulate most physiological processes. Adjustments to circadian time, called phase shifts, are necessary following international travel and on a more frequent basis for individuals who work non-traditional schedules such as rotating shifts. As the disruption that results from frequent phase shifts is deleterious to both animals and humans, we sought to better understand the kinetics of resynchronization of the mouse circadian system to one of the most disruptive phase shifts, a 6-h phase advance. Mice bearing a luciferase reporter gene for mPer2 were subjected to a 6-h advance of the light cycle and molecular rhythms in suprachiasmatic nuclei (SCN), thymus, spleen, lung and esophagus were measured periodically for 2 weeks following the shift. For the SCN, the master pacemaker in the brain, we employed high-resolution imaging of the brain slice to describe the resynchronization of rhythms in single SCN neurons during adjustment to the new light cycle. We observed significant differences in shifting kinetics among mice, among organs such as the spleen and lung, and importantly among neurons in the SCN. The phase distribution among all Period2-expressing SCN neurons widened on the day following a shift of the light cycle, which was partially due to cells in the ventral SCN exhibiting a larger initial phase shift than cells in the dorsal SCN. There was no clear delineation of ventral and dorsal regions, however, as the SCN appear to have a population of fast-shifting cells whose anatomical distribution is organized in a ventral-dorsal gradient. Full resynchronization of the SCN and peripheral timing system, as measured by a circadian reporter gene, did not occur until after 8 days in the advanced light cycle.

Alterations in circadian rhythm generation may be related to the development of mood disorders. Although it has been reported that the most popular antidepressant, selective serotonin reuptake inhibitors (SSRIs) affect circadian phase, no data are available that describe the effects of SSRIs on other circadian parameters (period, amplitude and damping rate) in dissociated cells. In the present study we used real-time monitoring of bioluminescence in rat-1 fibroblasts expressing the Period1-luciferase transgene, and that in Period1-luciferase transgenic mouse suprachiasmatic nucleus (SCN) explants, in order to characterize the effects of SSRI on circadian oscillator function in vitro. We found that mRNA of the serotonin transporter (SERT), a target of SSRIs, was expressed in rat-1 fibroblasts. Sertraline, fluoxetine, fluvoxamine, citalopram and paroxetine all significantly shortened the period of Period1-bioluminescence rhythms in rat-1 fibroblasts. The amplitude was reduced by sertraline, and the damping rate was decreased by sertraline, fluoxetine, flvoxamine and paroxetine. The effect of sertraline was dose-dependent, and it also shortened the circadian period in the SCN. SERT is associated with lipid microdomains, which are required for efficient SERT activity. Indeed, cholesterol chelating reagent methyl-beta-cyclodextrin significantly reduced the period and the amplitude in rat-1 fibroblasts. Furthermore, lipid binding reagent xylazine significantly reduced the period. In summary our data present evidence that SSRIs affect circadian rhythmicity. The action of SSRIs is likely mediated by suppression of SERT activity. A better understanding of the relationship between mental illness and biological timing may yield new insight into disease etiology and avenues for treatment.

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.

Several studies have demonstrated that the mammalian retina contains an autonomous circadian clock. Dopaminergic and other inner retinal neurons express many of the clock genes, whereas some of these genes seem to be absent from the photoreceptors. This observation has led to the suggestion that in mammalian retina the circadian pacemaker driving retinal rhythms is located in the inner nuclear layer. However, other evidence points to the photoreceptor layer as the site of the mammalian retinal clock. The goal of the present study was to demonstrate the presence of a functional circadian clock in photoreceptors. First, using laser capture microdissection and reverse transcriptase-polymerase chain reaction, we investigated which of the clock genes are expressed in rat photoreceptors. We then prepared photoreceptor layer cultures from the retina to test whether these isolated cultures were viable and could drive circadian rhythms. Our data indicated that Per1, Per3, Cry1, Cry2, Clock, Bmal1, Rev-erb alpha, and Rora RNAs were present in the photoreceptors, whereas we were unable to amplify mRNA for Per2 and Npas2. Photoreceptor layers obtained from Period1-luciferase rats expressed a robust circadian rhythm in bioluminescence and melatonin synthesis. These results demonstrate that mammalian photoreceptors contain the circadian pacemaker driving rhythmic melatonin synthesis.

Daily rhythms in mammals are controlled by the circadian system, which is a collection of biological clocks regulated by a central pacemaker within the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Changes in SCN function have pronounced consequences for behaviour and physiology; however, few studies have examined whether individual differences in circadian behaviour reflect changes in SCN function. Here, PERIOD2::LUCIFERASE mice were exposed to a behavioural assay to characterize individual differences in baseline entrainment, rate of re-entrainment and free-running rhythms. SCN slices were then collected for ex vivo bioluminescence imaging to gain insight into how the properties of the SCN clock influence individual differences in behavioural rhythms. First, individual differences in the timing of locomotor activity rhythms were positively correlated with the timing of SCN rhythms. Second, slower adjustment during simulated jetlag was associated with a larger degree of phase heterogeneity among SCN neurons. Collectively, these findings highlight the role of the SCN network in determining individual differences in circadian behaviour. Furthermore, these results reveal novel ways that the network organization of the SCN influences plasticity at the behavioural level, and lend insight into potential interventions designed to modulate the rate of resynchronization during transmeridian travel and shift work.

Background Daily rhythms in mammals are programmed by a master clock in the suprachiasmatic nucleus (SCN). The SCN contains two main compartments (shell and core), but the role of each region in system-level coordination remains ill defined. Herein, we use a functional assay to investigate how downstream tissues interpret region-specific outputs by using in vivo exposure to long day photoperiods to temporally dissociate the SCN. We then analyze resulting changes in the rhythms of clocks located throughout the brain and body to examine whether they maintain phase synchrony with the SCN shell or core. Results Nearly all of the 17 tissues examined in the brain and body maintain phase synchrony with the SCN shell, but not the SCN core, which indicates that downstream oscillators are set by cues controlled specifically by the SCN shell. Interestingly, we also found that SCN dissociation diminished the amplitude of rhythms in core clock gene and protein expression in brain tissues by 50–75 %, which suggests that light-driven changes in the functional organization of the SCN markedly influence the strength of rhythms in downstream tissues. Conclusions Overall, our results reveal that body clocks receive time-of-day cues specifically from the SCN shell, which may be an adaptive design principle that serves to maintain system-level phase relationships in a changing environment. Further, we demonstrate that lighting conditions alter the amplitude of the molecular clock in downstream tissues, which uncovers a new form of plasticity that may contribute to seasonal changes in physiology and behavior. Electronic supplementary material The online version of this article (doi:10.1186/s12915-015-0157-x) contains supplementary material, which is available to authorized users.

Dividing cells, including human cancers, organize processes necessary for their duplication according to circadian time. Recent evidence has shown that disruption of central regulation of circadian rhythms can increase the rate at which a variety of cancers develop in rodents. In order to study circadian rhythms in liver tumors, we have chemically induced hepatocellular carcinoma in transgenic rats bearing a luciferase reporter gene attached to the promoter of a core circadian clock gene (Period 1). We explanted normal liver cells and hepatomas, placed them into short-term culture, and precisely measured their molecular clock function by recording light output. Results show that isolated hepatocellular carcinoma is capable of generating circadian rhythms in vitro. Temporally restricting food availability to either day or night altered the phase of the rhythms in both healthy and malignant tissue. However the hepatomas were much less sensitive to this signal resulting in dramatically different phase relationships between host and tumor tissue as a function of mealtime. These data support the conclusion that hepatoma is differentially sensitive to circadian timing signals, although it maintains the circadian organization of the non-malignant cells from which it arose. Because circadian clocks are known to modulate the sensitivity of many therapeutic cytotoxic targets, controlling mealtiming might be used to increase the efficacy of treatment. Specifically, meal and treatment schedules could be designed that take advantage of coincident times of greatest tumor sensitivity and lowest sensitivity of host tissue to damage.

Acute cardiovascular events exhibit a circadian rhythm in the frequency of occurrence. The mechanisms underlying these phenomena are not yet fully understood, but they may be due to rhythmicity inherent in the cardiovascular system. We have begun to characterize rhythmicity of the clock gene mPer1 in the rat cardiovascular system. Luciferase activity driven by the mPer1 gene promoter is rhythmic in vitro in heart tissue explants and a wide variety of veins and arteries cultured from the transgenic Per1-luc rat. The tissues showed between 3 and 12 circadian cycles of gene expression in vitro before damping. Whereas peak per1-driven bioluminescence consistently occurred during the late night in the heart and all arteries sampled, the phases of the rhythms in veins varied significantly by anatomical location. Varying the time of the culture procedure relative to the donor animal's light:dark cycle revealed that, unlike some other rat tissues such as liver, the phases of in vitro rhythms of arteries, veins, and heart explants were affected by culture time. However, phase relationships among tissues were consistent across culture times; this suggests diversity in circadian regulation among components of the cardiovascular system.

Biological systems use internal circadian clocks to efficiently organize physiological and behavioral activity within the 24-hour time domain. In the absence of time cues, circadian periods vary slightly from 24 hours, but in nature, ambient light serves as the most salient synchronizer for these rhythms, fine-tuning them to exactly 24 hours each day. For some species, social cues can serve to synchronize circadian rhythms in the absence of other time cues or to amplify ambiguous light cues. This has been demonstrated to various degrees in fruit flies, degus, birds, fish, bats, beavers and humans; however, studies in rats and hamsters have shown that social cues are less salient time cues for these species. Social influences on circadian timing might function to tightly organize the social group, thereby decreasing the chances of predation and increasing the likelihood of mating.

Food-anticipatory activity (FAA) is the increase in locomotion and core body temperature that precedes a daily scheduled meal. It is driven by a circadian oscillator but is independent of the suprachiasmatic nuclei. Recent results that reveal meal-entrained clock gene expression in rat and mouse peripheral organs raise the intriguing possibility that the digestive system is the site of the feeding-entrained oscillator (FEO) that underlies FAA. We tested this possibility by comparing FAA and Per1 rhythmicity in the digestive system of the Per1-luciferase transgenic rat. First, rats were entrained to daytime restricted feeding (RF, 10 days), then fed ad libitum (AL, 10 days), then food deprived (FD, 2 days). As expected FAA was evident during RF and disappeared during subsequent AL feeding, but returned at the correct phase during deprivation. The phase of Per1 in liver, stomach and colon shifted from a nocturnal to a diurnal peak during RF, but shifted back to nocturnal phase during the subsequent AL and remained nocturnal during food deprivation periods. Second, rats were entrained to two daily meals at zeitgeber time (ZT) 0400 and ZT 1600. FAA to both meals emerged after about 10days of dual RF. However, all tissues studied (all five liver lobes, esophagus, antral stomach, body of stomach, colon) showed entrainment consistent with only the night-time meal. These two results are inconsistent with the hypothesis that FAA arises as an output of rhythms in the gastrointestinal (GI) system. The results also highlight an interesting diversity among peripheral oscillators in their ability to entrain to meals and the direction of the phase shift after RF ends.

The mammalian Per1 gene is an important component of the core cellular clock mechanism responsible for circadian rhythms. The rodent liver and other tissues rhythmically express Per1 in vitro but typically damp out within a few cycles. In the liver, the peak of this rhythm occurs in the late subjective night in an ad lib-fed rat, but will show a large phase advance in response to restricted availability of food during the day. The relationship between this shift in the liver clock and food-anticipatory activity (FAA), the circadian behavior entrained by daily feeding, is currently unknown. Insulin is released during feeding in mammals and could serve as an entraining signal to the liver. To test the role of insulin in the shift in liver Per1 expression and the generation of FAA, per-luciferase transgenic rats were made diabetic with a single injection of streptozotocine. Following 1 week of restricted feeding and locomotor activity monitoring, liver was collected for per-luc recording. In two separate experiments, FAA emerged and liver Per1 phase-shifted in response to daytime 8-h food restriction. The results rule out insulin as a necessary component of this system.

Various aspects of immune response exhibit 24-h variations suggesting that infection susceptibility and treatment efficacy may vary by time of day. Whether these 24-h variations are endogenous or evoked by changes in environmental or behavioral conditions is not known. We assessed the endogenous circadian control and environmental and behavioral influences on ex-vivo lipopolysaccharide stimulation of whole blood in thirteen healthy participants under 48. h of baseline conditions with standard sleep-wake schedules and 40-50. h of constant environmental and behavioral (constant routine; CR) conditions. Significant 24-h rhythms were observed under baseline conditions in Monocyte Chemotactic Protein, Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin 8 but not Tumor Necrosis Factor alpha whereas significant 24-h rhythms were observed in all four immune factors under CR conditions. The rhythm amplitudes, expressed as a percentage of mean, were comparable between immune factors and across conditions. In contrast, the acrophase time (time of the fitted peak) was different between immune factors, and included daytime and nighttime peaks and changes across behavioral conditions. These results suggest that the endogenous circadian system underpins the temporal organization of immune responses in humans with additional effects of external environmental and behavioral cycles. These findings have implications for understanding the adverse effects of recurrent circadian disruption and sleep curtailment on immune function. © 2014 Elsevier Inc..

Rats anticipate daily meals with increased approaches to a feeder and an increase in core body temperature. Food-anticipatory activity (FAA) is thought to be under the control of a feeding-entrained circadian oscillator (FEO). Ibotenic acid and electrolytic lesions in the region of the parabrachial nuclei (PBN) in the rat severely disrupt FAA (feeder approaches) and temperature rhythms. The PBN receive dense input from the area postrema (AP), which lacks a blood-brain barrier and thus has access to humoral factors in the systemic circulation. The present study assesses development and maintenance of FAA in rats with cautery lesions of the AP. The results demonstrate that AP lesions do not alter FAA. This experiment does not support the hypothesis that the AP in the caudal brainstem detects and relays circulating signals from the periphery that trigger FAA.

The immune and the circadian systems interact in a bidirectional fashion. The master circadian oscillator, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, responds to peripheral and local immune stimuli, such as proinflammatory cytokines and bacterial endotoxin. Astrocytes exert several immune functions in the CNS, and there is growing evidence that points toward a role of these cells in the regulation of circadian rhythms. The aim of this work was to assess the response of SCN astrocytes to immune stimuli, particularly to the proinflammatory cytokine TNF-a. TNF-α applied to cultures of SCN astrocytes from Per2 luc knockin mice altered both the phase and amplitude of PER2 expression rhythms, in a phase-dependent manner. Furthermore, conditioned media from SCN astrocyte cultures transiently challenged with TNF-a induced an increase in Per1 expression in NIH 3T3 cells, which was blocked by TNF-a antagonism. In addition, these conditioned media could induce phase shifts in SCN PER2 rhythms and, when administered intracerebroventricularly, induced phase delays in behavioral circadian rhythms and SCN activation in control mice, but not in TNFR-1 mutants. In summary, our results show that TNF-a modulates the molecular clock of SCN astrocytes in vitro, and also that, in response to this molecule, SCN astrocytes can modulate clock gene expression in other cells and tissues, and induce phase shifts in a circadian behavioral output in vivo. These findings suggest a role for astroglial cells in the alteration of circadian timing by immune activation. Fil: Duhart, José Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina Fil: Leone, Maria Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina Fil: Paladino, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina Fil: Evans, J. A.. Neuroscience Institute; Estados Unidos Fil: Castanon Cervantes, O.. Morehouse School of Medicine; Estados Unidos Fil: Davidson, A. J.. Morehouse School of Medicine; Estados Unidos Fil: Golombek, Diego Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina

Rats anticipate daily restricted meals with increased approaches to a feeder and an increase in core body temperature. Food anticipatory activity (FAA) is thought to be under the control of a feeding-entrained circadian oscillator. Although numerous forebrain lesions have failed to permanently abolish FAA, the hindbrain has not been investigated. The parabrachial nuclei (PBN) integrate information from visceral and gustatory afferents. This region is also innervated by neurons in the area postrema that have access to the peripheral circulation. Therefore, it is possible that this region plays a role in triggering FAA. In two experiments, a total of 19 rats were given ibotenic acid or electrolytic lesions targeted at the PBN. The PBN-lesioned animals showed a marked attenuation in anticipatory approaches to the food bin relative to sham-operated controls. Some animals did not anticipate the meal at all. In addition, the expected increase in core body temperature was severely attenuated in the PBN-lesioned animals compared with controls. The most likely interpretation of these data is that the PBN serve as a relay for information about the zeitgeber (food in the gut) or as a clock output pathway, but not as the site of the feeding-entrained circadian oscillator.

Several pituitary hormones are important in the regulation of metabolism, and their release appears to be controlled by a circadian clock. Consequently, they may be involved in feeding-entrained circadian rhythms. Hypophysectomized (Hypox) and sham-operated male Sprague-Dawley rats had access to food for 4 h each day. Food-anticipatory activity (FAA) and core body temperature (Tb) were monitored. Both groups entrained to the daily meal with an increase in activity in the 4 h preceding meal access and quickly reentrained after an 8-h phase advance of food access. FAA was not disrupted in either group after suprachiasmatic lesions were added. Core Tb increased in the sham-operated subjects before mealtime, but Hypox rats failed to show this effect. Rather, Tb declined during anticipation and throughout the food access period. Respiratory quotient (RQ), an indirect measure of metabolic rate, was measured for 24 h in some subjects. Sham-operated rats showed a dramatic downturn in RQ 1 h before mealtime, whereas Hypox rats showed a steadily decreasing RQ throughout the day. The results show that the pituitary hormones are not necessary for FAA and that in Hypox rats the anticipatory rise in Tb and changes in RQ become dissociated from anticipatory behavior, indicating that these functions are separate outputs of the food-entrainable circadian oscillator.

The circadian food entrainable oscillator (FEO) mediates an increase in activity preceding access to periodic meals. The FEO is anatomically independent of the suprachiasmatic nucleus (SCN), but its locus remains to be established. Whether the FEO is located in the central nervous system (CNS) or in the periphery, it seems reasonable to assume that there is a link of communication between the digestive system and the CNS because only nutritive meals entrain the FEO. Subdiaphragmatic vagotomy and visceral deafferentation with capsaicin do not eliminate food-anticipatory activity (FAA), indicating that a neural signal is not necessary. The present study investigates the hypothesis that humoral signals from the digestive system act upon the CNS to trigger or entrain FAA. Intact rats and rats with SCN lesions were entrained to daily meals and then sacrificed prior to FAA or during FAA, but before meal access. Average plasma concentrations of glucose and insulin were nearly identical in both groups. Plasma and duodenal mucosal motilin concentrations also were not different between the two times. Corticosterone was elevated during anticipation, but the difference was not statistically reliable. Glucagon concentration was decreased during FAA compared to concentration prior to FAA in both intact and SCN-lesioned subjects. This difference was not observed in control rats fasted for the same number of hours, but not previously entrained to a daily meal. Although the decrease in glucagon could be a signal that initiates FAA, a causal role remains to be established.

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.

Daily rhythms in behavior and physiology are programmed by a hierarchical collection of biological clocks located throughout the brain and body, known as the circadian system. Mounting evidence indicates that disruption of circadian regulation is associated with a wide variety of adverse health consequences, including increased risk for premature death, cancer, metabolic syndrome, cardiovascular dysfunction, immune dysregulation, reproductive problems, mood disorders, and learning deficits. Here we review the evidence for the pervasive effects of circadian disruption in humans and animal models, drawing from both environmental and genetic studies, and identify questions for future research.

Daily rhythms in behavior and physiology are programmed by a hierarchical collection of biological clocks located throughout the brain and body, known as the circadian system. Mounting evidence indicates that disruption of circadian regulation is associated with a wide variety of adverse health consequences, including increased risk for premature death, cancer, metabolic syndrome, cardiovascular dysfunction, immune dysregulation, reproductive problems, mood disorders, and learning deficits. Here we review the evidence for the pervasive effects of circadian disruption in humans and animal models, drawing from both environmental and genetic studies, and identify questions for future research.

The mammalian pacemaker in the suprachiasmatic nucleus (SCN) contains a population of neural oscillators capable of sustaining cell-autonomous rhythms in gene expression and electrical firing. A critical question for understanding pacemaker function is how SCN oscillators are organized into a coherent tissue capable of coordinating circadian rhythms in behavior and physiology. Here we undertake a comprehensive analysis of oscillatory function across the SCN of the adult PER2::LUC mouse by developing a novel approach involving multi-position bioluminescence imaging and unbiased computational analyses. We demonstrate that there is phase heterogeneity across all three dimensions of the SCN that is intrinsically regulated and extrinsically modulated by light in a region-specific manner. By investigating the mechanistic bases of SCN phase heterogeneity, we show for the first time that phase differences are not systematically related to regional differences in period, waveform, amplitude, or brightness. Furthermore, phase differences are not related to regional differences in the expression of arginine vasopressin and vasoactive intestinal polypeptide, two key neuropeptides characterizing functionally distinct subdivisions of the SCN. The consistency of SCN spatiotemporal organization across individuals and across planes of section suggests that the precise phasing of oscillators is a robust feature of the pacemaker important for its function.

Circadian rhythms modulate nearly every mammalian physiological process. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work.

BACKGROUND We have reported that human prostate cancer ARCaPE cells undertake epithelial to mesenchymal transition (EMT) when stimulated by certain soluble factors, and that EMT is regulated by surface receptor-elicited signaling pathways through protein phosphorylation. It is known that phorbol ester phorbol-12-myristate-13-acetate (PMA), a potent antagonist to both conventional and novel protein kinase C (PKC) isoenzymes, induces cancer cell scattering. METHODS To assess the effect of PMA on EMT, ARCaPE cells were treated with PMA and were assayed for EMT-related morphologic and behavioral changes. Specific inhibitors were used to investigate the PMA-induced EMT. RESULTS PMA at 100 nM induced EMT in a time-dependent manner, resulting in a complete change from epithelial to mesenchymal stromal morphology. Concurrently, PMA inhibited expression of epithelial marker E-cadherin and increased the level of stromal marker protein vimentin, while the treated cells showed increased migratory and invasive capacities. Using specific inhibitors, we confirmed that the effect of PMA was mediated by PKC, while isoenzymes of the novel PKC subfamily were implicated as the main mediator. Finally, we determined that the EMT was dependent on newly synthesized proteins, because inhibitors for gene transcription and protein translation could both inhibit the initiation of EMT. CONCLUSIONS Although PMA is well known for its effects on cell migration and tumor formation, this work is the first to define PMA as an EMT inducer in prostate cancer cells. Further investigation in this experimental model may reveal important regulatory mechanisms and additional molecular changes underlying EMT. Prostate 70: 1119–1126, 2010. © 2010 Wiley-Liss, Inc.

Behavior ablation remains a powerful, if not cutting-edge, approach for localization of function within the nervous system. The initial discovery of the suprachiasmatic nuclei as the site of the mammalian light-entrainable circadian pacemaker is owed to this approach. Food-anticipatory activity (FAA), an output of a putative feeding-entrainable circadian pacemaker, is a behavior that has been surprisingly resilient to elimination by surgical lesion. Here we review this literature, with particular attention paid to recent studies aimed at defining the role of the dorsomedial hypothalamus in the generation of FAA. This literature is fraught with examples of inconsistent results among lesion studies, which in some cases can be accounted for by varied endpoint measures. The site of the feeding-entrainable circadian pacemaker, if it resides in a discrete structure at all, remains unknown.

Daily rhythms are a ubiquitous feature of living systems. Generally, these rhythms are not just passive consequences of cyclic fluctuations in the environment, but instead originate within the organism. In mammals, including humans, the master pacemaker controlling 24 h rhythms is localized in the suprachiasmatic nuclei of the hypothalamus. This pacemaker, or circadian clock, has been shown to be responsible for the temporal organization of a wide variety of functions, ranging from sleep and food intake, to physiological measures such as body temperature, heart rate, and hormone release.