Circadian photoreception in vertebrates.

To be adaptively useful, internal circadian clocks must be entrained (synchronized) to daily rhythms in the external world. The entraining process adjusts the period of the internal clock to 24 hours and its phase to a value that determines the organism's temporal niche (e.g., diurnal and nocturnal). For most vertebrates, the dominant environmental synchronizer is light. All vertebrates employ specialized photoreceptor cells to perceive synchronizing light signals, but mammals and nonmammalian vertebrates do this differently. Mammals concentrate circadian photoreceptors in the retina, employing rods, cones, and a subset of retinal ganglion cells that are directly photosensitive and contain an unusual photopigment (melanopsin). Nonmammalian vertebrates use photoreceptors located deep in the brain and in the pineal gland as well as others in the retina. Such photoreceptor extravagance is difficult to explain. It seems likely that the different photoreceptor classes in this elaborate sensory system may have specialized roles in entrainment. There is some evidence that this is in fact the case. Furthermore, this nonvisual "circadian" photoreceptive system also controls acute behavioral responses to light (masking), pupillary constriction, and photoperiodic regulation of reproductive state. We review some of the early work on birds and describe new findings that indicate specific roles for retinal rods, cones, and photosensitive retinal ganglion cells in mammals.

Circadian clocks: 50 years on.

Since the first Cold Spring Harbor meeting on "Biological Clocks" in 1960, the field has progressed from the study of a fascinating but esoteric set of phenomena of interest primarily to a relatively small group of prescient biologists to become recognized as defining a centrally important aspect of biological organization. This change is the consequence of a profound increase in understanding of the mechanisms that generate and control circadian rhythmicity, coupled with the realization that circadian temporal organization is an important component of much of what most organisms do. As such, it impinges on human health, agriculture, and biological conservation, as well as on many more basic aspects of biology at every level. Many of the seminal discoveries of the last 47 years were presented and discussed at this exciting meeting.

Cardiovascular tissues contain independent circadian clocks.

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.

Is the food-entrainable circadian oscillator in the digestive system?

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.

Seizures induce phase shifts of rat circadian rhythms.

RATIONALE: Epileptic seizures may alter neuroendocrinological cycles. Light pulses induce phase shifts in circadian rhythms. Using hippocampal-kindled rats to ensure maximal clinical expression, we determined if seizures likewise induce phase shifts. METHODS: We monitored the circadian rhythm of temperature (CRT) with intraperitoneal radiotelemetry in rats (n=21) isolated from time cues and light for 3-week trials. Seizures were triggered with hippocampal electrical stimulation at different circadian phases. Optimized, least-error phase shifts were calculated from preictal and postictal CRTs. Induced seizures were referenced to CRT (t(max)=00:00, 24-h circadian cycle). RESULTS: Phase shifts (individual responses=57) differed across the circadian cycle. Rather than forming a clear phase-response curve, phase shifts were especially variable between 00:00 and 06:00 h. CONCLUSIONS: This study demonstrates that electrically-induced seizures induce advances and delays in CRT in a phase-dependent fashion but in a pattern different from typical light-induced phase shifts. Disorders of circadian regulation may contribute to some of the altered endogenous cycles associated with epilepsy.

Entrainment of the circadian clock in the liver by feeding.

Circadian rhythms of behavior are driven by oscillators in the brain that are coupled to the environmental light cycle. Circadian rhythms of gene expression occur widely in peripheral organs. It is unclear how these multiple rhythms are coupled together to form a coherent system. To study such coupling, we investigated the effects of cycles of food availability (which exert powerful entraining effects on behavior) on the rhythms of gene expression in the liver, lung, and suprachiasmatic nucleus (SCN). We used a transgenic rat model whose tissues express luciferase in vitro. Although rhythmicity in the SCN remained phase-locked to the light-dark cycle, restricted feeding rapidly entrained the liver, shifting its rhythm by 10 hours within 2 days. Our results demonstrate that feeding cycles can entrain the liver independently of the SCN and the light cycle, and they suggest the need to reexamine the mammalian circadian hierarchy. They also raise the possibility that peripheral circadian oscillators like those in the liver may be coupled to the SCN primarily through rhythmic behavior, such as feeding.

Induction of photosensitivity in neonatal rat pineal gland.

Pineal glands removed from neonatal rats at 5, 7, and 9 days of age and explanted into short-term culture, synthesized melatonin when stimulated with norepinephrine (NE); their melatonin synthesis could not be suppressed with bright white light. Dispersed pineal cell cultures or pineal explants prepared from 1-day-old neonates and held in culture for 7 or 9 days also synthesized melatonin when stimulated with NE, but in these cases melatonin synthesis was significantly suppressed by light, demonstrating that the pineals had become photosensitive while in culture. The development of photosensitivity in culture could be partially or completely abolished by the continuous presence of 1 or 10 microm of NE in the culture medium. The pineals of all nonmammalian vertebrates are photoreceptive, whereas those of mammals do not normally respond to light. We hypothesize that a mechanism to suppress pineal photosensitivity by using NE released from sympathetic nerve endings evolved early in the history of mammals.

Modeling the dual pacemaker system of the tau mutant hamster.

Circadian pacemakers in many animals are compound. In rodents, a two-oscillator model of the pacemaker composed of an evening (E) and a morning (M) oscillator has been proposed based on the phenomenon of "splitting" and bimodal activity peaks. The authors describe computer simulations of the pacemaker in tau mutant hamsters viewed as a system of mutually coupled E and M oscillators. These mutant animals exhibit normal type 1 PRCs when released into DD but make a transition to a type 0 PRC when held for many weeks in DD. The two-oscillator model describes particularly well some recent behavioral experiments on these hamsters. The authors sought to determine the relationships between oscillator amplitude, period, PRC, and activity duration through computer simulations. Two complementary approaches proved useful for analyzing weakly coupled oscillator systems. The authors adopted a "distinct oscillators" view when considering the component E and M oscillators and a "system" view when considering the system as a whole. For strongly coupled systems, only the system view is appropriate. The simulations lead the authors to two primary conjectures: (1) the total amplitude of the pacemaker system in tau mutant hamsters is less than in the wild-type animals, and (2) the coupling between the unit E and M oscillators is weakened during continuous exposure of hamsters to DD. As coupling strength decreases, activity duration (alpha) increases due to a greater phase difference between E and M. At the same time, the total amplitude of the system decreases, causing an increase in observable PRC amplitudes. Reduced coupling also increases the relative autonomy of the unit oscillators. The relatively autonomous phase shifts of E and M oscillators can account for both immediate compression and expansion of activity bands in tau mutant and wild-type hamsters subjected to light pulses.

Effects of circadian regulation and rest-activity state on spontaneous seizures in a rat model of limbic epilepsy.

PURPOSE: Circadian regulation via the suprachiasmatic nuclei and rest-activity state may influence expression of limbic seizures. METHODS: Male rats (n = 14) were made epileptic by electrical stimulation of the hippocampus, causing limbic status epilepticus and subsequent seizures. We monitored seizures with intrahippocampal electrodes in 12-12-h light/dark (LD) cycles and in continuous dark (DD). We used radiotelemetry monitoring of activity to measure state and body temperature to determine circadian phase. Cosinor analysis and chi2 tests determined whether seizures occurred rhythmically when plotted by phase. State was defined as inactive or active in 10-min epochs based on whether activity count was below or above a cut-off value validated from video observation. RESULTS: In LD, the peak seizure occurrence was 14:59 h after circadian temperature peak (95% confidence limit, 13:37-16:19). Phasic seizure occurrence persisted in DD for 14:05 (12:31-15:38), p < 0.0001, against uniform mean distribution. In LD, 14,787 epochs contained 1, 268 seizures; seizures preferentially occurred during inactive epochs (965 observed, 878 expected in proportion to the overall distribution of inactive versus active epochs; p < 0.001). In DD, 20, 664 epochs contained 1,609 seizures; seizures had no preferential occurrence by state (999 observed, 1,025 expected; p = 0.16). CONCLUSIONS: Limbic seizures occurred with an endogenous circadian rhythm. Seizures preferentially struck during inactivity during entrainment to the light-dark cycle.

Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau.

The tau mutation is a semidominant autosomal allele that dramatically shortens period length of circadian rhythms in Syrian hamsters. We report the molecular identification of the tau locus using genetically directed representational difference analysis to define a region of conserved synteny in hamsters with both the mouse and human genomes. The tau locus is encoded by casein kinase I epsilon (CKIepsilon), a homolog of the Drosophila circadian gene double-time. In vitro expression and functional studies of wild-type and tau mutant CKIepsilon enzyme reveal that the mutant enzyme has a markedly reduced maximal velocity and autophosphorylation state. In addition, in vitro CKIepsilon can interact with mammalian PERIOD proteins, and the mutant enzyme is deficient in its ability to phosphorylate PERIOD. We conclude that tau is an allele of hamster CKIepsilon and propose a mechanism by which the mutation leads to the observed aberrant circadian phenotype in mutant animals.

Resetting central and peripheral circadian oscillators in transgenic rats.

In multicellular organisms, circadian oscillators are organized into multitissue systems which function as biological clocks that regulate the activities of the organism in relation to environmental cycles and provide an internal temporal framework. To investigate the organization of a mammalian circadian system, we constructed a transgenic rat line in which luciferase is rhythmically expressed under the control of the mouse Per1 promoter. Light emission from cultured suprachiasmatic nuclei (SCN) of these rats was invariably and robustly rhythmic and persisted for up to 32 days in vitro. Liver, lung, and skeletal muscle also expressed circadian rhythms, which damped after two to seven cycles in vitro. In response to advances and delays of the environmental light cycle, the circadian rhythm of light emission from the SCN shifted more rapidly than did the rhythm of locomotor behavior or the rhythms in peripheral tissues. We hypothesize that a self-sustained circadian pacemaker in the SCN entrains circadian oscillators in the periphery to maintain adaptive phase control, which is temporarily lost following large, abrupt shifts in the environmental light cycle.

Circadian rhythm of ERG in Iguana iguana: role of the pineal.

In green iguanas, the pineal controls the circadian rhythm of body temperature but not the rhythm of locomotor activity. As part of a program to investigate the characteristics of this multioscillator circadian system, the authors studied the circadian rhythms of the electroretinographic response (ERG) and asked whether the pineal gland is necessary for the expression of this rhythm. ERGs from a total of 24 anesthetized juvenile iguanas were recorded under four different conditions: (a) complete darkness (DD), (b) dim light-dark cycles (dLD), (c) constant dim light (dLL), and (d) pinealectomized in DD. Results demonstrate that the b-wave component of the ERG shows a very clear circadian rhythm in DD and that this rhythm persists in dLL and entrains to dLD cycles. The ERG response is maximally sensitive during the subjective day. Pinealectomy does not abolish the circadian rhythm in ERG, demonstrating that the oscillator responsible for the ERG rhythm is located elsewhere.

Hypothalamic neuronal loss and altered circadian rhythm of temperature in a rat model of mesial temporal lobe epilepsy.

PURPOSE: Numerous dysfunctions in endogenous hypothalamic function have been associated with mesial temporal lobe epilepsy (MTLE). One endogenous activity is the circadian rhythm of temperature (CRT). In this study we examined whether hypothalamically mediated function is altered in the electrically induced, self-sustained, limbic status epilepticus model of MTLE. We then wished to determine whether there was a structural basis for regulatory alterations. METHODS: We measured CRT with peritoneal temperature telemetry obtained in light-entrained (LD) and in free-running, constant-dark (DD) conditions. CRT from epileptic and controls of normal animals and kindled animals were quantized by fast Fourier transform-nonlinear least squares analysis to determine rhythmic complexity. RESULTS: The circadian component of CRT was preserved in all animals. In DD, CRTs of epileptic animals were more complex than those of normal animals. CRT of kindled animals showed no increased complexity after electrically induced seizures. Neuronal density was decreased in regions of the anterior and posterior hypothalamus but not in the suprachiasmatic nuclei from the epileptic rats. CONCLUSIONS: Alterations in CRT due to the epileptic state were independent of isolated seizures. Altered circadian thermoregulation in epileptic rats corresponded to regional hypothalamic neuronal loss. Structural changes of the hypothalamus may explain alterations in endogenous rhythms in MTLE.

Circadian control of photoreceptor outer segment membrane turnover in mice genetically incapable of melatonin synthesis.

Vertebrate retinal photoreceptors periodically shed membrane from their outer segment distal tips; this material is phagocytosed and degraded by the retinal pigmented epithelium. Both a circadian oscillator and the daily light-dark cycle affect disk shedding, and the effects of both may be mediated by melatonin. To clarify melatonin's role in this process, we asked whether endogenous melatonin is required for rhythmic disk shedding in mouse retina. We analyzed disk shedding in two mouse strains: C3H, which produce melatonin in retina and pineal under the control of circadian oscillators, and C57BL/6, which do not produce melatonin. In cyclic light, both strains exhibited a robust cycle of disk phagosome content in the pigmented epithelium. Peak shedding occurred just after dawn, and trough levels occurred during the middle of the dark phase. In constant darkness, mice exhibited circadian rhythms of locomotor activity, the characteristics of which were similar between strains. Both strains also exhibited rhythmic disk shedding in constant darkness, although amplitudes of the rhythms were damped. Exogenous melatonin delivered once per day failed to reestablish high-amplitude cyclic shedding in mice held in constant darkness. Our results show that, while disk shedding in cyclic light is robustly rhythmic, neither rhythmic production of melatonin nor the circadian oscillator responsible for rhythmic locomotor activity is sufficient to drive high-amplitude rhythmic shedding in constant darkness. More importantly, melatonin is required neither for cyclic changes in the rate of disk shedding in cyclic light, nor for the circadian rhythm of disk shedding in constant darkness.

No evidence for extraocular photoreceptors in the circadian system of the Syrian hamster.

Campbell and Murphy reported recently that 3 h of bright light (13,000 lux) exposure to the area behind the knee caused phase shifts of the circadian rhythms of both body temperature and saliva melatonin in humans. The authors tested the hypothesis that extraocular photoreception is also involved in the circadian system of the Syrian hamster. Hamsters were bilaterally enucleated (eyes removed), and their backs were shaved. Hamsters with stable free-running rhythms in constant darkness were exposed to direct sunlight for 1 or 3 hours during their subjective night. Intact (control) animals showed phase shifts as expected, but the locomotor activity of enucleated animals was unaffected by the exposure to sunlight. The authors also measured the pineal melatonin content after exposure to sunlight. Pineal melatonin content in intact animals declined markedly as expected, but no decline was observed in the enucleated hamsters. The authors conclude that extraocular phototransduction is not capable of shifting the phase of the hamster's locomotor activity rhythm or of suppressing pineal melatonin synthesis.

Serotonin-containing cell bodies in novel brain locations: effects of light input.

Cell bodies staining positively for serotonin (5HT) appear in the suprachiasmatic nuclei (SCN) of hamsters that have been held in constant darkness (DD) for several months but are otherwise untreated. No such cell bodies are found in the SCN of animals that have been bilaterally enucleated for the same amount of time; however, in enucleated hamsters 5HT-containing cell bodies appear in the superior colliculus. These data provide the first indication that changes in sensory input can modulate 5HT levels in cells bodies outside of the raphe nuclei.

Free running circadian rhythms of melatonin, luteinizing hormone, and cortisol in Syrian hamsters bearing the circadian tau mutation.

The tau mutation of Syrian hamsters induces a robust reduction in the period of circadian activity rhythms, from 24 h (wild-type; tau++) to 22 h (heterozygote; tauS+) and 20 h (homozygous mutant, tauSS). Here, we examine the effect of this mutation on circadian rhythms of LH, melatonin, and cortisol in ovariectomized hamsters. Free running circadian rhythms were observed in all three hormones. In each genotype, endocrine rhythms were synchronized with concurrently assessed activity rhythms, suggesting a shared period around 20 h in tauSS, 22 h in tausS+, and 24 h in tau++. Phasing with respect to the activity rhythm was generally similar in tau++ and mutant genotypes. However, melatonin concentrations rose significantly earlier in tauSS than in tau++ animals. Explanted pineals from both genotypes exhibited a similar time course of response to norepinephrine administration, suggesting that the phase advance of melatonin production observed in tauSS in vivo is not a direct effect of the tau mutation within the pinealocyte. The demonstration of reduced period endocrine rhythms in the mutant genotypes extends previous behavioral studies and, together with recent work on rhythmicity in the isolated retina, suggests an ubiquitous influence of the tau mutation on the processes of circadian rhythm generation in this species.

Rhythmic properties of the hamster suprachiasmatic nucleus in vivo.

We recorded multiple unit neural activity [multiunit activity (MUA)] from inside and outside of the suprachiasmatic nucleus (SCN) in freely moving male golden hamsters housed in running-wheel cages under both light/dark cycles and constant darkness. The circadian period of MUA in the SCN matched the period of locomotor activity; it was approximately 24 hr in wild-type and 20 hr in homozygous tau mutant hamsters. The peak of MUA in the SCN always occurred in the middle of the day or, in constant darkness, the subjective day. There were circadian rhythms of MUA outside of the SCN in the ventrolateral thalamic nucleus, the caudate putamen, the accumbens nucleus, the medial septum, the lateral septum, the ventromedial hypothalamic nucleus, the medial preoptic region, and the stria medullaris. These rhythms were out-of-phase with the electrical rhythm in the SCN but in-phase with the rhythm of locomotor activity, peaking during the night or subjective night. In addition to circadian rhythms, there were significant ultradian rhythms present; one, with a period of approximately 80 min, was in antiphase between the SCN and other brain areas, and another, with a period of approximately 14 min, was in-phase between the SCN and other brain areas. The periods of these ultradian rhythms were not significantly different in wild-type and tau mutant hamsters. Of particular interest was the unique phase relationship between the MUA of the bed nucleus of the stria terminalis (BNST) and the SCN; in these two areas both circadian and ultradian components were always in-phase. This suggests that the BNST is strongly coupled to the SCN and may be one of its major output pathways. In addition to circadian and ultradian rhythms of MUA, neural activity both within and outside the SCN was acutely affected by locomotor activity. Whenever a hamster ran on its wheel, MUA in the SCN and the BNST was suppressed, and MUA in other areas was enhanced.