Ontogenetic development of the mammalian circadian system

This review summarizes the current knowledge about the ontogenetic development of the circadian system in mammals. The developmental changes of overt rhythms are discussed, although the main focus of the review is the underlying neuronal and molecular mechanisms. In addition, the review describes ontogenetic development, not only as a process of morpho-functional maturation. The need of repeated adaptations and readaptations due to changing developmental stage and environmental conditions is also considered. The review analyzes mainly rodent data, obtained from the literature and from the author's own studies. Results from other species, including humans, are presented to demonstrate common features and species-dependent differences. The review first describes the development of the suprachiasmatic nuclei as the central pacemaker system and shows that intrinsic circadian rhythms are already generated in the mammalian fetus. As in adult organisms, the period length is different from 24 h and needs continuous correction by environmental periodicities, or zeitgebers. The investigation of the ontogenetic development of the mechanisms of entrainment reveals that, at prenatal and early postnatal stages, non-photic cues deriving from the mother are effective. Light-dark entrainment develops later. At a certain age, both photic and non-photic zeitgebers may act in parallel, even though the respective time information is 12 h out of phase. That leads to a temporary internal desynchronization. Because rhythmic information needs to be transferred to effector organs, the corresponding neural and humoral signalling pathways are also briefly described. Finally, to be able to transform a rhythmic signal into an overt rhythm, the corresponding effector organs must be functionally mature. As many of these organs are able to generate their own intrinsic rhythms, another aspect of the review is dedicated to the development of peripheral oscillators and mechanisms of their entrainment. The latter includes control by the central pacemaker as well as by distinct environmental signals. Ecological aspects of the described developmental changes in the circadian system and some practical consequences are also briefly discussed.     

The Endogenous Melatonin (MT) Signal Facilitates Reentrainment of the Circadian System to Light-Induced Phase Advances by Acting Upon MT2 Receptors

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors. (Author correspondence: M.Pfeffer@em.uni-frankfurt.de)     

Circadian modulation in photic induction of Fos-like immunoreactivity in the suprachiasmatic nucleus cells of diurnal chipmunk,Eutamias asiaticus

Photic induction of immediate early genes including c-fos in the suprachiasmatic nucleus (SCN) has been well demonstrated in the nocturnal rodents. On the other hand, in diurnal rodents, no data is available whether the light can induce c-fos or Fos in the SCN. We therefore examined whether 60 min light exposure induces Fos-like immunoreactivity (Fos-lir) in the SCN cells of diurnal chipmunks and whether the induction is phase dependent, comparing with the results in nocturnal hamsters. We also examined an effect of light on the locomotor activity rhythm under continuous darkness. Fos-lir was induced in the chipmunk SCN. The induction was clearly phase dependent. The light during the subjective night induced strong expression of Fos-lir. This phase dependency is similar to that in hamsters. However, unlike in hamsters, the Fos-lir was induced in some SCN cells of chipmunks exposed to light during the subjective day. In the locomotor rhythm, on the other hand, the light pulse failed to induce the phase shift at phases at which the Fos-lir was induced. These results suggest that the photic induction of Fos-lir in the diurnal chipmunks is gated by a circadian oscillator as well as in the nocturnal hamsters. However, the functional role of Fos protein may be different in the diurnal rodents from in the nocturnal rodents.     

Effects of Prolonged Exposure to Darkness on Circadian Photic Responsiveness in the Mouse

Circadian rhythms in mammals are generated by an endogenous pacemaker but are modulated by environmental cycles, principally the alternation of light and darkness. Although much is known about nonparametric effects of light on the circadian system, little is known about other effects of photic stimulation. In the present study, which consists of a series of five experiments in mice, various manipulations of photic stimulation were used to dissect the mechanisms responsible for a variation in the magnitude of light-induced phase-shifts that results from prolonged exposure to darkness. The results confirmed previous observations that prolonged exposure to darkness causes an increase in the magnitude of phase shifts (both phase advances and phase delays) evoked by discrete light pulses. The results also indicated that the increase in responsiveness results from the lack of exposure to light per se and not from collateral effects of exposure to constant darkness such as the lack of previous entrainment. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system but with a much slower temporal course. The results suggest that circadian dark adaptation may take place at the retinal level, but it is not clear whether it involves a change in the sensitivity or maximal responsiveness of the system.     

Dim Nighttime Illumination Interacts With Parametric Effects of Bright Light to Increase the Stability of Circadian Rhythm Bifurcation in Hamsters

The endogenous circadian pacemaker of mammals is synchronized to the environmental day by the ambient cycle of relative light and dark. The present studies assessed the actions of light in a novel circadian entrainment paradigm where activity rhythms are bifurcated following exposure to a 24-h light:dark:light:dark (LDLD) cycle. Bifurcated entrainment under LDLD reflects the temporal dissociation of component oscillators that comprise the circadian system and is facilitated when daily scotophases are dimly lit rather than completely dark. Although bifurcation can be stably maintained in LDLD, it is quickly reversed under constant conditions. Here the authors examine whether dim scotophase illumination acts to maintain bifurcated entrainment under LDLD through potential interactions with the parametric actions of bright light during the two daily photophases. In three experiments, wheel-running rhythms of Syrian hamsters were bifurcated under LDLD with dimly lit scotophases, and after several weeks, dim scotophase illumination was either retained or extinguished. Additionally, “full” and “skeleton” photophases were employed under LDLD cycles with dimly lit or completely dark scotophases to distinguish parametric from nonparametric effects of bright light. Rhythm bifurcation was more stable in full versus skeleton LDLD cycles. Dim light facilitated the maintenance of bifurcated entrainment under full LDLD cycles but did not prevent the loss of rhythm bifurcation in skeleton LDLD cycles. These studies indicate that parametric actions of bright light maintain the bifurcated entrainment state; that dim scotophase illumination increases the stability of the bifurcated state; and that dim light interacts with the parametric effects of bright light to increase the stability of rhythm bifurcation under full LDLD cycles. A further understanding of the novel actions of dim light may lead to new strategies for understanding, preventing, and treating chronobiological disturbances. (Author correspondence: jevans@msm.edu)     

Investigating the Endogenous Component of Human Circadian Rhythms: A Review of Some Simple Alternatives to Constant Routines

Several types of constant routine are accepted as an important means by which the endogenous component of circadian rhythms can be studied. Nevertheless, they are impracticable to perform and unsuitable for routine use in many individuals. We describe a group of simple methods with which rhythms measured in normal circumstances can be dissociated into the components due to masking and the internal clock. Each method is best suited to a particular type of experimental condition. Results from a variety of protocols are analysed by these and conventional methods to assess the validity of the new methods.     

An Investigation of Natural Genetic Variation in the Circadian System of Drosophila melanogaster: Rhythm Characteristics and Methods of Quantification

Variation in four characteristics of the circadian locomotor activity rhythm was investigated in 24 true-breeding strains of Drosophila melanogaster with a view to establishing methods of phenotypic measurement sufficiently robust to allow subsequent biometric analysis. Between them, these strains formed a representative sample of the genetic variability of a natural population. Period, phase, definition (the degree to which a rhythmic signal was obscured by noise), and rhythm waveform were all found to vary continuously among the strains, although within each strain the rhythm phenotype was remarkably consistent. Each characteristic was found to be sufficiently robust to permit objective measurement using several different methods of quantification, which were then compared.     

An Investigation of Natural Genetic Variation in the Circadian System of Drosophila melanogaster: Rhythm Characteristics and Methods of Quantification

Variation in four characteristics of the circadian locomotor activity rhythm was investigated in 24 true-breeding strains of Drosophila melanogaster with a view to establishing methods of phenotypic measurement sufficiently robust to allow subsequent biometric analysis. Between them, these strains formed a representative sample of the genetic variability of a natural population. Period, phase, definition (the degree to which a rhythmic signal was obscured by noise), and rhythm waveform were all found to vary continuously among the strains, although within each strain the rhythm phenotype was remarkably consistent. Each characteristic was found to be sufficiently robust to permit objective measurement using several different methods of quantification, which were then compared.     

Illuminating the Impact of Habitual Behaviors in Depression

Researchers have hypothesized that habitual behaviors are zeitgebers for the circadian clock. However, few studies have examined the relationship between habitual behaviors and light, the strongest zeitgeber. Depression is an ideal model in which to explore this relationship because depression is a disorder associated with disruptions in circadian biological activity, sleep, and social rhythms (or patterns of habitual behaviors). We hypothesized that individuals with fewer habitual behaviors have less average exposure to light from morning rise time to evening bedtime and that a reduction in light exposure increases the likelihood of depression. Thirty‐nine depressed and 39 never‐depressed participants wore an ambulatory light monitor and completed the Social Rhythm Metric over the course of 2 weeks. Linear and logistic regression techniques were used to calculate regression coefficients, and confidence limits based on the distribution of the product of two normal random variables were computed to test the significance of the mediation effect. Infrequent habitual behaviors were associated with a decrease in average levels of light exposure, and low levels of light increased the likelihood of depression. This mediation effect was partial; the overall number of habitual behaviors had a direct relationship with depression above and beyond the association with light exposure. Longitudinal studies are needed to empirically demonstrate the direction of relationships between each of the variables tested.     

Circadian rhythms in the blowfly, Phormia terraenovae: control of phase within the range of entrainment

ABSTRACT Locomotor activity of individual blowflies, Phormia (= Protophormia) terraenovae R.D. (Diptera, Calliphoridae) was recorded by means of running wheels. A few days after emergence, adult flies were placed in the wheels and exposed to at least two of four light-dark cycles (LD) differing in cycle duration T (LD 11:11, 12:12, 13:13 and 14:14 h). The intensity of illumination was 400 lux in L and 2 lux in D. From the actograms, phase-angle differences were read off between onset of activity and light-on (ψ/_onset), and between end of activity and light-off (ψend)- Within the range of entrainment, ψ changed systematically from negative values in T =22 h to positive values in T =28 h: the mean change in ψ per hour change in T , expressed in degree of the full circadian cycles, was 20^o. Standard deviations of ψ around its mean were computed for ten-cycle intervals; in ψ_onset and in ψ_end standard deviation was minimal when 4ψ was close to zero, and increased steadily with increasing negative or positive ψ -values.     

Entrainment of Circadian Wheel-Running Rhythms of the Northern Brown Bandicoot, Isoodon macrourus, by Daily Restricted Feeding Schedules

Northern brown bandicoots (Isoodon macrourus) were subjected to restricted feeding for 3 h in the middle of the light period of a 14: 10 light/dark cycle and immediately following this in constant dark. When feeding was restricted to the middle of the light period of the light/dark cycle, all bandicoots maintained a nocturnal activity rhythm. In addition to the nocturnal rhythm, a few bandicoots showed meal-anticipatory activity during the light period. In bandicoots that did not show meal-anticipatory activity, diurnal activity was sometimes evident either during or shortly after the daily meal time. The observation of meal-anticipatory activity in some bandicoots suggests that this species may have a mechanism separate from the light-entrainable mechanism that allows the daily anticipation of periodically available food sources. In the next stage of the experiment, which was in constant dark, the meal was presented at the same time of day as it had been in the previous stage. In all bandicoots, the previously light-entrained component of activity free-ran and was eventually affected by the restricted feeding schedule to some degree. Bandicoots showed weak entrainment and relative coordination, suggesting that restricted feeding is a weak zeitgeber in this species. Evidence also suggesting that two separate but coupled pacemakers control the activity rhythms of the bandicoot was that (a) bandicoots simultaneously showed free-running light-entrainable rhythms and meal-entrained anticipatory rhythms; (b) in several bandicoots, the light-entrainable rhythm was phase advanced when it free-ran through the meal time; and (c) in one bandicoot, meal-entrained anticipatory activity was forced away from the meal time when the previously light-entrained component of activity free-ran through it.     

Circadian and Ultradian (12 H) Rhythms of Hepatic Thiosulfate Sulfurtransferase (Rhodanese) Activity in Mice During the First Two Months of Life

Thiosulfate sulfurtransferase (TST) is an important ‘enzyme of protection,’ that accelerates the detoxification of cyanide, converting it into thiocyanate. The TST physiological rhythm was investigated at wks 2, 4, and 8 of post‐natal development (PND) in the mouse. The results revealed a statistically significant gender‐related difference, with the highest activity in females, at all the documented PND stages. In the second week of PND (pre‐weaning time), the circadian rhythm of the enzyme activity was associated with ultradian components. The prominent circadian rhythm (τ=24 h) peaked at the beginning of the light span, more precisely ～3 HALO (Hours After Light Onset). A week after weaning (wk 4 of PND), an impairment of the rhythm, with the peak shifted toward the second half of photophase, was recorded. Four to 6 wks later, about wk 8 of PND, the circadian rhythm pattern was stabilized, with its peak then located at the beginning of the dark span (13 HALO). The obtained results showed a 12 h phase‐shift of the circadian TST peak time during PND, suggesting that the rhythm stabilization is age‐dependent.     

Influence of a Dorsomedial Hypothalamus Lesion on the Circadian Changes in the Enzyme Development and Activity of Intestinal Brushborder Membranes in the Rat

Circadian changes in activities and electrophoretic pattern of the intestinal brushborder enzymes alkaline phosphatase (EC 3.1.3.1), isomaltase (EC 3.2.1.10) and sucrase (EC 3.2.1.26) of normal rats and of rats with a lesion of the dorsomedial hypothalamus (DMH-rats) were studied. In contrast to the rhythm found in normal rats, there are no significant differences between the average enzyme activities in the light or dark period for the DMH-rats. The peak ratio in the electrophoretic pattern for sucrase-isomaltase (SI) over pro-sucrase-isomaltase (pro-SI) changes over 24 hr with higher values during the active (dark) period of the normal rat. In DMH-rats this ratio is much higher, but no significant difference could be demonstrated between the values near the acrophase in these rats.     

Evidence for Circadian Variations of Thyroid Hormone Concentrations and Type II 5'-Iodothyronine Deiodinase Activity in the Rat Central Nervous System

Abstract: The 24-h patterns of tissue thyroid hormone concentrations and type II 5'- and type III 5-iodothyronine deiodinase (5'D-II and 5D-III, respectively) activities were determined at 4-h intervals in different brain regions of male euthyroid rats entrained to a regular 12-h light/12-h dark cycle (lights on at 6:00 a.m.). Activity of 5'D-II, which catalyzes the intracellular conversion of thyroxine (T₄) to 3,3',5-triiodo- l -thyronine (T₃) in the CNS, and the tissue concentrations of both T₄ and T₃ exhibited significant daily variations in all brain regions examined. Periodic regression analysis revealed significant circadian rhythms with amplitudes ranging from 9 to 23% (for T₃) and from 15 to 40% (for T₄ and 5'D-II) of the daily mean value. 5'D-II activity showed a marked nocturnal increase (1.3–2.1-fold vs. daytime basal value), with a maximum at the end of the dark period and a minimum between noon and 4:00 p.m. 5D-III did not exhibit circadian patterns of variation in any of the brain tissues investigated. Our results disclose circadian rhythms of 5'D-II activity and thyroid hormone concentrations in discrete brain regions of rats entrained to a regular 12:12-h light-dark cycle and reveal that, in the rat CNS, T₃ biosynthesis is activated during the dark phase of the photoperiod. For all parameters under investigation, the patterns of variation observed were in part regionally specific, indicating that different regulatory mechanisms may be involved in generating the observed rhythms.     

Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock

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Ontogeny of the Circadian System During Embryogenesis in Rainbow Trout (Oncorhynchus mykyss) and the Effect of Prolonged Exposure to Continuous Illumination on Daily Rhythms of per1, clock,and aanat2 Expression

It is widely held that the development of the circadian system during embryogenesis is important for future survival of an organism. Work in teleosts has been, to date, limited to zebrafish, which provides little insight into the diversity of this system within such a large vertebrate class. In this study, the authors analyzed the diel expression of per1, clock, and aanat2 in unfertilized rainbow trout oocytes and embryos maintained under either a 12:12-h light:dark (LD) cycle or continuous illumination (LL) from fertilization. 24-h profiles in expression were measured at fertilization as well as 8, 21 42, and 57 days postfertilization (dpf). Both per1 and clock were expressed in unfertilized oocytes and all embryonic stages, whereas aanat2 expression was only measureable from 8 dpf. A reduction in both per1 and clock mean expression levels between unfertilized oocytes/0–1 dpf embryos and 8–9 dpf embryos was suggestive of a transition from maternal RNA to endogenous mRNA expression. Although aanat2 expression was not clearly associated with photic conditions, photoperiod treatment did alter the expression of per1 and clock expression/rhythmicity from as early as 8 dpf (per1), which could suggest the presence and functionality of an as yet unidentified “photoreceptor.” As a whole, this work demonstrates that clock systems are present and functional during embryonic development in rainbow trout. Further studies of their expression and regulation will help understand how the environment interacts with embryonic development in the species. (Author correspondence: andrew.davie@stir.ac.uk)