Darkness during early postnatal development is required for normal circadian patterns in the adult rat.

Early light experience influences the brain during development. Perinatal light exposure has an important effect on the development of the circadian system, although the role of quantity versus quality of light in this process is still unclear. We tested the development of the circadian rhythm of locomotor activity under constant bright light from the day of weaning, of six groups of rats raised under different light conditions during suckling. Results indicated that when rats received daily darkness during suckling (rats reared under constant darkness or light-dark cycles with dim or bright light) became arrhythmic when exposed to continuous bright light after weaning. However, those rats reared in the absence of darkness (constant dim or bright light, or alternating dim and bright light) developed a circadian rhythm, which was stronger and had a shorter period depending on the quantity of light received during suckling. Vasointestinal polypeptide immunoreactivity in the suprachiasmatic nucleus (SCN) was higher in those rats with weaker rhythms. However, no apparent differences among these groups were found in the melanopsin-expressing retinal ganglion cells, which provide the SCN with light input in the photoentrainment process. When bright light was shifted to dim light in three of the groups on day 57 after weaning, all of them generated a circadian rhythm with a longer period in those rats previously arrhythmic. Our results indicate the importance of the amount of light received at the early stages of life in the development of the circadian system and suggest that darkness is needed for the normal development of circadian behaviour.

Social interaction and sex differences influence rat temperature circadian rhythm under LD cycles and constant light.

Circadian rhythms produce an efficient organization of animal behaviour over the 24h day. In some species, social cues have been found to have a role as synchronizers of these rhythms. Here, the influence of social interaction on rat circadian behaviour was investigated, addressing the question of whether cohabitation would produce a delay in the appearance of arrhythmicity under constant light conditions. To this end, the circadian rhythms of male and female rat body temperature were studied for 10days under light-dark conditions, followed by 33days under constant bright light. Half of the animals were maintained in individual cages, whilst the others were maintained in larger cages in groups of three rats of the same sex. Results showed that individual circadian rhythms under 24hour light-dark (LD) cycles were more stable and with higher amplitude in grouped than in isolated animals, and higher in males than in females. During the first days under constant light (LL), the stability of the rhythm was also higher in males than in females, but there were no differences according to the group. Moreover, we did not find significant differences in the time of circadian rhythm loss under LL, since high individual variability was found for this variable. On the other hand, female rats living in isolation showed a delayed acrophase in the circadian rhythm under LD conditions compared with those living in groups. These results suggest that cohabitation increases the internal coherence of circadian behaviour, and could be interpreted as indicating that living in isolation may induce a level of stress that disturbs manifestation of the circadian rhythm, especially in females, which are also more reactive than males to external signals.

Motor activity rhythms of forced desynchronized rats subjected to restricted feeding.

Although light is the strongest zeitgeber for the circadian pacemaker, other stimuli can also produce entrainment. In the rat, periodic restricted feeding (RF) is a weak stimulus that may act as a zeitgeber. We tested the effect of RF on the motor activity rhythms of rats subjected to forced dissociation. In this situation two components, supposed to be related with the ventrolateral and dorsomedial areas of the suprachiasmatic nucleus, are detected in their motor activity. One component is entrained to the external light-dark cycle (Light Dependent Component, LDC) and thus has the same period, while the other has a period longer than 24 h (Non-Light Dependent Component, NLDC). This experiment examined whether RF can act on one or both of these two rhythms. Rats were maintained under the light-dark cycles of 22 h (T22) or 23 h (T23) for 44 days with food available for four hours per day. Afterwards the rats received food ad libitum, to test the effect of the previous RF condition. Results show that RF modifies the manifestation of the two initial rhythms, being this effect stronger under T23 than under T22. However RF does not affect the NLDC period. The results reveal that the animal can manifest simultaneously several rhythmic patterns.

Arterial elasticity measurement in renal transplant patients under anticalcineurin immunosuppression.

INTRODUCTION: Calcineurin inhibitors may be associated with decreased arterial elasticity and increased vascular risk. We measured pulse wave velocity (PWV) in large or small arteries as an index of elasticity. The aim of our study was to determine aortic and radial arterial elasticity in 30 stable kidney transplant patients treated with calcineurin inhibitor immunosuppression. PATIENTS AND METHODS: In stable kidney transplant patients we determined the usual biochemical parameters as well as lipid profiles, 24-hour blood pressure (BP) monitoring (CBPM) using a chronobiological program (Garapa), and PWV with a HDI-PWV CR-2000 monitor. RESULTS: Sixteen patients received cyclosporine (CsA, G-1) and 14 tacrolimus (G-2) immunosuppression. There were no baseline differences regarding age (G-1: 56 +/- 12 years, G-2: 56 +/- 14 years), renal transplant follow-up (G-1: 7 +/- 3 years, G-2: 7.5 +/- 3 years), Systolic BP, pulse pressure or plasma creatinine (G-1: 163 +/- 35 umol/L, G-2: 173 +/- 26 umol/L). Patients in the G-1 showed higher diastolic BP (79 +/- 11 vs 74 +/- 8 mm Hg), greater proteinuria (1.26 +/- 0.4 vs 0.6 +/- 0.2 g/d, P < .05), total cholesterol (5.51 +/- 1.2 mmol/L) and low-density lipoprotein (3.08 +/- 0.3 vs 2.99 +/- 0.3 mmol/L, P = NS). Aortic arterial elasticity was decreased in G-1 patients (10.4 +/- 6 vs 14.3 +/- 2 mL/mm Hg x10, P < .05) as well as that in the radial artery (G-1: 5.52 +/- 1 vs 5.57 +/- 1.2 mL/mm Hg x100, P = NS). Almost 100% of the patients presented normal diurnal BP with high nocturnal BP in a nondipper pattern in both groups. CONCLUSION: Calcineurin immunosuppression may contribute to arterial stiffness in kidney transplant patients. No differences between CsA or tacrolimus were observed in our study. CBPM and PWV are useful tools to evaluate subclinical atherosclerosis in renal transplant patients.

The manifestation of the motor activity circadian rhythm of blinded rats depends on the lighting conditions during lactation.

The purpose of this experiment was to examine the effect of different lighting conditions during lactation on the functioning of the circadian pacemaker in the adult rat in absence of the retinal input. We reared one group of rats under constant light (LL-rats) and the other under constant darkness (DD-rats). After weaning they were placed under light-dark cycles of 24h period for 29 days to eliminate the aftereffects of the previous lighting. All the animals were then binocularly enucleated and motor activity was recorded. Results reveal that, before and after the enucleation, the expression of the circadian rhythm was stronger in DD- than in the LL-rats. Our results indicate that lighting conditions during lactation modify the functioning of the circadian pacemaker.

Effect of light during lactation on the phasic and tonic responses of the rat pacemaker.

The circadian system in mammals generates endogenous circadian rhythms and entrains them to external cycles. Here, we examine whether the lighting conditions under which rats are reared affect the properties of the circadian pacemaker. We maintained three groups of rats under constant darkness (DD-rats), constant bright light (LL-rats) or light-dark cycles of 24 hours (LD-rats) during lactation. We then studied motor activity rhythm under constant light of four intensities, and under seven light-dark cycles with periods ranging between 22 and 27 hours. Results show that neither the tau nor the phase angle to the external cycle differed between groups. Differences were found in the amplitude of the circadian rhythm and in the number of rats that became arrhythmic under LL. We conclude that the light received during lactation affects the strength of the circadian pacemaker and its sensitivity to light.

Métodos de análisis cuantitativo de los registros ambulatorios de presión arterial / Cuantitative analytical methods for evaluating ambulatory blood pressure regristries

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Effect of short light-dark cycles on young and adult TGR(mREN2)27 rats.

Animals placed under short light-dark (LD) cycles show a dissociation of their circadian rhythms. However, this effect has only been studied in Wistar rats and with the motor activity (MA) rhythm. Thus, in the present experiment, we studied in TGR(mREN2)27 (TGR) rats, a strain of hypertensive rats, the effect of a short LD cycle on the circadian rhythms of MA, heart rate (HR), and blood pressure (BP). Our aim was (1) to investigate whether the exposure of TGR rats to a short LD cycle induced a dissociation of their circadian rhythms, (2) to study the effect of short LD cycles on the development of the circadian rhythms of TGR rats, and (3) to compare the effect of short LD cycles on young and adult TGR rats. One group of TGR rats was maintained under LD cycles of 22h periods (group G22). The progress in time of their rhythms was compared to that of TGR rats of the same age that had been kept under LD cycles of 24h periods (group G24). For the third point, the rhythms of a group of 5-week-old TGR rats kept under LD 22h cycles (young rats) were compared to those of a group of 11-week-old TGR rats (adult rats). Results showed that there is a dissociation of the circadian rhythms of all the variables monitored in TGR rats maintained under LD 22h cycles, independent of age. We have also found that group G22 showed a higher increase in BP with age and a higher mortality due to malignant hypertension compared to group G24. Finally, it seems that it is harder for young rats to entrain to short LD cycles than for adult rats, and young rats have a higher mortality due to malignant hypertension than adult rats. In conclusion, we demonstrated that short LD cycles produce a dissociation in the HR, BP, and MA circadian rhythms. The results of this experiment, compared to those previously obtained in Wistar rats, suggest that the light perception, the responses of the circadian system to light, or both are altered in the TGR rats.

Effect of light on the development of the circadian rhythm of motor activity in the mouse.

In previous experiments, we found that rats raised in constant light (LL) manifested a more robust circadian rhythm of motor activity in LL and showed longer phase shifts after a light pulse in constant darkness (DD) than chose raised under constant darkness. In addition, we observed that the effects produced by constant light differed depending on the time of postnatal development in which it was given. These results suggest that both sensitivity to light and the functioning of the circadian pacemaker of the rat could be affected by the environmental conditions experienced during postembryonic development. Thus, the present experiment aimed to study whether postnatal exposure to light could also affect the circadian system of the mouse. Three groups of mice were formed: One group was raised under constant darkness during lactation (DD group), the second under constant light (LL group), and the third under light-dark cycles (LD group). After lactation, the three groups were submitted first to constant light of high intensity, then to LD cycles, and finally to constant darkness. In the DD stage, a light pulse was given. Finally, mice were submitted to constant light of low intensity. We observed that the circadian rhythm of the DD group was more disturbed under constant light than the rhythm of the LL group, and that, when light intensity increased, the period of the rhythm of the DD group lengthened more than that of the LL group. No significant differences among the groups were found in the phase shift induced by the light pulse. Therefore, it appears that DD mice are more sensitive to light than their LL counterparts. However, at present there is no evidence to affirm that the light environment experienced by the mouse during postnatal development affects the circadian pacemaker.

Functioning of the rat circadian system is modified by light applied in critical postnatal days.

Lighting conditions influence biological clocks. The present experiment was designed to test the presence of a critical window of days during the lactation stage of the rat in which light has a decisive role on the development of the circadian system. Rats were exposed to 4, 8, or 12 days of constant light (LL) during the first days of life. Their circadian rhythm was later studied under LL and constant darkness. The response to a light pulse was also examined. Results show that the greater the number of LL days during lactation, the stronger the rhythm under LL and the smaller the phase shift due to the light pulse. These responses are enhanced when rats are exposed to LL days around postnatal day 12. A mathematical model was built to explain the responses of the circadian system with respect to the timing of LL during lactation, and we deduced that between postnatal days 10 to 20 there is a critical period of sensitivity to light; consequently, exposure to LL during this time modifies the circadian organization of the motor activity.

Entrainment of the rat motor activity rhythm: effects of the light-dark cycle and physical exercise.

The circadian system is believed to be composed of a population of oscillators that couple together and generate a single rhythm. If this coupling is not strong enough, the circadian system can be dissociated into two or more groups of oscillators, and this is manifested in a dissociation of the overt rhythm into at least two circadian components. This study aims to examine the influence of factors, such as the difference in impact between T and tau, light intensity, and access to a running wheel, on the distribution of motor activity throughout the light-dark (LD) cycle and the dissociation of the rhythm. Rats were submitted to LD cycles of 23 h (T23) or 25 h. For each such cycle, half the rats were submitted to high light intensity and the other half to low light intensity. For each of these conditions, half the rats were kept in small cages, and the other half were in cages with a running wheel. Rats were maintained first under LD cycles and afterwards under constant darkness (DD). Motor activity was recorded throughout the whole experiment by means of activity meters with infrared beams. Results show that the distribution of motor activity throughout the cycle and the after effects observed in the rhythm under DD depended on light intensity and access to the wheel. Moreover, under T23, some rats showed two simultaneous circadian components whose manifestation also depended on the experimental conditions. The results indicate that the strength of circadian entrainment to LD cycles in the rat depends on three factors: the period length of the LD cycle, light intensity used during the light phase, and access to a running wheel.

Bright light during lactation alters the functioning of the circadian system of adult rats.

To examine the role of light in the maturation of the circadian pacemaker, twelve groups of rats were raised in different conditions of exposure to constant bright light (LL) during lactation: both duration and timing of LL were varied. We studied the motor activity rhythm of the rats after weaning, first under LL and then under constant darkness (DD). In DD, two light pulses [at circadian time 15 (CT15) and CT22] were applied to test the response of the pacemaker. Greater exposure to LL days during lactation increased the number of rhythmic animals and the amplitude of their motor activity rhythm in the LL stage and decreased the phase delay due to the light pulse at CT15. The timing of LL during lactation affected these variables too. Because the response of the adult to light depended on both the number and timing of LL days during lactation, the exposure to light at early stages may influence the development of the circadian system by modifying it structurally or functionally.

Period length of the light-dark cycle influences the growth rate and food intake in mice.

The adaptation of the endogenous rhythm of an organism to external cycles may be critical for the development of physiological processes in which energy is expended. We sought to determine whether growth rate depends on the degree of tuning between the external cycle and the manifestation of the circadian rhythms. To do so, we studied the growth rate and the food intake of mice (seven groups of 20 animals each) kept under symmetric LD cycles with different periods (T) of 21, 22, 23, 24, 25, 26, and 27 h, respectively, for 80 days. The mice were then kept in constant darkness for a further 80 days. Throughout the experiment, motor activity was recorded every 15 min for each mouse by means of an actimeter that used crossed infrared beams. Several variables related to the circadian motor activity rhythm were calculated, and correlated with body weight, food intake, and the efficiency of food for growth (food efficiency) calculated as: 100 x body weight increase/ amount of food intake. Results show that these three variables seem to be influenced by the number of circadian cycles that the animal has experienced, but also, and more significantly, by the degree to which the alpha phase of the individual rhythm and the dark phase of the external cycle coincide. Therefore, circadian rhythms would seem to affect the physiological processes that regulate growth and energy expenditure.

Constant bright light (LL) during lactation in rats prevents arrhythmicity due to LL.

Light has a strong effect on the circadian system. Light-dark (LD) cycles are the main zeitgebers for practically all organisms, and the exposure of animals to constant bright light (LL) alters the manifestation of circadian rhythms. In rats, exposure to LL in adulthood produces an arrhythmic pattern in their motor activity, with a large number of ultradian components. In previous experiments, we found that rats born and kept under LL during lactation develop, after weaning, a circadian rhythm which is maintained for at least a couple of months. Here, we examined motor activity rhythms under LL of two groups of rats which differed in the lighting conditions under which they were kept during lactation: 1) rats kept under LL during lactation (LL-rats), which manifested a circadian rhythm after weaning, and 2) rats kept under constant darkness (DD-rats), which were arrhythmic after weaning. We investigated whether the presence of rhythmicity under LL in LL-rats is a transitory effect or whether it persists throughout most of the life of the rat. Moreover, we examined motor activity rhythms of both groups of rats under different lighting conditions to find out other possible differences in the manifestation of their circadian rhythms. Results showed that there are no differences in the capacity of entrainment of both groups of rats to LD cycles or in the rhythm that rats show under DD. Most of the LL-rats maintained their circadian rhythms for the duration of the experiment (1 year), although we found differences in the rhythms manifested between males and females. We found that most of the LL-males became arrhythmic; consequently, at the end of the experiment, there were no differences in the number of males showing circadian rhythm in the LL- and DD-groups. Most of the females in the LL-group showed a clear circadian rhythm under LL during the entire experiment. Thus, LL during lactation has a protective effect against the disruptive effect of LL on the circadian rhythm, although it is only clearly manifested in females.

Dissociation of the rat motor activity rhythm under T cycles shorter than 24 hours.

Since the suprachiasmatic nuclei (SCN) were identified as the principal mammalian circadian clock, many studies describing their morphology and physiology have been carried out. Today, the multioscillatory nature of the SCN, which explains the dissociation of the circadian rhythms under some experimental conditions, is widely accepted. Here, we study the simultaneous presence of two circadian rhythms in the motor activity of the rat when exposed to symmetric light-dark (LD) cycles shorter than 24 h (T21, T21.5, T22, T22.5, T23, and T23.5). One rhythmic component was entrained by the external LD cycle whereas the other ran free with a period longer than 24 h. The results show that two circadian rhythms were present only when T was shorter than T23, whereas at T23.5 only one entrained component was manifested. The manifestation of the two circadian components depends quantitatively on the period of the external cycle--i.e., the strength of the entrained rhythm increases when the external T is closer to 24 h--whereas that of the nonentrained rhythm decreases. The dissociation of the motor activity rhythm and the gradual appearance of the two components are explained by considering the entrainment of a multioscillatory system as not taking place as a whole but rather in a partial manner, in such a way that some oscillators may entrain but not others. The effect of the entrained oscillators is added to the masking effect of the LD cycles.

Manifestation of circadian rhythm under constant light depends on lighting conditions during lactation.

Adult rats transferred to continuous illumination (LL) show a disruption of circadian rhythms, although the mechanisms underlying this effect are not yet well known. In previous experiments, we found that when rats were born and raised under LL they showed an ultradian pattern during the first 10 days after weaning, but afterward they generated a circadian rhythm that was maintained until adulthood. It was not clear whether this evolution was attributable to the influence of the rhythm of the mother or to the effect of constant light. Here, we have studied the motor activity rhythm of young rats maintained under LL after weaning, taking into account the conditions to which they were exposed during lactation [LL or continuous darkness (DD)]. To check the possible effect of the rhythm of the dam, on the day of delivery some of the dams were blinded, others were subjected to a restricted feeding schedule of 3 h/day, and the others were used as controls. For each rat, the period of the circadian rhythm and the percentage of variance explained by this rhythm were calculated. Results show that all rats maintained under LL during lactation expressed a circadian rhythm in their motor activity. However, rats maintained under DD during lactation did not. This effect did not seem to be dependent on the type of dam. These results suggest that the rhythm of the dams does not affect the manifestation of the rhythm of the pups and that the expression of circadian rhythmicity under constant bright light depends on the lighting conditions under which the animals were maintained during lactation, which could affect the development of the circadian pacemaker or the retina.

Simultaneous manifestation of free-running and entrained rhythms in the rat motor activity explained by a multioscillatory system.

Seven groups of 6 young male rats were exposed for 60 calendar days to symmetrical light/dark cycles with different periods (T = 22, 23, 24, 25, 26, 27, and 28 h), and subsequently to constant darkness for 30 days. During exposure to the light/dark cycles, all the animals in all groups presented a motor activity component that was entrained to the external cycles, although the animals subjected to light/dark cycles with periods shorter than 25 hours also presented a nonentrained circadian component. Moreover, in all cases, the effect of masking was present, manifested as a reactivity to the light-dark transitions and as a reduction of activity induced by light. Our results demonstrate that masking, entrainment, and free-running rhythm can be present at the same time. The simultaneous presence of the light-entrained component and the nonentrained component can be explained in terms of a multioscillatory system, in which some oscillators could be entrained to the light/dark cycles while other oscillators could be nonentrained at the same time. Thus, the present study indicates that the circadian system is not necessarily entirely entrained, and that the degree of entrainment may depend on the number of oscillators involved in generating the entrained rhythm.

Dissociation of motor activity circadian rhythm in rats after exposure to LD cycles of 4-h period.

For > 30 days Wistar rats were subjected to six dark pulses per day (T4 cycles; 3 h light, 1 h dark) to study the possibility of dissociating their motor activity rhythm into distinct circadian components. Rats of both sexes were used, one-half of which were pinealectomized to examine the effect of the pineal gland on the entrainment process. Results show that when rats were maintained under T4 a 4-h rhythm in their motor activity was present. Rats showed anticipatory activity to dark phases, suggesting that the motor activity components are actually entrained to the external light/dark (LD) cycles. When rats were left under constant darkness after T4, some motor activity components coming from the dark phases free ran for several days with different circadian periods. This suggests that the motor activity pattern is generated by several circadian oscillators. Moreover, the free-running components of motor activity after T4 were more evident when T4 was applied after exposure to constant light than after exposure to constant darkness. These results support the hypothesis that the circadian system of the rat is formed by several circadian oscillators, whose degree of coupling depends on light conditions. In constant light, bright light may inhibit internal coupling within the system, making it subsequently more susceptible to the T4 cycles. No differences were observed between pinealectomized and sham-operated animals, although females were more sensitive to T4 cycles than males.

Sound does not entrain the motor activity circadian rhythm of rats.

The effect of sound/silence cycles on the motor activity rhythm of the rat was studied. The daily motor activity pattern was studied in two groups of rats of both sexes kept first under DD and later under LL. One group (16 rats) was subjected to a cycle of sound and silence and the other (8 rats) was used as control. The study was performed under two different light regimes, to consider the possibility that a weak zeitgeber (such as sound might be), would exert an effect on the circadian pacemaker. This effect would depend on the stability of the circadian system, which could be modified by the lighting conditions. Results show no statistically significant differences between the two groups, neither in the motor activity patterns nor in the period of the rhythm. It was concluded that sound is not a zeitgeber for the motor activity rhythm of rats.