Effects of Pregnancy and Parturition on Free-Running Circadian Activity Rhythms in the Rat

Alterations in circadian rhythms have previously been associated with estrous and seasonal changes in reproductive state. In the present study we explored the effects of the reproductive events of pregnancy and parturition on free-running circadian activity rhythms in the rat. Free-running rhythms were monitored before mating, during pregnancy, and following parturition and removal of pups. Systematic and long-lasting alterations of the period of the free-running activity rhythm were seen following parturition. The effects of estrous, seasonal, and gestational reproductive states on circadian rhythms may be mediated by the endocrine events which accompany these states.     

Circadian Rhythms of Locomotor Activity in the Nile Tilapia Oreochromis niloticus

Behavioral rhythms of the Nile tilapia were investigated to better characterize its circadian system. To do so, the locomotor activity patterns of both male and female tilapia reared under a 12:12 h light-dark (LD) cycle were studied, as well as in males the existence of endogenous rhythmicity under free-running conditions (DD and 45 min LD pulses). When exposed to an LD cycle, the daily pattern of activity differed between individuals: some fish were diurnal, some nocturnal, and a few displayed an arrhythmic pattern. This variability would be typical of the plastic circadian system of fish. Moreover, reproductive events clearly affected the behavioral rhythms of female tilapia, a mouth-brooder teleost species. Under DD, 50% (6 of 12) of male fish showed circadian rhythms with an average period (τ) of 24.1±0.2 h, whereas under the 45 min LD pulses, 58% (7 of 12) of the fish exhibited free-running activity rhythms with an average τ of 23.9±0.5 h. However, interestingly in this case, activity was always confined to the dark phase. Furthermore, when the LD cycle was reversed, a third of the fish showed gradual resynchronization to the new phase, taking 7–10 days to be completely re-entrained. Taken together, these results suggest the existence of an endogenous circadian oscillator that controls the expression of locomotor activity rhythms in the Nile tilapia, although its anatomical localization remains unknown.     

Interseasonal variation in the circadian rhythms of locomotor activity and temperature selection in sleepy lizards, <Emphasis Type="Italic">Tiliqua rugosa</Emphasis>

Few studies in non-mammalian vertebrates have examined how various effectors of the circadian system interact. To determine if the daily locomotor and behavioural thermoregulatory rhythms of Tiliqua rugosa are both controlled by the circadian system in different seasons, lizards were tested in laboratory thermal gradients in four seasons and in constant darkness. Circadian rhythmicity for both rhythms was present in each season, being most pronounced in spring and summer and least evident in autumn. Most lizards displayed a unimodal locomotor activity pattern across all seasons. However, some individuals presented a bimodal locomotor activity pattern in spring and summer. Seasonal variations in the phase relationships of both rhythms to the light:dark (LD) cycle were demonstrated. No seasonal differences in the free-running period lengths of either rhythm were detected, raising the possibility that a single circadian pacemaker drives both rhythms in this species. Our present results demonstrate that both rhythms are similarly controlled by the circadian system in each season. Although seasonal variations in the thermal preferences of reptiles both in the field and laboratory have previously been well documented, this study is the first to demonstrate circadian rhythms of temperature selection in a reptile species in each season.     

Shortened seasonal photoperiodic cycles accelerate aging of the diurnal and circadian locomotor activity rhythms in a primate

The gray mouse lemur (Microcebus murinus), a prosimian primate, exhibits seasonal rhythms strictly controlled by photoperiodic variations. Previous studies indicated that longevity can be altered by long-term acceleration of seasonal rhythms, providing a model for assessing various aspects of aging. To assess the effect of aging and accelerated aging on the circadian system of this primate, we compared the circadian rhythm of the locomotor activity in adult mouse lemurs (2-4.5 years, n = 9), aged mouse lemurs (5-9 years, n = 10), and adult mouse lemurs that had been exposed from birth to a shortened seasonal photoperiodic cycle (2-4.5 years, n = 7). Compared to adult animals, aged mouse lemurs showed a significant increase in intradaily variability and an advanced activity onset. Aging was characterized by a decrease in amplitude, with both a decrease in nocturnal activity and an increase in daytime activity. When maintained in constant dim red light, aged animals exhibited a shortening of the free-running period (22.8 +/- 0.1 h) compared to adult animals (23.5 +/- 0.1 h). A 3- to 5-year exposure to an accelerated seasonal photoperiodic rhythm ("annual" duration of 5 months) in accelerated mouse lemurs produced disturbances of the locomotor activity rhythm that resembled those of aged mouse lemurs, whether animals were studied in entrained or in free-running conditions. The present study demonstrated a weakened and fragmented locomotor activity rhythm during normal aging in this primate. Increasing the number of expressed seasonal cycles accelerated aging of parameters related to circadian rhythmicity in adult animals.     

Circadian rhythms of locomotor activity and temperature selection in sleepy lizards, <Emphasis Type="Italic">Tiliqua rugosa</Emphasis>

This study examined whether the daily rhythms of locomotor activity and behavioural thermoregulation that have previously been observed in Australian sleepy lizards (Tiliqua rugosa) under field conditions are true circadian rhythms that persist in constant darkness (DD) and whether these rhythms show similar characteristics. Lizards held on laboratory thermal gradients in the Australian spring under the prevailing 12-hour light : dark (LD) cycle for 14 days displayed robust daily rhythms of behavioural thermoregulation and locomotor activity. In the 13-day period of DD that followed LD, most lizards exhibited free-running circadian rhythms of locomotor activity and behavioural thermoregulation. The predominant activity pattern displayed in LD was unimodal and this was retained in DD. While mean levels of skin temperature and locomotor activity were found to decrease from LD to DD, activity duration remained unchanged. The present results demonstrate for the first time that this species’ daily rhythm of locomotor activity is an endogenous circadian rhythm. Our results also demonstrate a close correlation between the circadian activity and thermoregulatory rhythms in this species indicating that the two rhythms are controlled by the same master oscillator(s). Future examination of seasonal aspects of these rhythms, may, however, cause this hypothesis to be modified.     

The circadian timing system and reproduction in mammals.

Circadian systems in a wide variety of organisms all appear to include three basic components: 1) biological oscillators that maintain a self-sustained circadian periodicity in the absence of environmental time cues; 2) input pathways that convey environmental information, especially light cues, that can entrain the circadian oscillations to local time; and 3) output pathways that drive overt circadian rhythms, such as the rhythms of locomotor activity and a variety of endocrine rhythms. In mammals, the circadian system is employed in the regulation of reproductive physiology and behavior in two very important ways. 1) In some species, there is a strong circadian component in the timing of ovulation and reproductive behavior, ensuring that these events will occur at a time when the animal is most likely to encounter a potential mate. 2) Many mammals exhibit seasonal reproductive rhythms that are largely under photoperiod regulation; in these species, the circadian system and the pineal gland are crucial components of the mechanism that is used to measure day length. The rhythm of pineal melatonin secretion is driven by a neural pathway that includes the circadian oscillator(s) in the suprachiasmatic nuclei. Melatonin is secreted at night in all mammals, and the duration of each nocturnal episode of melatonin secretion is inversely related to day length. The pineal melatonin rhythm appears to serve as an internal signal that represents day length and that is capable of regulating a variety of seasonal variations in physiology and behavior.     

Probing the circadian oscillator of a mammal by two-pulse perturbations

When organisms are maintained under constant conditions of light and temperature, their endogenous circadian rhythms free run, manifesting their intrinsic period. The phases of these free-running rhythms can be shifted by stimuli of light, temperature, and drugs. The change from one free-running steady state to another following a perturbation often involves several transient cycles (cycles of free-running rhythm drifting slowly to catch up with the postperturbation steady state). Although the investigation of oscillator kinetics in circadian rhythms of both insects and mammals has revealed that the circadian pacemaker phase shifts instantaneously, the phenomenon of transient cycles has remained an enigma. We probed the phases of the transient cycles in the locomotor activity rhythm of the field mouse Mus booduga, evoked by a single light pulse (LP), using LPs at critically timed phases. The results of our experiments indicate that the transient cycles generated during transition from one steady state to another steady state do not represent the state of the circadian pacemaker (basic oscillator) controlling the locomotor activity rhythm in Mus booduga. (Chronobiology International, 17(2), 129-136, 2000)     

Lithium and bipolar disorder: Impacts from molecular to behavioural circadian rhythms

Bipolar disorder (BD) is a severe and common psychiatric disorder. BD pathogenesis, clinical manifestations and relapses are associated with numerous circadian rhythm abnormalities. Lithium (Li) is the first-line treatment in BD, and its therapeutic action has been related to its ability to alter circadian rhythms. We systematically searched the PubMed database until January 2016, aiming to critically examine published studies investigating direct and indirect effects of Li on circadian rhythms. The results, from the 95 retained studies, indicated that Li: acts directly on the molecular clocks; delays the phase of sleep–wakefulness rhythms and the peak elevation of diurnal cycle body temperature; reduces the amplitude and shortens the duration of activity rhythms and lengthens free-running rhythms. Chronic Li treatment stabilizes free-running activity rhythms, by improving day-to-day rhythmicity of the activity, with effects that appear to be dose related. Pharmacogenetics demonstrate several associations of Li’s response with circadian genes (NR1D1, GSK3β, CRY1, ARNTL, TIM, PER2). Finally, Li acts on the retinal-hypothalamic pineal pathway, influencing light sensitivity and melatonin secretion. Li is a highly investigated chronobiologic agent, and although its chronobiological effects are not completely understood, it seems highly likely that they constitute an inherent component of its therapeutic action in the treatment of mood disorders.     

Pineal and hypothalamic pacemakers: Their role in regulating circadian rhythmicity in Japanese quail

Summary Neither pinealectomy nor administration of melatoninvia silastic capsules had any effect on free-running circadian rhythms of locomotor activity in Japanese quail (Coturnix coturnix japonica). The quail, like the chicken, therefore differs from sparrows and starlings in which pinealectomy dramatically disrupts free-running rhythms. Nevertheless, it seems unlikely that there are fundamental differences in circadian organisation within the Class Aves. The effects of lesions within the supraoptic region (SOR) of the hypothalamus were similar to those which follow the ablation of the suprachiasmatic nuclei (SCN) in sparrows, rats and hamsters, causing the breakdown of free-running rhythms of locomotor activity, but not necessarily an arrhythmic state. The SOR and SCN appear then to have homologous functions in birds and mammals. Differences in circadian organisation, such as the degree of influence of the pineal gland and the particular photoreceptors used for entrainment, may therefore be modifications peripheral to the fundamental components of the circadian clock.Abbreviations POR preoptic area - SOR supraoptic region - SCN suprachiasmatic nuclei     

Photoentrainment, pharmacology, and phase shifts of the circadian rhythm in the rat pineal.

Photoentrainment of circadian rhythms in mammals is mediated by the retinohypothalamic projection to the suprachiasmatic nucleus of the hypothalamus. It should therefore be possible to mimic or block the effects of light on the circadian pacemaker with appropriate pharmacological agents. Such agents and their effects should be useful in identifying the neurotransmitters involved in photoentrainment and their mechanisms of action on the circadian pacemaker. The effects of light on the circadian rhythm in rat pineal serotonin N-acetyltransferase activity are described. Carbachol, a cholinergic agonist, was found to mimic the effects of light on this rhythm, including the acute reduction of nocturnal activity and phase-shifting of the free-running rhythm. These results raise the possibility that acetylcholine is involved in the photoentrainment of mammalian circadian rhythms.     

Food-Entrained Feeding and Locomotor Circadian Rhythms in Rats Under Different Lighting Conditions

It has been suggested that two endogenous timekeeping systems, a light-entrainable pacemaker (LEP) and a food-entrainable pacemaker (FEP), control circadian rhythms. To understand the function and interaction between these two mechanisms better, we studied two behavioral circadian rhythmicities, feeding and locomotor activity, in rats exposed to two conflicting zeitgebers, food restriction and light-dark cycles. For this, the food approaches and wheel-running activity of rats kept under light-dark (LD) 12:12, constant darkness (DD), or constant light (LL) conditions and subjected to different scheduled feeding patterns were continuously recorded. To facilitate comparison of the results obtained under the different lighting conditions, the period of the feeding cycles was set in all three cases about Ih less than the light-entrained or free-running circadian rhythms. The results showed that, depending on the lighting conditions, some components of the feeding and wheel-running circadian rhythms could be entrained by food pulses, while others retained their free-running or light-entrained state. Under LD, food pulses had little influence on the light-entrained feeding and loco-motor rhythms. Under DD, relative coordination between free-running and food-associated rhythms may appear. In both cases, the feeding activity associated with the food pulses could be divided into a prominent phase-dependent peak of activity within the period of food availability and another afterward. Wheel-running activity mainly followed the food pulses. Under LL conditions, the food-entrained activity consisted mainly of feeding and wheel-running anticipatory activity. The results provide new evidence that lighting conditions influence the establishment and persistence of food-entrained circadian rhythms in rats. The existence of two coupled pacemakers, LEP and FEP, or a multioscillatory LEP may both explain our experimental results.     

Food-Entrained Feeding and Locomotor Circadian Rhythms in Rats Under Different Lighting Conditions

It has been suggested that two endogenous timekeeping systems, a light-entrainable pacemaker (LEP) and a food-entrainable pacemaker (FEP), control circadian rhythms. To understand the function and interaction between these two mechanisms better, we studied two behavioral circadian rhythmicities, feeding and locomotor activity, in rats exposed to two conflicting zeitgebers, food restriction and light-dark cycles. For this, the food approaches and wheel-running activity of rats kept under light-dark (LD) 12:12, constant darkness (DD), or constant light (LL) conditions and subjected to different scheduled feeding patterns were continuously recorded. To facilitate comparison of the results obtained under the different lighting conditions, the period of the feeding cycles was set in all three cases about Ih less than the light-entrained or free-running circadian rhythms. The results showed that, depending on the lighting conditions, some components of the feeding and wheel-running circadian rhythms could be entrained by food pulses, while others retained their free-running or light-entrained state. Under LD, food pulses had little influence on the light-entrained feeding and loco-motor rhythms. Under DD, relative coordination between free-running and food-associated rhythms may appear. In both cases, the feeding activity associated with the food pulses could be divided into a prominent phase-dependent peak of activity within the period of food availability and another afterward. Wheel-running activity mainly followed the food pulses. Under LL conditions, the food-entrained activity consisted mainly of feeding and wheel-running anticipatory activity. The results provide new evidence that lighting conditions influence the establishment and persistence of food-entrained circadian rhythms in rats. The existence of two coupled pacemakers, LEP and FEP, or a multioscillatory LEP may both explain our experimental results.     

PROBING THE CIRCADIAN OSCILLATOR OF A MAMMAL BY TWO-PULSE PERTURBATIONS

When organisms are maintained under constant conditions of light and temperature, their endogenous circadian rhythms free run, manifesting their intrinsic period. The phases of these free-running rhythms can be shifted by stimuli of light, temperature, and drugs. The change from one free-running steady state to another following a perturbation often involves several transient cycles (cycles of free-running rhythm drifting slowly to catch up with the postperturbation steady state). Although the investigation of oscillator kinetics in circadian rhythms of both insects and mammals has revealed that the circadian pacemaker phase shifts instantaneously, the phenomenon of transient cycles has remained an enigma. We probed the phases of the transient cycles in the locomotor activity rhythm of the field mouse Mus booduga, evoked by a single light pulse (LP), using LPs at critically timed phases. The results of our experiments indicate that the transient cycles generated during transition from one steady state to another steady state do not represent the state of the circadian pacemaker (basic oscillator) controlling the locomotor activity rhythm in Mus booduga. (Chronobiology International, 17(2), 129–136, 2000)     

Influence of the estrous cycle on clock gene expression in reproductive tissues: Effects of fluctuating ovarian steroid hormone levels

Circadian rhythms in physiology and behavior are known to be influenced by the estrous cycle in female rodents. The clock genes responsible for the generation of circadian oscillations are widely expressed both within the central nervous system and peripheral tissues, including those that comprise the reproductive system. To address whether the estrous cycle affects rhythms of clock gene expression in peripheral tissues, we first examined rhythms of clock gene expression (Per1, Per2, Bmal1) in reproductive (uterus, ovary) and non-reproductive (liver) tissues of cycling rats using quantitative real-time PCR (in vivo) and luminescent recording methods to measure circadian rhythms of PER2 expression in tissue explant cultures from cycling PER2::LUCIFERASE (PER2::LUC) knockin mice (ex vivo). We found significant estrous variations of clock gene expression in all three tissues in vivo, and in the uterus ex vivo. We also found that exogenous application of estrogen and progesterone altered rhythms of PER2::LUC expression in the uterus. In addition, we measured the effects of ovarian steroids on clock gene expression in a human breast cancer cell line (MCF-7 cells) as a model for endocrine cells that contain both the steroid hormone receptors and clock genes. We found that progesterone, but not estrogen, acutely up-regulated Per1, Per2, and Bmal1 expression in MCF-7 cells. Together, our findings demonstrate that the timing of the circadian clock in reproductive tissues is influenced by the estrous cycle and suggest that fluctuating steroid hormone levels may be responsible, in part, through direct effects on the timing of clock gene expression.     

τ Changes After Single Nonphotic Events

Changes in the free-running period of the circadian rhythms of hamsters occur after single nonphotic events such as a 3-h pulse of running induced by being put in a novel wheel. These changes are mostly in the direction of longer periods, and can exceed 0.2 h; the magnitude of the effect depends on the circadian phase of the pulse. The phase response curves for period changes do not match up with those for phase shifts of the rhythms. Data on free-running rhythms after anisomycin injections and after novelty-induced wheel running in τ mutant hamsters support the view that period changes and phase shifts can occur independently of one another.     

Testicular hormones modulate circadian rhythms of the diurnal rodent, Octodon degus

Sex differences have been identified in a variety of circadian rhythms, including free-running rhythms, light-induced phase shifts, sleep patterns, hormonal fluctuations, and rates of reentrainment. In the precocial, diurnal rodent Octodon degus, sex differences have been found in length of free-running rhythm (tau), phase response curves, rates of reentrainment, and in the use of social cues to facilitate reentrainment. Although gonadal hormones primarily organize circadian rhythms during early development, adult gonadal hormones have activational properties on various aspects of circadian rhythms in a number of species examined. Gonadectomy of adult female O. degus did not influence tau, phase angle of entrainment, or activity patterns in previous experiments. The present experiment examined the role of gonadal hormones in adult male degus' circadian wheel-running rhythms. We predicted that male gonadal hormones would have an activational effect on some aspects of circadian rhythms, particularly those in which we see sex differences. Phase angles of entrainment, tau, length of the active period (alpha), maximum and mean activity levels, and activity amplitude were examined for intact and castrated males housed in LD 12:12. Responses to light pulses while housed in constant darkness (DD) were also compared. Castration had no significant effect on tau or light-induced phase shifts. However, castration significantly increased phase angle of entrainment and decreased activity levels. The data indicate that adult gonadal steroids are not responsible for the sex differences in endogenous circadian mechanisms of O. degus (tau, PRC), although they influence activity level and phase angle of entrainment. This is most likely due to masking properties of testosterone, similar to the activity-increasing effects of estrogen during estrus in O. degus females.     

Circadian rhythms of photorefractory siberian hamsters remain responsive to melatonin

Short day lengths increase the duration of nocturnal melatonin (Mel) secretion, which induces the winter phenotype in Siberian hamsters. After several months of continued exposure to short days, hamsters spontaneously revert to the spring-summer phenotype. This transition has been attributed to the development of refractoriness of Mel-binding tissues, including the suprachiasmatic nucleus (SCN), to long-duration Mel signals. The SCN of Siberian hamsters is required for the seasonal response to winter-like Mel signals, and becomes refractory to previously effective long-duration Mel signals restricted to this area. Acute Mel treatment phase shifts circadian locomotor rhythms of photosensitive Siberian hamsters, presumably by affecting circadian oscillators in the SCN. We tested whether seasonal refractoriness of the SCN to long-duration Mel signals also renders the circadian system of Siberian hamsters unresponsive to Mel. Males manifesting free-running circadian rhythms in constant dim red light were injected with Mel or vehicle for 5 days on a 23.5-h T-cycle beginning at circadian time 10. Mel injections caused significantly larger phase advances in activity onset than did the saline vehicle, but the magnitude of phase shifts to Mel did not differ between photorefractory and photosensitive hamsters. Similarly, when entrained to a 16-h light/8-h dark photocycle, photorefractory and photosensitive hamsters did not differ in their response to Mel injected 4 h before the onset of the dark phase. Activity onset in Mel-injected hamsters was masked by light but was revealed to be significantly earlier than in vehicle-injected hamsters upon transfer to constant dim red light. The acute effects of melatonin on circadian behavioral rhythms are preserved in photorefractory hamsters.     

Maternal entrainment of tau mutant hamsters.

Maternal entrainment of the circadian wheel-running activity rhythm was examined in Syrian hamsters heterozygous for a single gene mutation (tau) that affects the free-running period of circadian rhythms. Heterozygous tau pups were born to and raised by wild-type mothers under constant dim light. The pups' wheel-running activity was recorded after weaning on postnatal day 18 or 24. Pups weaned on day 18 had an average free-running period of 21.70 hr, demonstrating that the tau phenotype was fully expressed at this age. Using the activity onset of the postnatal free-running rhythms as a phase reference, we estimated the phase relationships between the pups and their mothers on days 18 and 24. In contrast to results with wild-type pups, the activity rhythms of tau pups were not in phase with the rhythms of their wild-type mothers; that is, activity onsets of mothers and pups did not coincide. The pups did, however, show synchrony among themselves, indicating that they had been exposed to a synchronizing signal sometime during development. It is likely that this synchronizing signal was provided by the mothers, since pups from different litters showed phase relationships similar to those of their mothers. Thus the mothers provided a signal that was sufficient to cause entrainment, despite the 2-hr difference in free-running period between the mothers and pups. Although the pups' activity rhythms appeared to have been entrained by the mothers, they were clearly free-running by postnatal day 18. The mechanism for entrainment is lost during the course of development, despite continued interaction between the mothers and pups.     

Aschoff's rule in retinally degenerate mice

Both retinally degenerate and wildtype mice lengthened the period of their free-running circadian rhythms and reduced the amount of wheel running when exposed to increasing levels of constant illumination, in accordance with Aschoff's rule. Decreased locomotor activity may contribute toward lengthening of period in bright light. However, the known effects of activity on free-running period are small compared to those obtained by changing illumination. This suggests that Aschoff's rule in mice is not dependent on changes in nonphotic input, but results from a direct effect of light on the circadian system. The sparing of Aschoff's rule in retinally degenerate mice is further evidence that circadian photoreception depends on mechanisms other than rods and cones.     

The Effect of Old Age on the Free-Running Period of Circadian Rhythms in Rat

The free-running period is regarded to be an exclusive feature of the endogenous circadian clock. Changes during aging in the free-running period may therefore reflect age-related changes in the internal organization of this clock. However, the literature on alterations in the free-running period in aging is not unequivocal. In the present study, with various confounding factors kept to a minimum, it was found that the free-running periods for active wakefulness, body temperature, and drinking behavior were significantly shorter (by 12-17 min) in old than in young rats. In addition, it was found that the day-to-day stability of the different sleep states was reduced in old rats, whereas that of the drinking rhythm was enhanced. Transient cycles were not observed, nor were there any age-related differences in daily totals of the various sleep-wake states. The amplitudes of the circadian rhythms of active wakefulness, quiet sleep, and temperature were reduced, whereas those of paradoxical sleep and quiet wakefulness remained unchanged.