Circadian rhythms of locomotor activity in the Lesotho mole-rat, Cryptomys hottentotus subspecies from Sani Pass, South Africa

The Lesotho mole-rat is a social subterranean rodent that occurs at altitude in the Drakensberg mountain range. As a consequence of living permanently underground these animals rarely if ever are exposed to light. The visual system of African mole-rats is particularly regressed whereas the circadian system is proportionately conserved. This study investigated the locomotor activity patterns of 12 Lesotho mole-rats maintained under a range of different lighting regimes. The majority (91.7%) of mole-rats entrained their activity patterns to a LD photoperiod of 12L/12D. The mole-rats displayed a monophasic nocturnal activity preference. Under constant dark (DD) most of the mole-rats (83.3%) showed a free running circadian activity pattern with a tau of 23.8 h to 24.4 h (mean+/-S.E.M.: 24.07 h+/-0.07 h; n=10). The phase of the activity rhythms each mole-rat exerted during the previous LD-cycle did not change when the animals started free-running after being placed in constant conditions. The duration of re-entrainment to a second bout of LD 12:12 amounted to 9.4+/-2.03 days (mean+/-S.E.M., n=10). Eleven mole-rats (91.7%) adjusted their locomotor activity rhythms to an inversed light regime DL 12:12 and displayed significant nocturnal activity preference. The animals required 9.73+/-2.01 days (mean+/-S.E.M., n=11) to adjust to the DL-photoperiod. The Lesotho mole-rat thus possesses a functional circadian clock that responds to a photic zeitgeber.     

Circadian rhythms of locomotor activity in the subterranean Mashona mole rat, Cryptomys darlingi

The Mashona mole rat, Cryptomys darlingi, is a social, subterranean African rodent that is rarely, if ever, exposed to light, and that exhibits a regressed visual system. This study investigated locomotor activity patterns of Mashona mole rats (n=12) under different light cycles. Activity was measured using either infrared captors (n=8) or running wheels (n=4). The mole rats entrained their activity to a standard (LD 12:12) photoperiod. They displayed either a nocturnal or diurnal activity preference with one bout of activity and one bout of rest. Therefore, as a species, the Mashona mole rat did not show a clear nocturnal or diurnal activity preference. When the LD (12:12) light cycle was inversed, the animals switched their activity, too. Under constant dark (DD), most mole rats (73%) showed a free-running circadian activity rhythm, but under constant light (LL), only some (36%) did. The free-run period of the rhythm (tau) ranged from 23.83 to 24.10 h. The remaining animals were arrhythmic. There was large interindividual and intraindividual variations in the rate and extent of entrainment, time of activity preference, and activity patterns. Possible reasons for the observed variations are discussed. It is concluded that the Mashona mole rat has an endogenous activity rhythm which approximates 24 h, that the mole rat can distinguish between light and dark, and that the endogenous clock utilises this photic information as a zeitgeber.     

Effects of restricted food access on diurnal fluctuation of behaviors and biochemical functions in hereditary microphthalmic rats.

The characteristics in circadian rhythms of spontaneous locomotor activity, and some metabolic properties were examined in microphthalmic mutant rats of the Donryu strain under ad lib or restricted food access conditions. The growth of microphthalmic rats was retarded compared to that of normal-sighted rats from the same strain. Under a 12:12-h light:dark (LD) cycle with free access to food, normal-sighted rats showed basically nocturnal patterns of the locomotor activity rhythms, but most of microphthalmic rats manifested free-running rhythms and a few of them showed arrhythmic. When food access was restricted only for 6 h in the light period of the LD cycle, the normal and hereditary blind rats generated gradually new patterns of the locomotor activities in which the animals showed to be more active in the light period. Plasma glucose concentration in normal rats showed a peak after food consumption, but microphthalmic mutants exhibited no periodic changes of the glucose levels. Responses of the biochemical parameters of protein and mineral metabolism to restricted food access in the mutants did not differ from those in normal rats. These results suggest that microphthalmic mutant rats show the free-running circadian rhythm of locomotor activity due to a complete lack of their optic nerve and visual input to the circadian clock, but the mutants maintained the ability to shift their circadian phase induced by restricted food access similar to that in control rats, and also that the mutants have almost normal properties of biochemical and physiological functions except for glucose metabolism.     

Effects of restricted food access on circadian fluctuation of serotonin N-acetyltransferase activities in hereditary microphthalmic rats

The characteristics in diurnal fluctuation of serotonin N-acetyltransferase activity were examined in normal and microphthalmic mutant rats of the Donryu strain under ad lib or restricted feeding conditions. Under a 12:12-h light:dark (12-h LD) cycle with free access to food, normal-sighted rats exhibited typical nocturnal increases in the activity of pineal serotonin N-acetyltransferase, being more than 50-fold higher in the dark period than that in the light period, but hereditary blind rats showed nonperiodic change in the pineal enzyme activity in the average, suggesting that the rhythms in individuals have become free-running, asynchronous. When the subjective night or subjective day of the mutants was discerned by active or inactive in the locomotor activity, the pineal enzyme activities in the mutants increased at the subjective night but depressed at the subjective daytime. When food access was restricted only for 6 h in the light period of the LD cycle, normal rats still showed the nocturnal increases in the pineal enzyme activity, but hereditary blind rats manifested a blunt peak in the activity of the pineal enzyme at eating time in the light period. The results suggest that microphthalmic mutant rats maintain the ability to shift and to synchronize their circadian phases induced by restricted access to food, even if they completely lack their optic nerve and visual input to the circadian clock.     

Individual variability and photic entrainment of circadian rhythms in golden spiny mice

Golden spiny mice are diurnally active in most of their natural habitat. Their diurnal activity is ascribed to non-photic cues: competitive exclusion from the nocturnal niche, or thermoregulatory considerations. Here we studied the entrainment of golden spiny mice to light. In the laboratory, golden spiny mice were primarily nocturnal and displayed an unusual variety of rhythm patterns, with activity bursts occurring during both activity and rest periods. Spontaneous shifts of activity rhythms between light phases were sometimes recorded. In all cases but one, body temperature shifted in parallel with activity. Under DD conditions, the free running period (tau) of all individuals but one was shorter than 24 h, and in all individuals but the same one it was shorter than tau under LL conditions. In response to a 6 h phase delay, all individuals entrained to the new LD cycle in a relatively uniform way. During phase advance four out of the twelve individuals further delayed their activity and body temperature rhythms, and eight individuals advanced their activity rhythm, but the re-entrainment took them over twice as long as to re-entrain to the phase delay. We suggest that the golden spiny mouse is a nocturnal rodent whose circadian system developed the flexibility to be nocturnal or diurnal according to environmental conditions, or a nocturnal rodent in the process of turning diurnal, and that it has low sensitivity to the immediate masking effect of light on activity.     

Nighttime dim light exposure alters the responses of the circadian system

The daily light dark cycle is the most salient entraining factor for the circadian system. However, in modern society, darkness at night is vanishing as light pollution steadily increases. The impact of brighter nights on wild life ecology and human physiology is just now being recognized. In the present study, we tested the possible detrimental effects of dim light exposure on the regulation of circadian rhythms, using CD1 mice housed in light/dim light (LdimL, 300 lux:20 lux) or light/dark (LD, 300 lux:1 lux) conditions. We first examined the expression of clock genes in the suprachiasmatic nucleus (SCN), the locus of the principal brain clock, in the animals of the LD and LdimL groups. Under the entrained condition, there was no difference in PER1 peak expression between the two groups, but at the trough of the PER 1 rhythm, there was an increase in PER1 in the LdimL group, indicating a decrease in the amplitude of the PER1 rhythm. After a brief light exposure (30 min, 300 lux) at night, the light-induced expression of mPer1 and mPer2 genes was attenuated in the SCN of LdimL group. Next, we examined the behavioral rhythms by monitoring wheel-running activity to determine whether the altered responses in the SCN of LdimL group have behavioral consequence. Compared to the LD controls, the LdimL group showed increased daytime activity. After being released into constant darkness, the LdimL group displayed shorter free-running periods. Furthermore, following the light exposure, the phase shifting responses were smaller in the LdimL group. The results indicate that nighttime dim light exposure can cause functional changes of the circadian system, and suggest that altered circadian function could be one of the mechanisms underlying the adverse effects of light pollution on wild life ecology and human physiology.     

Restricted feeding: a nonphotic zeitgeber in the rabbit.

The free-running circadian rhythms of five behavioral functions of the rabbit were masked by unsignalled restricted food access (RF). The rhythms were reorganized immediately, a large part of events being assembled around the end of food availability. In addition to masking a slower process of entrainment was running: a component of anticipatory activity (AA) was established 1-3 h before food access. AA consolidated in continuation of the camouflaged free-running rhythm. The time of AA establishment correlated significantly with the phase angle difference (PAD) between free-running rhythm and RF: it decreased with decreasing positive PAD. With the consolidation of AA, the rhythms had attained a stable phase relation to RF. At the termination of RF, the circadian rhythms started to free-run again, the phase being dependent from that of the preceding RF schedule. The period length of the RF zeitgeber (TRF) had some impact on tau of the circadian rhythm. This aftereffect was most evident after termination of TRF less than 24 h: the free-running rhythm continued for up to 49 days with a tau less than 24 h and turned longer thereafter. The results demonstrate the entrainment of circadian rhythms of the rabbit by RF in addition to masking.     

Locomotor, feeding and melatonin daily rhythms in sharpsnout seabream (Diplodus puntazzo)

Sharpsnout seabream is a marine teleost of increasing interest for Mediterranean aquaculture, but there is still a lack of information regarding its circadian organization. In this study, we have investigated sharpsnout seabream locomotor activity, feeding and plasma melatonin daily rhythms under a 12:12-h LD cycle, as well as the persistence of locomotor activity circadian rhythmicity under constant light (LL) conditions. When submitted to an LD cycle, most sharpsnout seabream displayed a diurnal locomotor pattern, with an average 74% of activity recorded during daytime. However, along the experiment 40% of fish spontaneously changed their locomotor rhythm phasing and became nocturnal. Feeding behaviour, nevertheless, remained strictly diurnal in all cases, with 97% of food demands being made during the light period. Free-running locomotor rhythms were recorded in one third of the fish kept under LL. Daily plasma melatonin levels displayed a rhythmic profile, with low daytime values (111 pg/ml) and high nighttime concentrations (791 pg/ml). Taken together, these results evidence a high degree of plasticity for sharpsnout seabream activity patterns, as well as phasing independence of locomotor and feeding rhythms. Finally, the existence of a well-defined daily rhythm of plasma melatonin was found.     

Splitting of the locomotor activity rhythm in rats by exposure to continuous light

Female rats exposed to low intensities (0.1-1.5 lx) of continuous light (LL), displayed regular estrous cycles and free-running circadian rhythms of locomotor activity. In most rats, as the intensity of LL was increased to greater than 2.0 lx, components within the active portion (alpha) of the locomotor rhythm remained synchronized as the periodicity of the rhythm lengthened. However, in a few rats alpha split into two components; one of which free-ran with a period shorter than 24 h, while the other free-ran with a period longer than 24 h. As soon as the two components became maximally separated they spontaneously rejoined. In most rats, estrous cycles ceased shortly after the intensity of LL was increased to greater than 2.0 lx even though the locomotor activity rhythm retained its unsplit free-running nature. These observations suggest that the multiple oscillators that control the rhythms of locomotor activity and the estrous cycle are normally coupled to one another. In certain intensities of LL, these oscillators uncouple and free-run with different periodicities, a condition which causes estrous cycles to cease and sometimes produces a split locomotor activity rhythm.     

Synchronisation to light and feeding time of circadian rhythms of spawning and locomotor activity in zebrafish

Reproduction in most fish is a seasonal phenomenon, since spawning occurs at a precise moment of the year to ensure maximal survival of the offspring. Nevertheless, fish reproduction cannot be considered an exclusively annual phenomenon, since spawning might also show daily rhythmicity. In this study, we used an automatic programmable egg collector to investigate the existence of circadian spawning and activity rhythms in zebrafish (Danio rerio L.), and their synchronization to different light and feeding cycles. Under 14L:10D, the results showed a diurnal spawning rhythm with an acrophase at ZT3 (lights went on at ZT0). Activity rhythms were also diurnal (74.4% of the total daily activity occurring during daytime), peaking immediately after lights on, in anticipation of spawning. Feeding at night did not change the diurnal spawning rhythm, but altered the daily pattern of activity, whose diurnal percentage dropped to 49.6%. When applying 1 h of darkness at ZT3, fish shifted the time of spawning to ZT7, while 1 h of darkness applied at ZT7 resumed spawning to ZT3. Under continuous light, locomotor activity rhythms persisted with τ = 22.3 h and the spawning rhythm maintained its phase relationship with them, with an acrophase at CT3. In short, these findings revealed the existence of circadian spawning and locomotor rhythms in zebrafish. The two rhythms are in phase with each other and both are synchronized by light, though only locomotion is influenced by feeding time.     

Splitting of the circadian rhythm of body temperature in the golden hamster

The circadian rhythm of hamster locomotor activity “splits” into two distinct circadian components during conditions of constant illumination. To determine if the circadian rhythm of body temperature also splits under these conditions, body temperature and locomotor activity were monitored concurrently in animals housed in constant illumination. Splitting of the body temperature rhythm into two circadian components was observed in animals manifesting split activity rhythms. Concurrent splitting of both rhythms suggests a common mechanism either for the generation or coupling of these rhythms.     

Disturbed sleep/wake rhythms and neuronal cell loss in lateral hypothalamus and retina of mice with a spontaneous deletion in the ubiquitin carboxyl-terminal hydrolase L1 gene

Many neurodegenerative disorders including Parkinson's disease (PD) and Alzheimer's disease (AD) are associated with sleep disturbances with presumably multifactorial etiology. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is involved in the pathophysiology of PD and AD. In the present study, we analyzed locomotor rhythms, orexin A-immunoreaction (Ir) in the lateral hypothalamus (LH) and melanopsin-Ir in the retina of gracile axonal dystrophy (gad) mice with a spontaneous deletion in the Uch-l1 gene. In constant darkness, gad mice showed circadian rhythms in locomotor activity, indicating the integrity of the endogenous circadian rhythm generator. However, gad mice showed an increased activity during subjective day and a decreased number of orexin A-immunoreactive neurons in the LH compared with the wild type (WT). In addition, gad mice showed increased locomotor activity in the light period when kept in a standard photoperiod and entrainment to phase shifts was significantly slower than in WT. Moreover, melanopsin-Ir was significantly reduced in the retina of gad mice, suggesting an impairment of circadian light perception in gad mice.     

Mutual influence of rats having different circadian rhythm of adrenocortical activity

The possibility of a mutual influence between male adult rats having different circadian adrenocortical rhythms was studied under two different lighting conditions. Intact and optic-enucleated rats were housed together in the same cage in various ratios of intact to blinded rats. Twenty-four- or forty-eight-hour patterns of plasma corticosterone levels were determined individually at 4-h intervals. Under diurnal light-dark alternation, the circadian periodicity of the intact rats always synchronized with light, whereas single blinded rats in cages with three intact rats demonstrated free-running rhythms throughout the experimental period of 15 wk. Under constant light the circadian rhythm free-ran in both intact and blinded rats for the first 2 wk. A phase reversal of the rhythm was observed in the intact rats at the end of the 2nd wk, and between 5 and 8 wk after binding in the blinded rats. These results indicate that the circadian adrenocortical rhythms in intact and blinded rats are not entrained to each other, even when the two types of rats live together in the same cage.     

Effects of histamine and alpha-fluoromethylhistidine injections on circadian phase of free-running rhythms

Sequential IP injections of alpha-fluoromethylhistidine at CT-6 and CT-18 elicited transient delay during the injection period in the onset of activity in the free-running circadian rhythms of locomotor and drinking activities. Histamine injection ICV resulted in various changes, permanent or transient, in the circadian phase of the free-running rhythms depending on the subjective time of the injection: it caused transient advance in onset of activity when injected at CT-0, 3, 6, 9, 18 or 21, whereas it caused permanent phase-delay when injected at CT-12 or 15. These permanent effects were plotted in a phase-response curve. Histamine administration at CT-6 and CT-18 resulted in transient, reciprocal changes of almost equivalent amplitude changes to those of histamine depletion at the corresponding subjective times. Although this transient shift may represent a masking effect of the drug, HA may modulate the circadian synchronization mechanism(s) during the advance sensitive phase. Moreover, HA seems to elicit phase-delay by affecting the light-relaying tract.     

Development of circadian rhythms in rats with lesions of serotonergic system

In order to assess the role of the serotonergic system in the development of overt circadian rhythms in the rat, serotonin neurons in the brain were destroyed either by thermocoagulation of the median raphe (MRL) or by an intracerebroventricular injection of the neurotoxin, 5,7-dihydroxytryptamine (DHT). The reductions in serotonin content induced by two manipulations with MRL and DHT were 41% and 100% in the striatum, 40% and 66% in the hypothalamus, and 62% and 88% in the hippocampus, respectively. Neither manipulation eliminated the expression of circadian rhythms in corticosterone (CS) secretion, locomotor activity and drinking behavior, and changed the phase relationship in the overt CS rhythm. Also, 5,7-DHT treatments did not significantly affect the free-running period in locomotor activity. However, the emergence of CS circadian rhythm was delayed for one week in both MRL and DHT groups compared to the intact control ones. These results suggested that a serotonergic system would not be essential for the generation of the endogenous rhythm and the photoentrainment of overt circadian rhythms, but seems to participate in the only development of CS rhythms during the early stage of life.     

PLEIOTROPIC EFFECTS OF CRYPTOCHROMES 1 AND 2 ON FREE-RUNNING AND LIGHT-ENTRAINED MURINE CIRCADIAN RHYTHMS

Cryptochromes function in both light entrainment of circadian rhythms and in a peripheral circadian clock mechanism in Drosophila . Mice have two closely related cryptochrome genes ( mCry1 and mCry2 ). To further understand the roles of mammalian cryptochromes, we bred mice to carry all possible combinations of wild-type and cryptochrome knockout alleles, and tested these mice for free-running and entrained circadian rhythmicity. We find that a single wild-type copy of mCry1 , but not mCry2 , is sufficient for free running circadian rhythmicity; however, these mice show markedly variable free-running periods. Two wild-type copies of either mCry1 or mCry2 are sufficient to establish a stable free-running clock. A subset of mCry1 ? / mCry ? ; mCry2 ? / mCry2 ? mice have a diurnal activity preference, suggesting that cryptochromes function in light-dependent behavioral masking. We also analyzed mice lacking both cryptochromes and carrying the homozygous rd retinal degeneration mutation. These mice have markedly depressed behavioral photoresponses in light-dark conditions, despite having an anatomically intact retinohypothalamic tract and normal expression of melanopsin. These results suggest that, similar to insect cryptochromes, mammalian cryptochromes function pleiotropically in both circadian rhythm generation and in photic entrainment and behavioral responses.     

Pleiotropic effects of cryptochromes 1 and 2 on free-running and light-entrained murine circadian rhythms

Cryptochromes function in both light entrainment of circadian rhythms and in a peripheral circadian clock mechanism in Drosophila. Mice have two closely related cryptochrome genes (mCry1 and mCry2). To further understand the roles of mammalian cryptochromes, we bred mice to carry all possible combinations of wild-type and cryptochrome knockout alleles, and tested these mice for free-running and entrained circadian rhythmicity. We find that a single wild-type copy of mCry1, but not mCry2, is sufficient for free running circadian rhythmicity; however, these mice show markedly variable free-running periods. Two wild-type copies of either mCry1 or mCry2 are sufficient to establish a stable free-running clock. A subset of mCry1-/mCry-; mCry2-/mCry2- mice have a diurnal activity preference, suggesting that cryptochromes function in light-dependent behavioral masking. We also analyzed mice lacking both cryptochromes and carrying the homozygous rd retinal degeneration mutation. These mice have markedly depressed behavioral photoresponses in light-dark conditions, despite having an anatomically intact retinohypothalamic tract and normal expression of melanopsin. These results suggest that, similar to insect cryptochromes, mammalian cryptochromes function pleiotropically in both circadian rhythm generation and in photic entrainment and behavioral responses.     

Effects of restricted feeding schedules on circadian organization in squirrel monkeys

Free running circadian rhythms of motor activity, food-motivated lever-pressing, and either drinking (N = 7) or body temperature (N = 3) were recorded from 10 squirrel monkeys maintained in constant illumination with unlimited access to food. Food availability was then restricted to a single unsignaled 3-hour interval each day. The feeding schedule failed to entrain the activity rhythms of 8 monkeys, which continued to free-run. Drinking was almost completely synchronized by the schedule, while body temperature showed a feeding-induced rise superimposed on a free-running rhythm. Nonreinforced lever-pressing showed both a free-running component and a 24-hour component that anticipated the time of feeding. At the termination of the schedule, all recorded variables showed free-running rhythms, but in 3 animals the initial phase of the postschedule rhythms was advanced by several hours, suggesting relative coordination. Of the remaining 2 animals, one exhibited stable entrainment of all 3 recorded rhythms, while the other appeared to entrain temporarily to the feeding schedule. These results indicate that restricted feeding schedules are only a weak zeitgeber for the circadian pacemaker generating free-running rhythms in the squirrel monkey. Such schedules, however, may entrain a separate circadian system responsible for the timing of food-anticipatory changes in behavior and physiology.     

Constant light housing attenuates circadian rhythms of mPer2 mRNA and mPER2 protein expression in the suprachiasmatic nucleus of mice

Constant light (LL) or constant dark (DD) environmental lighting conditions cause a free-running period and activity reduction in the rodent behavioral circadian rhythm. In order to understand the molecular process underlying behavioral rhythms in LL or DD housing conditions, we examined the circadian profile of mPer2 mRNA and mPER2 in the suprachiasmatic nucleus (SCN), a main oscillator, of free-running mice. The circadian expression rhythm of mPer2 in the SCN was dampened under 7-day LL conditions, whereas that of mPER2 protein was moderately attenuated and its expression peak delayed. The circadian expression of mPer2 and its product was slightly attenuated and advanced by 7-day DD conditions. With arrhythmic behavioral activity caused by long-term LL housing, mPER2 protein lost its rhythmicity in the SCN. On the other hand, LL or DD housing did not affect the mPer2 gene and its product in the cerebral cortex. The present results suggest that mPER2 circadian expression in the SCN corresponds well with behavioral circadian oscillation under LL or DD conditions. Thus, the behavioral circadian rhythm seems to correlate with molecular clock works in the SCN.     

Lesions of the suprachiasmatic nucleus disrupt circadian locomotor rhythms in the mouse

Entrained and free-running rhythms of wheel-running activity were recorded in male BALB/cByJ mice with electrolytic lesions of the suprachiasmatic nucleus (SCN), site of a circadian pacemaker in mammals. Complete ablation of the nucleus abolished the circadian locomotor rhythm; in some cases, wheel-running was synchronized by a light-dark cycle, but the phase relationship of this activity to the cycle was often aberrant. Unilateral lesions or those missing the SCN did not eliminate rhythmicity.