Circadian Adrenocortical Periodicity Changes in Adult Rats Stimulated During Early Development—I. Effect of Prednisolone

The aim of this study was to test the feasibility of manipulating the adrenocortical circadian rhythm in adult rats by early postnatal prednisolone treatment. Prednisolone, when injected at 7-9 or 17-19 days after birth, produced a permanent suppression of the circadian rhythm of the basal levels of plasma 11-OH-corticosteroids and the rhythm of its responsiveness to stress. The administration of prednisolone at age of 2-4 or 12-14 days did not affect the circadian adrenocortical patterns in adults. Evidence was obtained for the existence of two critical periods during early development. Stimulation with prednisolone during these periods caused a profound modification of circadian periodicity in the performance of the pituitary-adrenocortical system. This modification was not related to changes in adrenal cortex ACTH responsiveness and also to altered stress reactivity of the pituitary-adrenocortical system. It was the presumable consequence of a blockage of a regulatory central mechanism initiating circadian variations in the pituitary-adrenocortical function. The existence of two distinct critical periods suggests that some prednisolone-sensitive links of this central pacemaker mechanism mature asynchronously during early postnatal life.     

Circadian Adrenocortical Periodicity Changes in Adult Rats Stimulated During Early Development—II. Effect of Cold Stress

Rats were cooled at different time points of early postnatal development to study the effects of short-term stress on the parameters of the circadian rhythm of the adrenocortical function in adults. It was found that rats stressed at the age of 2-4 or 17-19 days exhibit modified tuning in the circadian periodicity of the adrenocortical function as adults. Cold stimulation during early development does not affect the function of isolated adrenal glands in vitro. It is concluded that the effect of early stress on the adrenocortical rhythmicity in adults is mediated through central regulatory mechanisms. This effect depends on the age when the rat pups are exposed to cold stress.     

Circadian Adrenocortical Periodicity Changes in Adult Rats Stimulated During Early Development—II. Effect of Cold Stress

Rats were cooled at different time points of early postnatal development to study the effects of short-term stress on the parameters of the circadian rhythm of the adrenocortical function in adults. It was found that rats stressed at the age of 2-4 or 17-19 days exhibit modified tuning in the circadian periodicity of the adrenocortical function as adults. Cold stimulation during early development does not affect the function of isolated adrenal glands in vitro. It is concluded that the effect of early stress on the adrenocortical rhythmicity in adults is mediated through central regulatory mechanisms. This effect depends on the age when the rat pups are exposed to cold stress.     

Role of brain noradrenergic mechanisms in disturbing the circadian rhythm of the functioning hypothalamo-hypophyseo-adrenal cortex system in white rats in early ontogeny

The effects of hormone action and disturbance in catecholamine synthesis in the early postnatal ontogenesis on the circadian rhythm in the hypothalamic-hypophysial-adrenocortical system function were compared in the adult albino rat males. Injection of prednisolone on the 17-19th days of life blocked completely the diurnal rhythm of the corticosterone basal level in blood, the rhythm of adrenocortical response to an emotional stressor and to injection of noradrenaline into the brain lateral ventricle in 3-4 month old animals. Injection of an inhibitor of tyrosine hydroxylase, alpha-methyl-p-tyrosine, at the same period resulted in disappearance of the diurnal rhythm of the corticosterone basal level in adult animals, although the rhythm of response to an emotional stressor or injection of noradrenaline into the brain remained unchanged. A conclusion has been reached that disturbances in catecholamine synthesis in the early postnatal period induces long-term changes of predominantly tonic corticosterone secretion, while the hormone action on the circadian rhythm of the corticosterone basal level and stress response is only partly due to changes in noradrenergic regulation of the hypothalamic-hypophysial-adrenocortical system.     

Effect of prednisolone injections in early postnatal ontogeny on the circadian rhythm of corticosteroid function in adult rats

The effect of prednisolone injections during the early postnatal ontogenesis on the diurnal rhythm of corticosteroid function and stress reactivity in adult animals has been studied in rats. The injections of prednisolone to 7--9 days old rats resulted in the subsequent disturbance of diurnal rhythmicity in the suprarenal cortex functioning: the diurnal fluctuations of the content of 11-oxycorticosteroids in the blood plasma are smoothed out; the diurnal changes in the hormonal reaction to the stress stimulation disappear. This disturbance appears to be due to changes in the central mechanisms of the control of hypophysis-suprarenal system. The reactivity of this system per se suffered no changes.     

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.     

Hypothalamic-pituitary-adrenal axis following glucocorticoid prophylaxis against acute mountain sickness.

The pituitary-adrenocortical and adrenomedullary response to high altitude (HA) stress was studied following daily single dose administration of prednisolone as a prophylaxis against altitude-induced acute mountain sickness (AMS). Forty healthy men, randomly divided into two groups of twenty, received placebo or prednisolone 20 mg once a day at 08.00 h for two days prior to induction to HA and during an initial three days stay at an altitude of 3450 m. The AMS score and circulatory levels of ACTH, cortisol, epinephrine and norepinephrine were measured at sea level (SL) and during residency at HA. The sensitivity of the hypothalamic-pituitary-adrenal axis in subjects receiving prednisolone therapy was evaluated at SL and on day four of stay at HA. Administration of prednisolone significantly (p < 0.01) decreased the severity of AMS in all the subjects. The steroid dose used did not inhibit endogenous secretion of ACTH, cortisol, epinephrine or norepinephrine, as HA response to adrenocortical and adrenomedullary hormones was identical in placebo and prednisolone treated subjects. The integrity of the hypothalamic-pituitary-adrenal axis was maintained well in subjects receiving low dose prednisolone therapy. These observations suggest that short-term administration of prednisolone is able to curtail AMS without causing suppression of the hypothalamic-pituitary-adrenal axis.     

Effect of chronic intracerebroventricular infusion of corticotropin-releasing factor on circadian corticosterone rhythm in the rat

The effects of chronic (14 day) intracerebroventricular infusion of various amounts of ovine corticotropin-releasing factor (oCRF) on the circadian blood corticosterone rhythm in male rats were examined. Control (saline-infused) rats showed distinct blood corticosterone rhythms over 48 h with nadirs at 0900 h and peaks at 2100 h on days 6-7 and 13-14. oCRF at 3 pmol/h did not affect the circadian corticosterone rhythm on these days. When oCRF was infused at a rate of 12 pmol/h, blood corticosterone was increased throughout the 48 h periods. A significant circadian rhythm remained at days 6-7, but continuous infusion for an additional 7 days disrupted the rhythm. Higher doses of oCRF (48 and 240 pmol/h) obliterated the rhythm during both periods; the disruption was characterized by an increase in corticosterone during the lights-on period without a substantial change in the evening maximum. Thus, the blood corticosterone concentration was eventually confined within a narrow range, not exceeding the normal circadian peak, over a wide dose range of centrally administered CRF. Significant effects of oCRF on body and adrenal weight were observed only at the two highest doses used. These findings may provide some insight into the state of the hypothalamic-pituitary-adrenal axis in animals exposed to chronic stress and in patients with depression.     

Functional diversities of two activity components of circadian rhythm in genetical splitting mice (CS strain)

CS mice, an inbred strain, showed two distinctive characteristics in the circadian rhythm of locomotor activity: (1) large variation in the freerunning period, and (2) spontaneous rhythm splitting under continuous darkness. In the splitting rhythm there was a positive correlation between the freerunning period of the evening component and the activity time of the morning component. The phase-shifting effect of a 15-min light pulse was examined on the two activity components of the splitting rhythm. There were significant differences in the amount of light-induced phase response between the two components. A light pulse during the late subjective night induced a phase advance shift only in the morning component, while a light pulse during the early subjective night induced a phase delay shift only in the evening component. These results indicate functional diversities of the two activity components in the circadian locomotor rhythm of CS mice, and suggest that the circadian system in CS mice consists of two mutually coupled oscillators which have different circadian periods and different responsiveness to light. The CS mouse is a useful model to explore a genetic background of oscillator coupling in the circadian system of nocturnal rodents. Accepted: 19 November 1998     

Postnatal development of the circadian rhythm of circulating blood cells in rats

In the present study the variations in the leukocyte and erythrocyte counts were investigated in the peripheral blood within the light and darkness periods during the postnatal development of rats. The animals were kept under an artificial illumination regime (12 hours light, 12 hours darkness), and examined at the ages of 7, 14, 21, 28, 35 and 45 days, and in maturity (56 days). Erythrocyte counts exhibited no light-dark rhythm. The circadian rhythm of leukocyte counts developed in male and female laboratory rats in the fourth week of age.     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8-10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Human cone light sensitivity and melatonin rhythms following 24-hour continuous illumination

This study investigates the possibility of an endogenous circadian rhythm in retinal cone function in humans. A full-field cone electroretinogram (ERG) was performed every 2 h for 24 h under continuous rod-saturating ambient white light (53 ± 30 lux; pupils dilated) in nine healthy subjects. Distinct circadian variations were superimposed upon a gradual decrease in cone responsiveness to light, demonstrated most reliably in the implicit times of b-wave and oscillatory potentials, and to a lesser extent in amplitude and a-wave implicit times. After mathematical correction of the linear trend, the cone response was found to be greatest around 20:00 h and least around 06:00 h. The phase of the ERG circadian rhythm was not synchronized with the phase of the salivary melatonin rhythm measured the previous evening. Melatonin levels measured under constant light on the day of ERG assessments were suppressed by 53% on average compared to melatonin profiles obtained previously under near-total darkness in seven participants. The progressive decline in cone responsiveness to light over the 24 h may reflect an adaptation of the cone-driven retinal system to constant light, although the mechanism is unclear. The endogenous rhythm of cone responsiveness to light may be used as an additional index of central or retinal circadian clock time.     

Central nervous system mediation of pituitary adrenal rhythmicity.

Evidence favoring the view that there is a dissociation between the two aspects of pituitary adrenal operation, the stress and the rhythm modes, is as follows 1) it is possible to abolish rhythmic function by anterior hypothalamic lesions without altering the responses to stress; 2) during maturation of the rat CNS, the stress response appears prior to weaning while pituitary-adrenal rhythm appears after weaning; 3) appropriate neuropharmacologic treatment can block circadian rhythm without altering the stress response; 4) responsiveness to ether or immobilization may be independent of the diurnal rhythm; 5) the stress response to a very strong stimulus, such as immobilization, habituates while the circadian rhythm does not. On the other hand there is some evidence in favor of interdependence between the two modes of operation: 1) input channels for stress and rhythm may overlap since strong synchronizers of rhythm, such as food and water restriction and light can become stress stimuli; 2) steroid feedback can alter the timing of the circadian rhythm; 3) circadian variation has been observed in the responses to certain stimuli; 4) neurotransmitters which are prominently implicated in the circadian rhythm also appear involved in the stress response.     

Postnatal Methylmercury Exposure Induces Hyperlocomotor Activity and Cerebellar Oxidative Stress in Mice: Dependence on the Neurodevelopmental Period

During the early postnatal period the central nervous system (CNS) is extremely sensitive to external agents. The present study aims at the investigation of critical phases where methylmercury (MeHg) induces cerebellar toxicity during the suckling period in mice. Animals were treated with daily subcutaneous injections of MeHg (7 mg/kg of body weight) during four different periods (5 days each) at the early postnatal period: postnatal day (PND) 1–5, PND 6–10, PND 11–15, or PND 16–20. A control group was treated with daily subcutaneous injections of a 150 mM NaCl solution (10 ml/kg of body weight). Subjects exposed to MeHg at different postnatal periods were littermate. At PND 35, behavioral tests were performed to evaluate spontaneous locomotor activity in the open field and motor performance in the rotarod task. Biochemical parameters related to oxidative stress (levels of glutathione and thiobarbituric acid reactive substances, as well as glutathione peroxidase and glutathione reductase activity) were evaluated in cerebellum. Hyperlocomotor activity and high levels of cerebellar thiobarbituric acid reactive substances were observed in animals exposed to MeHg during the PND 11–15 or PND 16–20 periods. Cerebellar glutathione reductase activity decreased in MeHg-exposed animals. Cerebellar glutathione peroxidase activity was also decreased after MeHg exposure and the lowest enzymatic activity was found in animals exposed to MeHg during the later days of the suckling period. In addition, low levels of cerebellar glutathione were found in animals exposed to MeHg during the PND 16–20 period. The present results show that the postnatal exposure to MeHg during the second half of the suckling period causes hyperlocomotor activity in mice and point to this phase as a critical developmental stage where mouse cerebellum is a vulnerable target for the neurotoxic and pro-oxidative effects of MeHg.     

Interaction of the retina with suprachiasmatic pacemakers in the control of circadian behavior

The suprachiasmatic nucleus (SCN) is the central circadian pacemaker governing the circadian rhythm of locomotor activity in mammals. The mammalian retina also contains circadian oscillators, but their roles are unknown. To test whether the retina influences circadian rhythms of locomotor behavior, the authors compared the activity of bilaterally enucleated hamsters with the activity of intact controls held in constant darkness (DD). Enucleated hamsters showed a broader range of free-running periods (tau) than did intact hamsters held for the same length of time in DD. This effect was independent of the age at enucleation (on postnatal days 1, 7, or 28). The average tau of intact animals kept in DD from days 7 or 28 was significantly longer than that of intact animals kept in DD from day 1 or any of the enucleated groups. This indicates that early exposure to light-dark cycles lengthens the tau and that the eye is required to maintain this effect even in DD. These data suggest that hypothalamic circadian pacemakers may interact continuously with the retina to determine the tau of locomotor activity. Enucleation caused a large decrease in glial fibrillary acidic protein in the SCN but has no (or slight) effects on calbindin, neuropeptide Y, vasopressin, or vasoactive intestinal polypeptide, which suggests that enucleation does not produce major damage to the SCN, an interpretation that is supported by the fact that enucleated animals retain robust circadian rhythmicity. The presence of an intact retina appears to contribute to system-level circadian organization in mammals perhaps as a consequence of interaction between its circadian oscillators and those in the SCN.     

Periodic fluctuations of the blood pressure and heart contraction rate in premature newborn infants

To study the postnatal development of circadian rhythm of the blood pressure and the heart rate these parameters were monitored automatically during 24-72 hours at 5h intervals. Fifty infants were investigated at the age 1, 2, 3, and 4 weeks. The results were compared with the cosine curves of different periods (1-48 h) by the IBM PC XT. The fluctuations with all mentioned periods including circadian could be determined in each infant, dominating period being of any duration. In traditional approach the expressiveness of periodical fluctuations is evaluated by the amplitude of cosine curve. Application of the criterion has shown that only amplitude values exceeding 8 mm Hg should be taken into account. Hence, the analysis of BP and HR time series has demonstrated that about 45-50% of the infants have noncircadian rhythms, 20-20% have no significant periodical fluctuations and only 25-30% have circadian rhythm.     

Circadian Rhythms in Stomatal Responsiveness to Red and Blue Light

Stomata of many plants have circadian rhythms in responsiveness to environmental cues as well as circadian rhythms in aperture. Stomatal responses to red light and blue light are mediated by photosynthetic photoreceptors; responses to blue light are additionally controlled by a specific blue-light photoreceptor. This paper describes circadian rhythmic aspects of stomatal responsiveness to red and blue light in Vicia faba. Plants were exposed to a repeated light:dark regime of 1.5:2.5 h for a total of 48 h, and because the plants could not entrain to this short light:dark cycle, circadian rhythms were able to "free run" as if in continuous light. The rhythm in the stomatal conductance established during the 1.5-h light periods was caused both by a rhythm in sensitivity to light and by a rhythm in the stomatal conductance established during the preceding 2.5-h dark periods. Both rhythms peaked during the middle of the subjective day. Although the stomatal response to blue light is greater than the response to red light at all times of day, there was no discernible difference in period, phase, or amplitude of the rhythm in sensitivity to the two light qualities. We observed no circadian rhythmicity in net carbon assimilation with the 1.5:2.5 h light regime for either red or blue light. In continuous white light, small rhythmic changes in photosynthetic assimilation were observed, but at relatively high light levels, and these appeared to be attributable largely to changes in internal CO2 availability governed by stomatal conductance.     

Dietary sodium manipulation during critical periods in development sensitize adult offspring to amphetamines

This study examined critical periods in development to determine when offspring were most susceptible to dietary sodium manipulation leading to amphetamine sensitization. Wistar dams (n = 6-8/group) were fed chow containing low (0.12% NaCl; LN), normal (1% NaCl; NN), or high sodium (4% NaCl; HN) during the prenatal or early postnatal period (birth to 5 wk). Offspring were fed normal chow thereafter until testing at 6 mo. Body weight (BW), blood pressure (BP), fluid intake, salt preference, response to amphetamine, open field behavior, plasma adrenocorticotropin hormone (ACTH), plasma corticosterone (Cort), and adrenal gland weight were measured. BW was similar for all offspring. Offspring from the prenatal and postnatal HN group had increased BP, NaCl intake, and salt preference and decreased water intake relative to NN offspring. Prenatal HN offspring had greater BP than postnatal HN offspring. In response to amphetamine, both prenatal and postnatal LN and HN offspring had increased locomotor behavior compared with NN offspring. In a novel open field environment, locomotion was also increased in prenatal and postnatal LN and HN offspring compared with NN offspring. ACTH and Cort levels 30 min after restraint stress and adrenal gland weight measurement were greater in LN and HN offspring compared with NN offspring. These results indicate that early life experience with low- and high-sodium diets, during the prenatal or early postnatal period, is a stress that produces long-term changes in responsiveness to amphetamines and to subsequent stressors.     

Forced Desynchrony of Orcadian Rhythms of Body Temperature and Activity in Rats

The daily rhythm in body temperature is thought to be the result of the direct effects of activity and the effects of an endogenous circadian clock. Forced desynchrony (FD) is a tool used in human circadian rhythm research to disentangle endogenous and activity-related effects on daily rhythms. In the present study, we applied an FD protocol to rats. We subjected 8 rats for 5 days to a 20h forced activity cycle consisting of lOh of forced wakefulness and lOh for rest and sleep. The procedure aimed to introduce a lOh sleep/ lOh wake cycle, which period was different from the endogenous circadian (about 24h) rhythm. Of the variation in the raw body temperature data, 68-77% could be explained by a summation of estimated endogenous circadian cycle and forced activity cycle components of body temperature. Free-running circadian periods of body temperature during FD were similar to free-running periods measured in constant conditions. The applied forced activity cycle reduced clock-related circadian modulation of activity. This reduction of circadian modulation of activity did not affect body temperature. Also, the effects of the forced activity on body temperature were remarkably small.