Embryonic development of circadian oscillations in the mouse hypothalamus.

Circadian rhythms in mammals are regulated by the hypothalamic suprachiasmatic nucleus (SCN). The generation of circadian oscillations is a cell-autonomous property, and coupling among cells is essential for the SCN to function as a pacemaker. The development of SCN anatomy and cytology has been extensively studied, but the point in development when the SCN first has the capacity to generate circadian oscillations has not been established. We therefore examined the development of circadian oscillations using per2::luc mice in which bioluminescence tracks the expression of the circadian clock protein, PER2. In vitro, hypothalamic explants first expressed consistent oscillations when isolated between 15 and 16 days postfertilization (e15). Oscillations were more robust at later ages. Explants from other brain areas did not express oscillations, indicating that the development of oscillations is not a general property of embryonic tissue. SCN explants obtained on e14 did not initially express oscillations but developed them in vitro over 4 to 6 d. Although coupling among cells is required for the long-term expression of tissue-level oscillations, explants from mice lacking the coupling peptide vasoactive intestinal peptide still developed oscillations. In the mouse, the capacity to generate molecular oscillations on e15 coincides with the completion of neurogenesis and SCN-specific transcription factor expression. Thus, within a day of its genesis at an age approximately equivalent to the end of the first trimester in humans, the SCN develops the capacity to express circadian oscillations and autonomously develops mechanisms sufficient to couple and synchronize its cells.

A fear-inducing odor alters PER2 and c-Fos expression in brain regions involved in fear memory.

Evidence demonstrates that rodents learn to associate a foot shock with time of day, indicating the formation of a fear related time-stamp memory, even in the absence of a functioning SCN. In addition, mice acquire and retain fear memory better during the early day compared to the early night. This type of memory may be regulated by circadian pacemakers outside of the SCN. As a first step in testing the hypothesis that clock genes are involved in the formation of a time-stamp fear memory, we exposed one group of mice to fox feces derived odor (TMT) at ZT 0 and one group at ZT 12 for 4 successive days. A separate group with no exposure to TMT was also included as a control. Animals were sacrificed one day after the last exposure to TMT, and PER2 and c-Fos protein were quantified in the SCN, amygdala, hippocampus, and piriform cortex. Exposure to TMT had a strong effect at ZT 0, decreasing PER2 expression at this time point in most regions except the SCN, and reversing the normal rhythm of PER2 expression in the amygdala and piriform cortex. These changes were accompanied by increased c-Fos expression at ZT0. In contrast, exposure to TMT at ZT 12 abolished the rhythm of PER2 expression in the amygdala. In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group. TMT exposure at either time point did not affect PER2 or c-Fos in the SCN, indicating that under a light-dark cycle, the SCN rhythm is stable in the presence of repeated exposure to a fear-inducing stimulus. Taken together, these results indicate that entrainment to a fear-inducing stimulus leads to changes in PER2 and c-Fos expression that are detected 24 hours following the last exposure to TMT, indicating entrainment of endogenous oscillators in these regions. The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus.

Resilience of circadian pacemaker development in hamsters.

Disruptions of circadian rhythms have been linked to a wide range of pathologies from sleep disorders to cancer. The extent to which disruptions of circadian rhythms during development contribute to later conditions is not known. The present study tested the hypothesis that functional properties of the central circadian pacemaker, the suprachiasmatic nucleus (SCN), are affected by abnormal entrainment during development. The SCN is specialized for the generation of robust rhythms, for direct and indirect output to physiological and behavioral systems, and for entrainment to light/dark cycles via direct retinal input. It consists of thousands of neurons and glia with distinct phenotypes and has subdivisions delineated by both anatomical and functional criteria. In rodents, SCN rhythms develop within days after SCN cells are produced and before many other aspects of differentiation, such as synaptogenesis, are complete. We demonstrated that around the time of birth, the hamster SCN in vivo can undergo repeated phase shifts by a dopamine D(1) receptor agonist (SKF-38393). For 2 days before and 2 days after birth, one group of hamsters received regular exposure to the drug at the same time of day, while another group was exposed at varying times to induce repeated phase shifts. Free-running and entrained activity rhythms were compared between the groups at different ages after weaning. Repeated phase shifts during SCN development had a significant effect on free-running period measured immediately after weaning. This effect was eliminated by subsequent entrainment to a light/dark cycle, indicating that the effect was not permanent. These and other results suggest that SCN development required for functional properties such as free-running period is resilient to perturbation.

Chronic stimulation of the hypothalamic vasoactive intestinal peptide receptor lengthens circadian period in mice and hamsters.

Evidence suggests that circadian rhythms are regulated through diffusible signals generated by the suprachiasmatic nucleus (SCN). Vasoactive intestinal peptide (VIP) is located in SCN neurons positioned to receive photic input from the retinohypothalamic tract and transmit information to other SCN cells and adjacent hypothalamic areas. Studies using knockout mice indicate that VIP is essential for synchrony among SCN cells and for the expression of normal circadian rhythms. To test the hypothesis that VIP is also an SCN output signal, we recorded wheel-running activity rhythms in hamsters and continuously infused the VIP receptor agonist BAY 55-9837 in the third ventricle for 28 days. Unlike other candidate output signals, infusion of BAY 55-9837 did not affect activity levels. Instead, BAY 55-9837 lengthened the circadian period by 0.69 +/- 0.04 h (P < 0.0002 compared with controls). Period returned to baseline after infusions. We analyzed the effect of BAY 55-9837 on cultured SCN from PER2::LUC mice to determine if lengthening of the period by BAY 55-9837 is a direct effect on the SCN. Application of 10 muM BAY 55-9837 to SCN in culture lengthened the period of PER2 luciferase expression (24.73 +/- 0.24 h) compared with control SCN (23.57 +/- 0.26, P = 0.01). In addition, rhythm amplitude was significantly increased, consistent with increased synchronization of SCN neurons. The effect of BAY 55-9837 in vivo on period is similar to the effect of constant light. The present results suggest that VIP-VPAC2 signaling in the SCN may play two roles, synchronizing SCN neurons and setting the period of the SCN as a whole.

Differential expression of the circadian clock in maternal and embryonic tissues of mice.

BACKGROUND: Molecular feedback loops involving transcription and translation and several key genes are at the core of circadian regulatory cycles affecting cellular pathways and metabolism. These cycles are active in most adult animal cells but little is known about their expression or influence during development. METHODOLOGY/PRINCIPAL FINDINGS: To determine if circadian cycles are active during mammalian development we measured the expression of key circadian genes during embryogenesis in mice using quantitative real-time RT-PCR. All of the genes examined were expressed in whole embryos beginning at the earliest age examined, embryonic day 10. In contrast to adult tissues, circadian variation was absent for all genes at all of the embryonic ages examined in either whole embryos or individual tissues. Using a bioluminescent fusion protein that tracks translation of the circadian gene, per2, we also analyzed protein levels. Similar to mRNA, a protein rhythm was observed in adult tissue but not in embryonic tissues collected in-vivo. In contrast, when tissues were placed in culture for the continuous assay of bioluminescence, rhythms were observed in embryonic (E18) tissues. We found that placing embryonic tissues in culture set the timing (phase) of these rhythms, suggesting the importance of a synchronizing signal for the expression of circadian cycles in developing tissues. CONCLUSIONS/SIGNIFICANCE: These results show that embryonic tissues express key circadian genes and have the capacity to express active circadian regulatory cycles. In vivo, circadian cycles are not expressed in embryonic tissues as they are in adult tissues. Individual cells might express oscillations, but are not synchronized until later in development.

Circadian clock genes in reproductive tissues and the developing conceptus.

The circadian (near 24-h) clock is involved in the temporal organisation of physiological and biochemical activities of many organisms, including humans. The clock functions through the rhythmic transcription and translation of several genes, forming an oscillatory feedback loop. Genetic analysis has shown that the circadian clock exists in both a central circadian pacemaker (i.e. the suprachiasmatic nucleus of the hypothalamus), as well as in most peripheral tissues. In particular, the circadian clockwork genes are expressed in all female and male reproductive tissues studied so far, as well as in the conceptus itself. The current data clearly show a robust rhythm in female reproductive tissues, but whether rhythmicity also exists in male reproductive tissues remains uncertain. Although the conceptus also expresses most of the canonical circadian genes, the rhythmicity of their expression is still under investigation. Published data indicate that environmental and genetic manipulations influence reproductive function and fecundity, suggesting an important role for the circadian clock in reproduction, and possibly early development.

Behavioral effects of systemic transforming growth factor-alpha in Syrian hamsters.

The growth factor, transforming growth factor-alpha (TGF-alpha) is strongly expressed in the hypothalamic circadian pacemaker, the suprachiasmatic nucleus (SCN). TGF-alpha is one of several SCN peptides recently suggested to function as a circadian output signal for the regulation of locomotor activity rhythms in nocturnal rodents. When infused in the brain, TGF-alpha suppresses activity. TGF-alpha suppresses other behaviors as well including feeding, resulting in weight loss. Elevated TGF-alpha is correlated with some cancers, and it is possible the TGF-alpha and its receptor, the epidermal growth factor receptor (EGFR), mediate fatigue and weight loss associated with cancer. If true for cancers outside of the brain, then systemic TGF-alpha should also affect behavior. We tested this hypothesis in hamsters with intraperitoneal injections or week-long subcutaneous infusions of TGF-alpha. Both treatments suppressed activity and infusions caused reduced food consumption and weight loss. To identify areas of the brain that might mediate these effects of systemic TGF-alpha, we used immunohistochemistry to localize cells with an activated MAP kinase signaling pathway (phosphorylated ERK1). Cells were activated in two hypothalamic areas, the paraventricular nucleus and a narrow region surrounding the third ventricle. These sites could not only be targets of TGF-alpha produced in the SCN but could also mediate effects of elevated TGF-alpha from tumors both within and outside the central nervous system.

Development of the mouse suprachiasmatic nucleus: determination of time of cell origin and spatial arrangements within the nucleus.

The suprachiasmatic nucleus (SCN) in mammals functions as the principal circadian pacemaker synchronizing diverse physiological and behavioral processes to environmental stimuli. It consists of heterogeneous populations of cells with unique spatial organization that can vary among species, but are commonly discussed within a framework of two principal regions, the ventrolateral or dorsomedial halves of the nucleus or in other instances the core and shell. In both hamsters and rats, cells of different SCN regions have been shown to have different developmental histories. Using bromodeoxyuridine as a marker of cell division, the present study investigated the time of SCN cell origin in mice (C57BL/6) and their settling patterns within the nucleus. Results show that SCN cytogenesis occurs between embryonic days 12 and 15 and is complete 5 days prior to birth. Cells born on embryonic day 12 are mainly confined to a ventrolateral region of the mid-SCN, whereas cells produced later on embryonic days 13.5 and 14.5 form a cap around the cells produced first and extend into the posterior and anterior ends of the nucleus. These results suggest an ordered spatiotemporal program of SCN cytogenesis whereby a mid-SCN core is born first followed by a surrounding shell of later-born cells. Variations in cytogenesis could affect the relative sizes of different SCN regions and, thereby, affect its function. The relative contributions of a highly ordered program of cytogenesis and intercellular interactions after postmitotic cells leave the germinal epithelium remain to be determined.

Developmental expression of clock genes in the Syrian hamster.

Transcription/translation feedback loops consisting of multiple clock genes are thought to be essential for circadian oscillations at cellular, tissue and organismal levels. We examined the developmental expressions of three clock genes (Bmal1, Cry1 and Per1) in the Syrian hamster to probe the oscillatory properties of the suprachiasmatic nucleus (SCN) over the first 4 days after the completion of SCN neurogenesis. Samples were taken at the dam's circadian times 6, 12, and 18 daily over 4 days in constant dim light and processed for in situ hybridization using 35S-labeled RNA probes. Collection times were based on the phases of Bmal1 and Per1 rhythms in adult SCN and on an observed difference in Per1 mRNA at CT6 and 18 on postnatal day 2. For the developmental study, sections from each brain were processed in parallel for the three genes. Bmal1 was prominently expressed in the fetal SCN while Per1 and Cry1 were only weakly expressed. Transcripts of all three genes showed higher abundance just after birth. At subsequent ages, Bmal1 showed a significant decrease, while Per1 continued to be greater than prenatal levels. Significant variation was detected across circadian times for Cry1, but no circadian variation was detected for Per1 and Bmal1. Molecular oscillations equivalent to those observed in adults were not present in the fetal SCN despite evidence for an entrainable pacemaker at that time. An absence of robust oscillations during early SCN development may in part explain the strong phase-setting effects of pharmacological agents on the fetal/neonatal clock.

Central administration of transforming growth factor-alpha and neuregulin-1 suppress active behaviors and cause weight loss in hamsters.

Transforming growth factor-alpha (TGF-alpha) is a candidate output signal of the hypothalamic circadian pacemaker. TGF-alpha is expressed in the suprachiasmatic nucleus (SCN) of rats, hamsters, and rhesus macaques [A. Kramer, F.C. Yang, P. Snodgrass, X. Li, T.E. Scammell, F.C. Davis and C.J. Weitz, Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signaling, Science, 294 (2001) 2511-5., X. Li, N. Sankrithi and F.C. Davis, Transforming growth factor-alpha is expressed in astrocytes of the suprachiasmatic nucleus in hamster: role of glial cells in circadian clocks, Neuroreport, 13 (2002) 2143-7., Y.J. Ma, M.E. Costa and S.R. Ojeda, Developmental expression of the genes encoding transforming growth factor alpha and its receptor in the hypothalamus of female rhesus macaques, Neuroendocrinology, 60 (1994) 346-59., Y.J. Ma, M.P. Junier, M.E. Costa and S.R. Ojeda, Transforming growth factor-alpha gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation, Neuron, 9 (1992) 657-70.]. TGF-alpha reversibly inhibits wheel-running activity during long-term infusions into the third ventricle of hamsters (2 weeks, intracerebroventricular or ICV) [A. Kramer, F.C. Yang, P. Snodgrass, X. Li, T.E. Scammell, F.C. Davis and C.J. Weitz, Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signaling, Science, 294 (2001) 2511-5.], and this effect appears to be mediated by the epidermal growth factor receptor (EGFR or ErbB-1) [A. Kramer, F.C. Yang, P. Snodgrass, X. Li, T.E. Scammell, F.C. Davis and C.J. Weitz, Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signaling, Science, 294 (2001) 2511-5.]. Here, we demonstrate that this inhibitory effect is not restricted to wheel-running behavior or to mediation by the EGFR. Using direct observation, we found the effects of long-term TGF-alpha infusion (ICV, 12 microl/day, 3.3 microM) to be more general than previously reported. Other active behaviors such as grooming and feeding were reversibly inhibited and hamsters showed dramatic weight loss as a result of reduced feeding (34% of body weight over 19 days). TGF-alpha did not disrupt a non-behavioral rhythm, the rhythm in pineal melatonin. Wheel-running activity was also inhibited by another epidermal growth factor-like (EGF-like) peptide, neuregulin (NRG-1), that binds to different ErbB receptors. Like TGF-alpha, NRG-1 caused a significant weight loss. We also show that an acute injection of TGF-alpha inhibits activity (ICV, 5 microl, 3.3 microM over 2 min), with inhibition and recovery occurring over a few hours. Although the results are consistent with the proposed [A. Kramer, F.C. Yang, P. Snodgrass, X. Li, T.E. Scammell, F.C. Davis and C.J. Weitz, Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signaling, Science, 294 (2001) 2511-5.] role for EGF-like peptides in the daily regulation of activity, the actions of these peptides might also contribute to the behavioral etiology of diseases in which EGF-like peptides are expressed.

Disruption of masking by hypothalamic lesions in Syrian hamsters.

Negative masking of locomotor activity by light in nocturnal rodents is mediated by a non-image-forming irradiance-detection system in the retina. Structures receiving input from this system potentially contribute to the masking response. The suprachiasmatic nucleus (SCN) regulates locomotor activity and receives dense innervation from the irradiance-detection system via the retinohypothalamic tract, but its role in masking is unclear. We studied masking in adult Syrian hamsters (Mesocricetus auratus) with electrolytic lesions directed at the SCN. Hamsters were exposed to a 3.5:3.5 ultradian light/dark cycle and their wheel-running activity was monitored. Intact hamsters showed robust masking, expressing less than 20% of their activity in the light even though light and dark occurred equally during their active times. In contrast, hamsters with lesions showed, on average, as much activity in the light as in the dark. Tracing of retinal projections using cholera toxin beta subunit showed that the lesions damaged retinal projections to the SCN and to the adjacent subparaventricular zone. Retinal innervation outside the hypothalamus was not obviously affected by the lesions. Our results indicate that retinohypothalamic projections, and the targets of these projections, to the SCN and/or adjacent hypothalamic areas play an important role in masking.

Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signalling.

The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behaviour by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor (TGF)alpha as a likely SCN inhibitor of locomotion. TGFalpha is expressed rhythmically in the SCN, and when infused into the 3rd ventricle it reversibly inhibits locomotor activity and disrupts circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors, which we identified on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibit excessive daytime locomotor activity and fail to suppress activity when exposed to light. These results implicate EGF receptor signalling in the daily control of locomotor activity, and they identify a neural circuit in the hypothalamus that likely mediates the regulation of behaviour both by the SCN and the retina.

Transforming growth factor-alpha is expressed in astrocytes of the suprachiasmatic nucleus in hamster: role of glial cells in circadian clocks.

Transforming growth factor-alpha (TGF-alpha) is abundantly expressed in the suprachiasmatic nucleus of several rodent species. It was recently suggested to be a clock output signal regulating the activity/rest rhythm. In this study we further characterized the cellular identity of TGF-alpha-expressing cells in the suprachiasmatic nucleus of the Syrian hamster (Mesocricetus auratus). Using confocal laser scanning fluorescence imaging on brain sections immuno-histologically processed for TGF-alpha and GFAP double staining, we observed that in the suprachiasmatic nucleus, TGF-alpha staining is located mainly in GFAP-positive cells, indicating suprachiasmatic nucleus astrocytes produce TGF-alpha. Glial expression of TGF-alpha was also observed in the 3rd ventricle tanycytes of the retrochiasmatic area. In other brain regions where the TGF-alpha message is abundant, such as in the piriform cortex, we observed that TGF-alpha staining is mainly located in neurons. Our results provide the first evidence that glial cells are involved in the regulation of output from the suprachiasmatic nucleus circadian clock through a diffusible mechanism.

Extensive and divergent circadian gene expression in liver and heart.

Many mammalian peripheral tissues have circadian clocks; endogenous oscillators that generate transcriptional rhythms thought to be important for the daily timing of physiological processes. The extent of circadian gene regulation in peripheral tissues is unclear, and to what degree circadian regulation in different tissues involves common or specialized pathways is unknown. Here we report a comparative analysis of circadian gene expression in vivo in mouse liver and heart using oligonucleotide arrays representing 12,488 genes. We find that peripheral circadian gene regulation is extensive (> or = 8-10% of the genes expressed in each tissue), that the distributions of circadian phases in the two tissues are markedly different, and that very few genes show circadian regulation in both tissues. This specificity of circadian regulation cannot be accounted for by tissue-specific gene expression. Despite this divergence, the clock-regulated genes in liver and heart participate in overlapping, extremely diverse processes. A core set of 37 genes with similar circadian regulation in both tissues includes candidates for new clock genes and output genes, and it contains genes responsive to circulating factors with circadian or diurnal rhythms.

Development of the Mouse Circadian Pacemaker: Independence from Environmental Cycles.

The freerunning period (τ) of the circadian pacemaker underlying the wheel-running activity rhythm of Mus musculus was found to be unaffected by the periods of environmental cycles (maternal and light/dark) under which the mice are raised. Mice born to mothers entrained to periods (T) of 28 or 20 h (ratio of light to dark of 14/10) and maintained on those cycle until beyond puberty showed only a temporary difference in freerunning period when placed into constant darkness. Such temporary 'after-effects ' of entrainment were shown, as had been previously, to occur in animals exposed to non-24-h cycles as adults only.After-effects on the ratio of activity to rest (α/ρ) were not even temporarily different in animals raised on T = 28 or T = 20.Rearing on T = 28 or T = 20 did not affect the abilities of animals to entrain to these cycles later in life.Measurements from young and old animals as well as remeasurement of the young animals later in their lives revealed several effects of age on the pacemaker: a) After-effects on freerunning period after T = 28 or T = 20 are not greater but last longer in older animals; b) Freerunning period is shorter in younger animals; and c) The ratio of activity to rest changes over time in constant darkness and is greater in young animals. Together these suggest that pacemaker 'plasticity' reflected in changes in τ and α/ρ over time in constant darkness decreases with age.The length of gestation measured in 'real' time was the same in mice entrained to T = 28 or T = 20, demonstrating that gestation is not measured in circadian cycles.