Family coping during critical illness

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus contain the master circadian pacemaker in mammals. On the occasion of the 25th anniversary of the discovery of the SCN as the circadian clock, Charles A. Czeisler and Steven M. Reppert organized a meeting to review milestones and recent developments in the study of the SCN. The discovery that the SCN contain tissue necessary for generation of circadian rhythmicity was established by lesion studies published in 1972. The second phase of study demonstrated unequivocally that the SCN contain an autonomous circadian pacemaker. The principal studies in this period showed the presence of metabolic and electrical activity rhythms in the SCN in vivo and progressed to studies showing that the SCN maintain rhythmicity in vitro, demonstrating that the transplanted SCN can restore circadian function following destruction of the host SCN and ultimately showing that single SCN "clock cells" exhibit independent rhythms in firing rate. The third phase of study, aimed at identifying the biochemical and molecular mechanisms responsible for rhythmicity within the SCN, has begun with the identification of circadian mutants (tau mutant hamsters and Clock mutant mice) and the isolation of the Clock gene. This report traces the important steps forward in our understanding of the suprachiasmatic circadian clock by recounting the information presented at the SCN Silver Anniversary Celebration.     

The tau mutation affects temperature compensation of hamster retinal circadian oscillators

Neural retinas of the golden hamster (Mesocricetus auratus) express circadian rhythms of melatonin synthesis when cultured in constant darkness. Retinas from wild-type hamsters synthesize melatonin with a period close to 24 h, while retinas obtained from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 h, synthesize melatonin with a period close to 20 h. The retinal circadian oscillators of both wild-type and tau mutant hamsters are temperature compensated; however, temperature compensation is adversely affected by the mutation.     

Mutagenesis and behavioral screening for altered circadian activity identifies the mouse mutant, Wheels

The molecular processes underlying the generation of circadian behavior in mammals are virtually unknown. To identify genes that regulate or alter circadian activity rhythms, a mouse mutagenesis program was initiated in conjunction with behavioral screening for alterations in circadian period (tau), a fundamental property of the biological clock. Male mice of the inbred BALB/c strain, treated with the potent mutagen N-ethyl-N-nitrosourea were mated with wild-type hybrids. Wheel-running activity of approximately 300 male progeny was monitored for 6-10 weeks under constant dark (DD) conditions. The tau DD of a single mouse (#187) was longer than the population mean by more than three standard deviations (24.20 vs. 23.32 +/- 0.02 h; mean +/- S.E.M.; n = 277). In addition, mouse #187 exhibited other abnormal phenotypes, including hyperactive bi-directional circling/spinning activity and an abnormal response to light. Heterozygous progeny of the founder mouse, generated from outcrossings with wild-type C57BL/6J mice, displayed lengthened tau DD although approximately 20% of the animals showed no wheel-running activity despite being quite active. Under light:dark conditions, all animals displaying circling behavior that ran in the activity wheels exhibited robust wheel-running activity at lights-ON and these animals also showed enhanced wheel-running activity in constant light conditions. The genetic dissection of the complex behavior associated with this mutation was facilitated by the previously described genetic mapping of the mutant locus causing circling behavior, designated Wheels (Whl), to the subcentromeric portion of mouse chromosome 4. In this report, the same locus is shown to be responsible for the abnormal responses to light and presumably for the altered circadian behavior. Characterization of the gene altered in the novel Whl mutation will contribute to understanding the molecular elements involved in mammalian circadian regulation.     

Circadian rhythms in cultured mammalian retina

Many retinal functions are circadian, but in most instances the location of the clock that drives the rhythm is not known. Cultured neural retinas of the golden hamster (Mesocricetus auratus) exhibited circadian rhythms of melatonin synthesis for at least 5 days at 27 degrees celsius. The rhythms were entrained by light cycles applied in vitro and were free-running in constant darkness. Retinas from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 hours, showed a shortened free-running period of melatonin synthesis. The mammalian retina contains a genetically programmed circadian oscillator that regulates its synthesis of melatonin.     

Role of mouse cryptochrome blue-light photoreceptor in circadian photoresponses

Cryptochromes are photoactive pigments in the eye that have been proposed to function as circadian photopigments. Mice lacking the cryptochrome 2 blue-light photoreceptor gene (mCry2) were tested for circadian clock-related functions. The mutant mice had a lower sensitivity to acute light induction of mPer1 in the suprachiasmatic nucleus (SCN) but exhibited normal circadian oscillations of mPer1 and mCry1 messenger RNA in the SCN. Behaviorally, the mutants had an intrinsic circadian period about 1 hour longer than normal and exhibited high-amplitude phase shifts in response to light pulses administered at circadian time 17. These data are consistent with the hypothesis that CRY2 protein modulates circadian responses in mice and suggest that cryptochromes have a role in circadian photoreception in mammals.     

The role of the intergeniculate leaflet in entrainment of circadian rhythms to a skeleton photoperiod

Mammalian circadian rhythms are synchronized to environmental light/dark (LD) cycles via daily phase resetting of the circadian clock in the suprachiasmatic nucleus (SCN). Photic information is transmitted to the SCN directly from the retina via the retinohypothalamic tract (RHT) and indirectly from the retinorecipient intergeniculate leaflet (IGL) via the geniculohypothalamic tract (GHT). The RHT is thought to be both necessary and sufficient for photic entrainment to standard laboratory light/dark cycles. An obligatory role for the IGL-GHT in photic entrainment has not been demonstrated. Here we show that the IGL is necessary for entrainment of circadian rhythms to a skeleton photoperiod (SPP), an ecologically relevant lighting schedule congruous with light sampling behavior in nocturnal rodents. Rats with bilateral electrolytic IGL lesions entrained normally to lighting cycles consisting of 12 hr of light followed by 12 hr of darkness, but exhibited free-running rhythms when housed under an SPP consisting of two 1 hr light pulses given at times corresponding to dusk and dawn. Despite IGL lesions and other damage to the visual system, the SCN displayed normal sensitivity to the entraining light, as assessed by light-induced Fos immunoreactivity. In addition, all IGL-lesioned, free-running rats showed masking of the body temperature rhythm during the SPP light pulses. These results show that the integrity of the IGL is necessary for entrainment of circadian rhythms to a lighting schedule like that experienced by nocturnal rodents in the natural environment.     

Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain

The period (per) gene, controlling circadian rhythms in Drosophila, is expressed throughout the body in a circadian manner. A homolog of Drosophila per was isolated from rat and designated as rPer2. The rPER2 protein showed 39 and 95% amino acid identity with mPER1 and mPER2 (mouse homologs of per) proteins, respectively. A robust circadian fluctuation of rPer2 mRNA expression was discovered not only in the suprachiasmatic nucleus (SCN) of the hypothalamus but also in other tissues including eye, brain, heart, lung, spleen, liver, and kidney. Furthermore, the peripheral circadian expression of rPer2 mRNA was abolished in SCN-lesioned rats that showed behavioral arrhythmicity. These findings suggest that the multitissue circadian expression of rPer2 mRNA was governed by the mammalian brain clock SCN and also suggest that the rPer2 gene was involved in the circadian rhythm of locomotor behavior in mammals.     

The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering

The dominant late elongated hypocotyl (lhy) mutation of Arabidopsis disrupted circadian clock regulation of gene expression and leaf movements and caused flowering to occur independently of photoperiod. LHY was shown to encode a MYB DNA-binding protein. In wild-type plants, the LHY mRNA showed a circadian pattern of expression with a peak around dawn but in the mutant was expressed constantly at high levels. Increased LHY expression from a transgene caused the endogenous gene to be expressed at a constant level, suggesting that LHY was part of a feedback circuit that regulated its own expression. Thus, constant expression of LHY disrupts several distinct circadian rhythms in Arabidopsis, and LHY may be closely associated with the central oscillator of the circadian clock.     

An improved sample packing device for rapid freeze-trap electron paramagnetic resonance spectroscopy kinetic measurements

Circadian rhythms are generated by the suprachiasmatic nuclei (SCN) and synchronized (entrained) to environmental light-dark cycles by the retinohypothalamic tract (RHT), a direct pathway from the retina to the suprachiasmatic nuclei. In anophthalmic mice, the optic primordia are resorbed between embryonic days 11.5 and 13, before retinal ganglion cells emerge. Thus the retinohypothalamic tract, which is the primary "zeitgeber" for circadian rhythms in sighted animals, never forms, and there is no retinal or photic input to the circadian system. We have used wheel running activity, a highly consistent and reliable measure of circadian rhythmicity in rodents, to establish the properties of endogenous locomotor rhythms of anophthalmic mice. We have identified three subpopulations of anophthalmic mice: a) rhythmic with strong stable circadian period but significantly increased period length; b) rhythmic with unstable circadian period; and c) arrhythmic. Future correlation of locomotor rhythms with properties of the suprachiasmatic nuclei in these mice will clarify the relationship between generation and properties of circadian rhythms and the neuroanatomical, neurochemical, and molecular organization of the circadian clock.     

Photic entrainment of circadian rhythms in rodents

Photic entrainment of circadian rhythms occurs as a consequence of daily, light-induced adjustments in the phase and period of the suprachiasmatic nuclei (SCN) circadian clock. Photic information is acquired by a unique population of retinal photoreceptors, processed by a distinct subset of retinal ganglion cells, and conveyed to the SCN through the retinohypothalamic tract (RHT). RHT neurotransmission is mediated by the release of the excitatory amino acid glutamate and appears to require the activation of both NMDA- and non-NMDA-type glutamate receptors, the expression of immediate early genes (IEGs), and the synthesis and release of nitric oxide. In addition, serotonin appears to regulate the response of the SCN circadian clock to light through postsynaptic 5-HT1A or 5-ht7 receptors, as well as presynaptic 5-HT1B heteroreceptors on RHT terminals.     

Polysialic acid facilitates migration of luteinizing hormone-releasing hormone neurons on vomeronasal axons

Luteinizing hormone-releasing hormone (LHRH) neurons migrate from the olfactory placode to the forebrain in association with vomeronasal nerves (VNN) that express the polysialic acid-rich form of the neural cell adhesion molecule (PSA-NCAM). Two approaches were used to investigate the role of PSA-NCAM: injection of mouse embryos with endoneuraminidase N, followed by the analysis of LHRH cell positions, and examination of LHRH cell positions in mutant mice deficient in the expression of NCAM or the NCAM-180 isoform, which carries nearly all PSA in the brain. The enzymatic removal of PSA at embryonic day 12 significantly inhibited the migration of nearly half of the LHRH neuron population, without affecting the VNN tract itself. Surprisingly, the absence of NCAM or NCAM-180 did not produce this effect. However, a shift in the route of migration, resulting in an excess number of LHRH cells in the accessory olfactory bulb, was observed in the NCAM-180 mutant. Furthermore, it was found that PSA expressed by the proximal VNN and its distal branch leading to the accessory bulb, but not the branch leading to the forebrain, was associated with the NCAM-140 isoform and thus was retained in the NCAM-180 mutant. These results provide two types of evidence that PSA-NCAM plays a role in LHRH cell migration: promotion of cell movement along the VNN tract that is sensitive to acute (enzymatic), but not chronic (genetic), removal of PSA-NCAM, and a preference of a subset of migrating LHRH cells for a PSA-positive axon branch over a PSA-negative branch in the NCAM-180 mutant.     

Immortal time: circadian clock properties of rat suprachiasmatic cell lines

Cell lines derived from the rat suprachiasmatic nucleus (SCN) were screened for circadian clock properties distinctive of the SCN in situ. Immortalized SCN cells generated robust rhythms in uptake of the metabolic marker 2-deoxyglucose and in their content of neurotrophins. The phase relationship between these rhythms in vitro was identical to that exhibited by the SCN in vivo. Transplantation of SCN cell lines, but not mesencephalic or fibroblast lines, restored the circadian activity rhythm in arrhythmic, SCN-lesioned rats. Thus, distinctive oscillator, pacemaker, and clock properties of the SCN are not only retained but also maintained in an appropriate circadian phase relationship by immortalized SCN progenitors.     

Rapid resetting of the mammalian circadian clock

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. We used a two-pulse paradigm, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrated that resetting of the oscillator occurred within 2 hr. This was the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse showed resetting within 2 hr and in addition showed that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal.     

Neuropeptide Y phase shifts the circadian clock in vitro via a Y2 receptor

The suprachiasmatic nuclei (SCN) contain a circadian clock whose activity can be recorded in vitro for several days. This clock can be reset by the application of neuropeptide Y. In this study, we focused on determination of the receptor responsible for neuropeptide Y phase shifts of the hamster circadian clock in vitro. Coronal hypothalamic slices containing the SCN were prepared from Syrian hamsters housed under a 14 h:10 h light:dark cycle. Tissue was bathed in artificial cerebrospinal fluid (ACSF), and the firing rates of individual cells were sampled throughout a 12 h period. Control slices received either no application or application of 200 nl ACSF to the SCN at zeitgeber time 6 (ZT6; ZT12 was defined as the time of lights off). Application of 200 ng/200 nl of neuropeptide Y at ZT6 resulted in a phase advance of 3.4 h. Application of the Y2 receptor agonist, neuropeptide Y (3-36), induced a similar phase advance in the rhythm, while the Y1 receptor agonist, [Leu31, Pro34]-neuropeptide Y had no effect. Pancreatic polypeptide (rat or avian) also had no measurable phase-shifting effect. Neuropeptide Y applied at ZT20 or 22 had no detectable phase-shifting effect. These results suggest that the phase-shifting effects of neuropeptide Y are mediated through a Y2 receptor, similar to results found in vivo.     

Effects of suprachiasmatic lesions on circadian rhythms of blood pressure, heart rate and locomotor activity in the rat

To determine whether the circadian rhythms in blood pressure (BP), heart rate (HR) and locomotor activity are controlled by an internal biological clock located in the suprachiasmatic nucleus (SCN), we continuously measured these parameters in SCN-lesioned rats using a newly developed implantable radiotelemetry device and a computerized data collecting system. Although SCN-lesioned rats showed a weak but significant 24-h periodicity in BP and HR under light-dark (LD) cycles, BP, HR and locomotor activity became completely aperiodic under constant dark (DD) conditions. The amount of locomotor activity was significantly reduced in SCN-lesioned rats compared to that in intact rats. BP tended to be higher in SCN-lesioned rats, but the differences were significant only in the comparison of systolic blood pressure (SBP) under LD and DD (p < 0.05) and of mean blood pressure (MBP) under LD (p < 0.05). HR in SCN-lesioned rats was significantly lower under LD (p < 0.05), but not under DD. The standard deviation and the variation coefficient of MBP, as indices of short-term variability of this parameter, were significantly larger in SCN-lesioned rats than in intact rats, while those of HR and locomotor activity did not differ significantly between SCN-lesioned and intact rats. These results indicate that the SCN is important not only for generating circadian rhythms of BP, HR and locomotor activity, but also for buffering the short-term variability of BP in rats.     

Disruption of retinoid-related orphan receptor beta changes circadian behavior, causes retinal degeneration and leads to vacillans phenotype in mice

The orphan nuclear receptor RORbeta is expressed in areas of the central nervous system which are involved in the processing of sensory information, including spinal cord, thalamus and sensory cerebellar cortices. Additionally, RORbeta localizes to the three principal anatomical components of the mammalian timing system, the suprachiasmatic nuclei, the retina and the pineal gland. RORbeta mRNA levels oscillate in retina and pineal gland with a circadian rhythm that persists in constant darkness. RORbeta-/- mice display a duck-like gait, transient male incapability to sexually reproduce, and a severely disorganized retina that suffers from postnatal degeneration. Consequently, adult RORbeta-/- mice are blind, yet their circadian activity rhythm is still entrained by light-dark cycles. Interestingly, under conditions of constant darkness, RORbeta-/- mice display an extended period of free-running rhythmicity. The overall behavioral phenotype of RORbeta-/- mice, together with the chromosomal localization of the RORbeta gene, suggests a close relationship to the spontaneous mouse mutation vacillans described >40 years ago.