Photoperiod alters phase difference between activity onset in vivo and mPer2::luc peak in vitro

Photoperiod is a significant modulator of behavior and physiology for many organisms. In rodents changes in photoperiod are associated with changes in circadian period and photic resetting of circadian pacemakers. Utilizing rhythms of in vivo behavior and in vitro mPer2::luc expression, we investigated whether different entrainment photoperiods [light:dark (L:D) 16:8 and L:D 8:16] alter the period or phase relationships between these rhythms and the entraining light cycle in Per2::luc C57BL/6J mice. We also tested whether mPer2::luc rhythms differs in anterior and posterior suprachiasmatic nucleus (SCN) slices. Our results demonstrate that photoperiod significantly changes the timing of the mPer2::luc peak relative to the time of light offset and the activity onset in vivo. In both L:D 8:16 and L:D 16:8 the mPer2::luc peak maintained a more stable phase relationship to activity offset, while altering the phase relationship to activity onset. After the initial cycle in culture, the period, phase, and peaks per cycle were not significantly different for anterior vs. posterior SCN slices taken from animals within one photoperiod. After short-photoperiod treatment, anterior SCN slices showed increased-amplitude Per2::luc waveforms and posterior SCN slices showed shorter-duration peak width. Finally, the SCN tissue in vitro did not demonstrate differences in period attributable to photoperiod pretreatment, indicating that period aftereffects observed in behavioral rhythms after long- and short-day photoperiods are not sustained in Per2::luc rhythms in vitro. The change in phase relationship to activity onset suggests that Per2::luc rhythms in the SCN may track activity offset rather than activity onset. The reduced amplitude rhythms following long-photoperiod treatment may represent a loss of coupling of component oscillators.     

Period gene expression in mouse endocrine tissues

Circadian rhythms are generated by the oscillating expression of the Per1 and Per2 genes, which are expressed not only in the central brain pacemaker but also in peripheral tissues. Hormones are likely to coordinate physiological function in time. We performed in situ hybridization to localize mPer1 and mPer2 mRNA to particular cell types and tissue compartments in adrenal, thyroid, and testis. BALB/c mice maintained in a 12:12-h light-dark cycle expressed mPer1 in adrenal medulla, particularly in late afternoon and early night. mPer2 mRNA was more intensely expressed in adrenal cortex, especially in afternoon and evening. mPer1 mRNA was detected in thyroid. mPer1 was found in some but not all seminiferous tubules of each mouse at all times of day. Quantitation in C57BL/6 mice revealed a significant increase in the number of heavily labeled seminiferous tubules early in the night. Consistent with in situ hybridization, immunocytochemistry showed PER1 protein in spermatocytes and spermatids (spermatogenic stages VII-XII). Staining in spermatogonia and interstitial cells was inconsistent. Double labeling with 5'-bromodeoxyuridine showed PER1 expression first occurring 5 days after DNA replication. We conclude that mPeriod genes are expressed in peripheral endocrine glands. Central regulation, adenohypophyseal control, and functional importance of expression and phase remain to be elucidated.     

Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 had severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms were blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not of mPER1-deficient mice. Peak mPER and mCRY1 protein levels were reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resembled rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice were immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes.     

Differential effects of two period genes on the physiology and proteomic profiles of mouse anterior tibialis muscles

The molecular components that generate and maintain circadian rhythms of physiology and behavior in mammals are present both in the brain (suprachiasmatic nucleus; SCN) and in peripheral tissues. Examination of mice with targeted disruptions of either mPer1 or mPer2 has shown that these two genes have key roles in the SCN circadian clock. Here we show that loss of the clock gene mPer2 affects forced locomotor performance in mice without altering muscle contractility. A proteomic analysis revealed that the anterior tibialis muscles of the mPer2 knockout mice had higher levels of glycolytic enzymes such as triose phosphate isomerase and enolase than those of either the wild type or mPer1 knockout mice. In addition, the level of expression of HSP90 in the mPer2 mutant mice was also significantly higher than in wildtype mice. These results suggest that the reduced locomotor endurance of the mPer2 knockout mice reflects a greater dependence on anaerobic metabolism under stress conditions, and that the two canonical clock genes, mPer1 and mPer2, play distinct roles in the physiology of skeletal muscle.     

Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression

Circadian (ca. 24 hr) oscillations in expression of mammalian "clock genes" are found not only in the suprachiasmatic nucleus (SCN), the central circadian pacemaker, but also in peripheral tissues. Under constant conditions in vitro, however, rhythms of peripheral tissue explants or immortalized cells damp partially or completely. It is unknown whether this reflects an inability of peripheral cells to sustain rhythms, as SCN neurons can, or a loss of synchrony among cells. Using bioluminescence imaging of Rat-1 fibroblasts transfected with a Bmal1::luc plasmid and primary fibroblasts dissociated from mPer2(Luciferase-SV40) knockin mice, we monitored single-cell circadian rhythms of clock gene expression for 1-2 weeks. We found that single fibroblasts can oscillate robustly and independently with undiminished amplitude and diverse circadian periods. Cells were partially synchronized by medium changes at the start of an experiment, but due to different intrinsic periods, their phases became randomly distributed after several days. Closely spaced cells in the same culture did not have similar phases, implying a lack of functional coupling among cells. Thus, like SCN neurons, single fibroblasts can function as independent circadian oscillators; however, lack of oscillator coupling in dissociated cell cultures leads to a loss of synchrony among individual cells and damping of the ensemble rhythm at the population level.     

The circadian system of Trypanosoma cruzi-infected mice

The effects of Chagas disease on the mammalian circadian system were studied in Trypanosoma cruzi-infected C57-Bl6J mice. Animals were inoculated with CAI or RA strains of T. cruzi or vehicle, parasitism confirmed by blood specimen visualization and locomotor activity rhythms analyzed by wheel-running recording. RA-strain infected mice exhibited significantly decreased amplitude of circadian rhythms, both under light-dark and constant dark conditions, probably due to motor deficiencies. CAI-treated animals showed normal locomotor activity rhythms. However, in these mice, reentrainment to a 6h phase shift of the LD cycle took significantly longer than controls, and application of 15min light pulses in DD produced smaller phase delays of the rhythms. All groups exhibited light-induced Fos expression in the suprachiasmatic nuclei. We conclude that the main effect of T. cruzi infection on the circadian system is an impairment of the motor output from the clock toward controlled rhythms, together with an effect on circadian visual sensitivity.     

Effects of vasoactive intestinal peptide genotype on circadian gene expression in the suprachiasmatic nucleus and peripheral organs

The neuropeptide vasoactive intestinal polypeptide (VIP) has emerged as a key candidate molecule mediating the synchronization of rhythms in clock gene expression within the suprachiasmatic nucleus (SCN). In addition, neurons expressing VIP are anatomically well positioned to mediate communication between the SCN and peripheral oscillators. In this study, we examined the temporal expression profile of 3 key circadian genes: Per1, Per2 , and Bmal1 in the SCN, the adrenal glands and the liver of mice deficient for the Vip gene (VIP KO), and their wild-type counterparts. We performed these measurements in mice held in a light/dark cycle as well as in constant darkness and found that rhythms in gene expression were greatly attenuated in the VIP-deficient SCN. In the periphery, the impact of the loss of VIP varied with the tissue and gene measured. In the adrenals, rhythms in Per1 were lost in VIP-deficient mice, while in the liver, the most dramatic impact was on the phase of the diurnal expression rhythms. Finally, we examined the effects of the loss of VIP on ex vivo explants of the same central and peripheral oscillators using the PER2::LUC reporter system. The VIP-deficient mice exhibited low amplitude rhythms in the SCN as well as altered phase relationships between the SCN and the peripheral oscillators. Together, these data suggest that VIP is critical for robust rhythms in clock gene expression in the SCN and some peripheral organs and that the absence of this peptide alters both the amplitude of circadian rhythms as well as the phase relationships between the rhythms in the SCN and periphery.     

EXPRESSION PROFILING REVEALS A POSITIVE REGULATION BY MPER2 ON CIRCADIAN RHYTHM OF CYTOTOXICITY RECEPTORS: LY49C AND NKG2D

The mammalian circadian gene, mPer2, an indispensable component of the mammalian circadian clock, not only modulates endogenous circadian rhythms but also plays a crucial role in regulating innate immune function. Previously, we showed that mPer2 plays a crucial role in regulating cytotoxic response. To investigate the molecular mechanism for mPer2-controlled cytotoxic response, in the present study we conducted mRNA expression for 11 genes participating in cytotoxicity regulation in wild-type (WT) and mPer2 knockout (mPer2 ^{?;?;?/ ?;?;?}) mice bone marrow, that is, Dap-10, Ly49C, Ly49I, Rac1, Mapk1, Map2k1, Nkg2d, Shp-1, Pak1, Pik3ca, and Vav1. The mRNA levels of Ly49C (p?<?0.001), Ly49I (p?=?0.039), and Nkg2d (p?=?0.038) were significantly downregulated in mPer2 ^{?;?;?/ ?;?;?} mice. Time-dependence of expression profiling was then conducted for four core clock genes (Per1, Bmal1, Clock, Rev-erbα), and six out of these 11 cytotoxic regulation genes (Ly49C, Ly49I, Mapk1, Nkg2d, Shp-1, Pik3ca) in WT and mPer2 ^{?;?;?/ ?;?;?} entrained in light/dark (LD) or dark/dark (DD) cycles. Consistently, circadian oscillations were observed for Per1, Rev-erbα, Ly49C, and Nkg2d in WT mice under LD and DD cycles. However, these rhythmic expressions were either disrupted or dampened in mPer2 ^{?;?;?/ ?;?;?} mice. Comparison of gene expression between WT and mPer2 ^{?;?;?/ ?;?;?} mice showed that mPer2 knockout had systematically downregulated the mRNA expression of two cytotoxicity regulators, Ly49C and Nkg2d. FACS analysis further confirmed that the circadian expression of these genes was not due to the daily difference in cell numbers of NK, NKT, or T cells in bone marrow. Taken together, our results reveal that mPer2 is a critical clock component in modulating circadian rhythms in bone marrow. Furthermore, it implies that Ly49C and Nkg2d are two clock-controlled genes that may play an important role in mediating mPer2-controlled cytotoxic response. (Author correspondence: zsunusa@yahoo.com)     

Altered Entrainment to the Day/Night Cycle Attenuates the Daily Rise in Circulating Corticosterone in the Mouse

The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT_1B receptors, decreasing responsiveness to light. Consequently, 5-HT_1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light∶dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT_1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h) conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH) stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies.     

Photoperiod differentially regulates circadian oscillators in central and peripheral tissues of the Syrian hamster

In many seasonally breeding rodents, reproduction and metabolism are activated by long summer days (LD) and inhibited by short winter days (SD). After several months of SD, animals become refractory to this inhibitory photoperiod and spontaneously revert to LD-like physiology. The suprachiasmatic nuclei (SCN) house the primary circadian oscillator in mammals. Seasonal changes in photic input to this structure control many annual physiological rhythms via SCN-regulated pineal melatonin secretion, which provides an internal endocrine signal representing photoperiod. We compared LD- and SD-housed animals and show that the waveform of SCN expression for three circadian clock genes (Per1, Per2, and Cry2) is modified by photoperiod. In SD-refractory (SD-R) animals, SCN and melatonin rhythms remain locked to SD, reflecting ambient photoperiod, despite LD-like physiology. In peripheral oscillators, Per1 and Dbp rhythms are also modified by photoperiod but, in contrast to the SCN, revert to LD-like, high-amplitude rhythms in SD-R animals. Our data suggest that circadian oscillators in peripheral organs participate in photoperiodic time measurement in seasonal mammals; however, circadian oscillators operate differently in the SCN. The clear dissociation between SCN and peripheral oscillators in refractory animals implicates intermediate factor(s), not directly driven by the SCN or melatonin, in entrainment of peripheral clocks.     

Reorganization of the suprachiasmatic nucleus coding for day length

In mammals, the suprachiasmatic nucleus (SCN), the circadian pacemaker, receives light information via the retina and functions in the entrainment of circadian rhythms and in phasing the seasonal responses of behavioral and physiological functions. To better understand photoperiod-related alterations in the SCN physiology, we analyzed the clock gene expression in the mouse SCN by performing in situ hybridization and real-time monitoring of the mPer1::luc bioluminescence. Under long photoperiod (LP) conditions, the expression rhythms of mPer1 and Bmal1 in the caudal SCN phase-led those in the rostral SCN; further, within the middle SCN, the rhythms in the ventrolateral (VL)-like subdivision advanced compared with those in the dorsomedial (DM)-like subdivision. The mPer1::luc rhythms in the entire coronal slice obtained from the middle SCN exhibited 2 peaks with a wide peak width under LP conditions. Imaging analysis of the mPer1::luc rhythms in several subdivisions of the rostral, middle, caudal, and horizontal SCN revealed wide regional variations in the peak time in the rostral half of the SCN under LP conditions. These variations were not due to alterations in the waveform of a single SCN neuronal rhythm. Our results indicate that LP conditions induce phase changes in the rhythms in multiple regions in the rostral half of the SCN; this leads to different circadian waveforms in the entire SCN, coding for day length.     

Effect of Changing the Light/Dark Schedule, the Time of Onset of the Light or Dark Period, or the Daylength, on Rhythms of Epidermal Cell Proliferation

Rhythms of labeling and mitotic indices were studied in the hindlimb epidermis of the anuran tadpole Rana pipiens under different light/dark (LD) cycles and daylengths in order to examine the role of the various parameters of the lighting regimen in setting the periods of the rhythms and the timing of the cell proliferation peaks. Altering the time of, or inverting, the 12 h light period on a 24 h day resulted in phase shifting of basically bimodal circadian rhythms with peaks in the light and dark. Thus the cell proliferation rhythms were entrained to the LD cycle. These rhythms also entrained to noncircadian schedules since they lengthened on a 15L : 15D cycle and shortened on a 9L : 9D cycle, although the bimodal characteristic of a peak in the light and a peak in the dark remained. Studies of 18L: 6D and 6L : 18D cycles in which either the time of onset of light or dark was changed relative to the 12L: 12D control indicated that the onset of dark may regulate the timing of the labeling index peaks while the onset of light may determine the time of occurrence of mitotic index peaks. Control of the timing of labeling and mitotic index peaks by different parameters of the LD cycle suggests a mechanism for cell cycle regulation by the environmental lighting schedule. Analysis of the rhythms on all the cycles studied suggested that labeling index rhythms equal the length of, or twice the length of, the dark period. Mitotic index rhythms equal the daylfength or a multiple of the length of the dark period.     

A partial hepatectomy results in altered expression of clock-related and cyclic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes

The liver is among the peripheral organs that display a clear circadian rhythmicity. To investigate whether specific pathological conditions affect circadian rhythms in the liver, we examined the expression profiles of the clock-related and glyceraldehyde 3-phosphate dehydrogenase (GADPH) genes following a partial hepatectomy in the mouse. This surgical procedure causes dynamic proliferation of residual hepatocytes and within one day of the operation the hepatectomized mice demonstrated higher expression of both mPer1 and mPer2 genes in the remaining liver tissue when compared to control mice that had undergone a Sham-operation. In contrast, the mCry1 gene in hepatectomized mice displayed a circadian gene expression profile that was similar to the control group. In addition, GAPDH levels, that demonstrated no oscillations in Sham-hepatectomized mice, underwent daily alterations following a partial hepatectomy. These findings suggest that the regenerative state of the liver affects the expression not only of clock-related genes but also of genes that are constitutively expressed under steady state conditions.     

Bimodal clock gene expression in mouse suprachiasmatic nucleus and peripheral tissues under a 7-hour light and 5-hour dark schedule

Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of mPer1 bioluminescence on each side of the SCN, in parallel with maintaining synchronization between the left and right sides of the SCN under an artificial light:dark:light:dark (LDLD) 7:5:7:5 condition. In situ hybridization analysis of mPer1 and mBmal1 mRNA distribution in the SCN showed that in 1 photophase (morning photophase; M) of LDLD, the mPer1 level in the ventrolateral-like (VL-like) subdivision of the SCN was higher than that in the dorsomedial-like (DM-like) subdivision, and this regional distribution pattern was reversed in another photophase (evening photophase; E). In contrast, the mBmal1 level was higher in the DM-like subdivision than in the VL-like subdivision in the M phase, and this distribution changed in the E phase. The prokineticin 2 (PK2) mRNA that encodes an SCN output molecule that is thought to transmit the circadian locomotor rhythms was reduced in both the DM-like and VL-like SCN and did not clearly correlate with the activity under the LDLD condition. The expression of mPer1 and mPer2 in the liver was clearly bimodal, whereas the expressions of other clock genes were not synchronized to the LDLD condition. These results may provide important insights into the mechanism underlying the splitting or bimodal rhythms that may in turn facilitate the understanding of the ability to measure the seasonal day length in mammals.