Resynchronization of the Circadian Corticosterone Rhythm After a Light/Dark Shift in Juvenile and Adult Mice

Most of the extensive literature concerning the resynchronization of circadian rhythms after a Zeitgeber shift is devoted to the dependence of resynchronization on the mode of the shift and the strength of the Zeitgeber, as well as on the circadian function investigated. Ontogenetic influences have rarely been investigated. Therefore, we studied the resynchronization of several circadian rhythms in juvenile and adult female laboratory mice. We present here the results concerning the corticosterone rhythm. The daily rhythms were determined as transverse profiles (2-h intervals) before as well as 3, 7, and 14 days after an 8-h phase delay of the light/dark cycle produced by a single prolongation of dark time. The corticosterone concentration in serum was determined radioimmunologically. In the control animals the daily patterns were bimodal, with main maxima at the end of the light time and secondary ones just after lights on. Ontogenetic differences were small. In adult mice the amplitude was slightly increased due to an increase in the maximum values, and the time of highest hormone concentrations was slightly phase advanced. In juvenile mice, a distinct daily pattern with a phase position in relation to the light/dark cycle corresponding to that of control animals was present on the 3rd day after the Zeitgeber shift. The daily mean as well as the minimum and maximum values increased initially and reached the values of control animals during the second week. In adult animals, a pronounced daily rhythm with the normal phase position was present only at the 7th postshift day. The amplitude, daily mean, and maximum values were decreased, and the minimum values were increased. The initial values were not reached even after 2 weeks. The results show that resynchronization was faster in juvenile mice compared with adult mice. As a possible cause for the observed age-related differences, a not yet stabilized phase-coupling between various circadian rhythms is supposed.     

Restricted daytime feeding attenuates reentrainment of the circadian melatonin rhythm after an 8-h phase advance of the light-dark cycle

It is well established that in the absence of photic cues, the circadian rhythms of rodents can be readily phase-shifted and entrained by various nonphotic stimuli that induce increased levels of locomotor activity (i.e., benzodiazepines, a new running wheel, and limited food access). In the presence of an entraining light-dark (LD) cycle, however, the entraining effects of nonphotic stimuli on (parts of) the circadian oscillator are far less clear. Yet, an interesting finding is that appropriately timed exercise after a phase shift can accelerate the entrainment of circadian rhythms to the new LD cycle in both rodents and humans. The present study investigated whether restricted daytime feeding (RF) (1) induces a phase shift of the melatonin rhythm under entrained LD conditions and (2) accelerates resynchronization of circadian rhythms after an 8-h phase advance. Animals were adapted to RF with 2-h food access at the projected time of the new dark onset. Before and at several time points after the 8-h phase advance, nocturnal melatonin profiles were measured in RF animals and animals on ad libitum feeding (AL). In LD-entrained conditions, RF did not cause any significant changes in the nocturnal melatonin profile as compared to AL. Unexpectedly, after the 8-h phase advance, RF animals resynchronized more slowly to the new LD cycle than AL animals. These results indicate that prior entrainment to a nonphotic stimulus such as RF may "phase lock" the circadian oscillator and in that way hinder resynchronization after a phase shift.     

Circadian Activity Rhythms of Laboratory Mice during the Last Weeks of their Life

The aim of the present paper was a detailed analysis of changes of circadian activity rhythms immediately before natural death. Investigations were carried out on individually housed female laboratory mice. Locomotor activity was measured by passive infrared detectors starting with an age of about 75 weeks up to death. At the beginning all animals had pronounced circadian activity rhythms with a main maximum during the dark time and a secondary one just after light-on. As compared to adult mice the amount of activity and the circadian range of oscillation were lower. The main maximum was phase advanced in most of the animals, so that the percentage of activity during dark time accounted for less than 50% of the total 24-h activity. Towards death the amount of activity and the amplitude decreased even more. A circadian rhythm was preserved however as long as the animals were active, although its synchronization with LD-Zeitgeber deteriorated markedly. The phase position of the main maximum became more unstable, leading in some cases to complete uncoupling (free-run with t < 24 h). The secondary maximum in contrast was more stable in its phase and remained synchronized longer. The results show that in old age the mechanisms of synchronization break down earlier than the circadian rhythms. Therefore it seems possible to stabilize the circadian rhythms, e.g. by strengthening of Zeitgebers, which in turn may improve wellbeing and performance.     

Age‐depending effects of narcosis and transmitter implantation on circadian body temperature and activity rhythms in mice

Summary

The effects of narcosis and of telemetry transmitter implantation on core temperature and locomotor activity were investigated in female laboratory mice of various age (3, 15 and 52 weeks old). Following surgery a transient hypothermia was observed. The body temperatures measured 30 min after beginning of narcosis were lower in juvenile and in presenile mice (29.6° ±0.8°C resp. 30.0° ±0.2°C) than in adult animals (31.9° ±0.3°C). The following temperature increase was fastest in juvenile mice. Normal body temperature was reached after 6h 20’ already. Adult and presenile mice needed 8h 30’ resp. 7h 30’. The temperature increase seemed to be independent from activity behaviour of the animals. No substantial differences could be obtained whether the transmitters had room or body temperature before implantation and whether the animals were warmed after surgery by an infrared bulb or not. Probably, the temperature increase depended mainly on the elimination rate of the drug.

Normal circadian core temperature and activity rhythms reappeared on average within 5–6 days in juvenile mice and a little faster in adult (4–5 days) as well as in presenile ones (3–4 days). However, interindividual differences in recovery time were more pronounced than age‐dependent variations.

Circadian core temperature and activity patterns were quite similar in all three age classes investigated. Ontogenetic differences concern, besides changes in daily mean values, mainly a temperature amplitude increasing with age, as well as a high percentage of ultradian components in the activity pattern of juvenile mice compared to older ones.

Telemetry systems are widely used for long‐term measurements of core temperature in laboratory animals (Clement et al., 1989; Refinetti and Menaker, 1992). In our investigations of ontogenetic changes of the circadian temperature and activity rhythms in mice we used an integrated telemetry and data acquisition system (Dataquest, Data Sciences Inc., USA). It comprises implantable wireless transmitters, telemetry receivers, a consolidation matrix and a data acquisition system. The aim of a preliminary study was to analyse the effects of narcosis and transmitter implantation. The time required to recover normal values of body temperature and of locomotor activity as well as normal circadian rhythms was determined, considering also ontogenetic variations.     

Plasma Corticosterone, Motor Activity and Metabolic Circadian Patterns in Streptozotocin-Induced Diabetic Rats

Experiments were conducted in male rats to study the effects of streptozotocin-induced diabetes on circadian rhythms of (a) plasma corticosterone concentrations; (b) motor activity; and (c) metabolic patterns. Animals were entrained to LD cycles of 12: 12 hr and fed ad libitum.

A daily rhythm of plasma corticosterone concentrations was found in controls animals with peak levels at 2400 hr and low values during the remaining hours. This rhythm was statistically confirmed by the cosinor method and had an amplitude of 3.37μg/100 ml and the acrophase at 100 hr. A loss of the normal circadian variation was observed in diabetic animals, with a nadir at the onset of light period and high values throughout the remaining hours; cosinor analysis of these data showed no circadian rhythm, delete and a higher mean level than controls.

As expected, normal rats presented most of their motor activity during the dark period with 80+ of total daily activity; the cosinor method demonstrated a circadian rhythm with an amplitude of 60+ of the mean level and the acrophase at 0852 hr. Both diabetic and control rats showed a similar activity during the light phase, but diabetic animals had less activity than controls during the night and their percentage of total daily activity was similar in both phases of the LD cycle (50+ for each one). With the cosinor method we were able to show the persistence of a circadian rhythm in the motor activity of diabetic rats, but with a mesor and amplitude lower than in controls (amplitude rested at 60+ of the mean level) and its acrophase advanced to 0148 hr.

The metabolic activity pattern of diabetic rats also changed: whereas controls showed a greater metabolic activity during the night (70+ food; 82+ water; 54+ urine; 67+ faeces), diabetics did not show differences between both phases of the LD cycle. Water ingested and urine excreted by the diabetic group were higher than normal during light and dark periods; food consumed and faeces excreted were higher than controls only in the light phase.

These data suggest that alterations in circadian rhythms of plasma corticosterone and motor activity are consecutive to the loss of the feeding circadian pattern, due to polyphagia and polydipsia showed by these animals, which need to extend intakes during the light and dark phases.     

Dark-phase light contamination disrupts circadian rhythms in plasma measures of endocrine physiology and metabolism in rats

Dark-phase light contamination can significantly disrupt chronobiologic rhythms, thereby potentially altering the endocrine physiology and metabolism of experimental animals and influencing the outcome of scientific investigations. We sought to determine whether exposure to low-level light contamination during the dark phase influenced the normally entrained circadian rhythms of various substances in plasma. Male Sprague-Dawley rats (n = 6 per group) were housed in photobiologic light-exposure chambers configured to create 1) a 12:12-h light:dark cycle without dark-phase light contamination (control condition; 123 μW/cm(2), lights on at 0600), 2) experimental exposure to a low level of light during the 12-h dark phase (with 0.02, 0.05, 0.06, or 0.08 μW/cm(2) light at night), or 3) constant bright light (123 μW/cm(2)). Dietary and water intakes were recorded daily. After 2 wk, rats underwent 6 low-volume blood draws at 4-h intervals (beginning at 0400) during both the light and dark phases. Circadian rhythms in dietary and water intake and levels of plasma total fatty acids and lipid fractions remained entrained during exposure to either control conditions or low-intensity light during the dark phase. However, these patterns were disrupted in rats exposed to constant bright light. Circadian patterns of plasma melatonin, glucose, lactic acid, and corticosterone were maintained in all rats except those exposed to constant bright light or the highest level of light during the dark phase. Therefore even minimal light contamination during the dark phase can disrupt normal circadian rhythms of endocrine metabolism and physiology and may alter the outcome of scientific investigations.     

Acute Behavioral Responses to Light and Darkness in Nocturnal Mus musculus and Diurnal Arvicanthis niloticus

The term masking refers to immediate responses to stimuli that override the influence of the circadian timekeeping system on behavior and physiology. Masking by light and darkness plays an important role in shaping an organism's daily pattern of activity. Nocturnal animals generally become more active in response to darkness (positive masking) and less active in response to light (negative masking), and diurnal animals generally have opposite patterns of response. These responses can vary as a function of light intensity as well as time of day. Few studies have directly compared masking in diurnal and nocturnal species, and none have compared rhythms in masking behavior of diurnal and nocturnal species. Here, we assessed masking in nocturnal mice (Mus musculus) and diurnal grass rats (Arvicanthis niloticus). In the first experiment, animals were housed in a 12:12 light-dark (LD) cycle, with dark or light pulses presented at 6 Zeitgeber times (ZTs; with ZT0 = lights on). Light pulses during the dark phase produced negative masking in nocturnal mice but only at ZT14, whereas light pulses resulted in positive masking in diurnal grass rats across the dark phase. In both species, dark pulses had no effect on behavior. In the 2nd experiment, animals were kept in constant darkness or constant light and were presented with light or dark pulses, respectively, at 6 circadian times (CTs). CT0 corresponded to ZT0 of the preceding LD cycle. Rhythms in masking responses to light differed between species; responses were evident at all CTs in grass rats but only at CT14 in mice. Responses to darkness were observed only in mice, in which there was a significant increase in activity at CT 22. In the 3rd experiment, animals were kept on a 3.5:3.5-h LD cycle. Surprisingly, masking was evident only in grass rats. In mice, levels of activity during the light and dark phases of the 7-h cycle did not differ, even though the same animals had responded to discrete photic stimuli in the first 2 experiments. The results of the 3 experiments are discussed in terms of their methodological implications and for the insight they offer into the mechanisms and evolution of diurnality.     

Lesions of the Intergeniculate Leaflet Lead to a Reorganization in Circadian Regulation and a Reversal in Masking Responses to Photic Stimuli in the Nile Grass Rat

Light influences the daily patterning of behavior by entraining circadian rhythms and through its acute effects on activity levels (masking). Mechanisms of entrainment are quite similar across species, but masking can be very different. Specifically, in diurnal species, light generally increases locomotor activity (positive masking), and in nocturnal ones, it generally suppresses it (negative masking). The intergeniculate leaflet (IGL), a subdivision of the lateral geniculate complex, receives direct retinal input and is reciprocally connected with the primary circadian clock, the suprachiasmatic nucleus (SCN). Here, we evaluated the influence of the IGL on masking and the circadian system in a diurnal rodent, the Nile grass rat (Arvicanthis niloticus), by determining the effects of bilateral IGL lesions on general activity under different lighting conditions. To examine masking responses, light or dark pulses were delivered in the dark or light phase, respectively. Light pulses at Zeitgeber time (ZT) 14 increased activity in control animals but decreased it in animals with IGL lesions. Dark pulses had no effect on controls, but significantly increased activity in lesioned animals at ZT0. Lesions also significantly increased activity, primarily during the dark phase of a 12:12 light/dark cycle, and during the subjective night when animals were kept in constant conditions. Taken together, our results suggest that the IGL plays a vital role in the maintenance of both the species-typical masking responses to light, and the circadian contribution to diurnality in grass rats.     

Altered Entrainment to the Day/Night Cycle Attenuates the Daily Rise in Circulating Corticosterone in the Mouse

The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT_1B receptors, decreasing responsiveness to light. Consequently, 5-HT_1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light∶dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT_1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h) conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH) stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies.     

The circadian system of Trypanosoma cruzi-infected mice

The effects of Chagas disease on the mammalian circadian system were studied in Trypanosoma cruzi-infected C57-Bl6J mice. Animals were inoculated with CAI or RA strains of T. cruzi or vehicle, parasitism confirmed by blood specimen visualization and locomotor activity rhythms analyzed by wheel-running recording. RA-strain infected mice exhibited significantly decreased amplitude of circadian rhythms, both under light-dark and constant dark conditions, probably due to motor deficiencies. CAI-treated animals showed normal locomotor activity rhythms. However, in these mice, reentrainment to a 6h phase shift of the LD cycle took significantly longer than controls, and application of 15min light pulses in DD produced smaller phase delays of the rhythms. All groups exhibited light-induced Fos expression in the suprachiasmatic nuclei. We conclude that the main effect of T. cruzi infection on the circadian system is an impairment of the motor output from the clock toward controlled rhythms, together with an effect on circadian visual sensitivity.     

Running wheel size influences circadian rhythm period and its phase shift in mice

Running wheels are widely used in studies on biological rhythms. In mice wheel diameters have ranged from 11 cm to 23 cm. We provided mice with running wheels of two different sizes: 15 cm diameter and 11 cm diameter. The amount of running in the 12-h light:12-h dark condition and the endogenous period of wheel running in constant darkness was determined over 40 days. On the 1st day in constant darkness all animals were exposed to a 15-min light pulse at circadian time 13. The animals in the small wheel ran significantly less both in 12 h light: 12 h dark and constant darkness, and showed a longer endogenous period in constant darkness compared to animals in the large wheel. Moreover, after the light pulse at circadian time 13, mice in the small wheel showed a significantly smaller phase delay in running wheel activity than mice in the larger wheels. The data suggest that the magnitude of a photic phase shift depends on the amount and timing of activity the animals display in relation to this stimulus. It can be concluded that technical features of the running wheel can influence the circadian period of wheel running.     

Effect of Behavioural Feedback on Circadian Clocks of the Nocturnal Field Mouse Mus booduga

The effect of 'novel running wheels' on circadian clocks of the nocturnal field mouse Mus booduga was investigated during free-running and entrained conditions. In order to find out whether daily access to novel running wheels can entrain the locomotor activity rhythms experimental animals (n = 6) were provided with 'novel running wheels' at a fixed time of the day. The control animals (n = 5) were handled similar to the experimental animals but were not given access to novel running wheels. The results show that daily access to novel running wheels entrained the free-running locomotor activity rhythm of these mice. The post-entrainment free-running period (τ) of the experimental animals was significantly shorter than the pre-entrainment τ, whereas the pre- and post-treatment τ of the control animals did not differ significantly. In separate set of experiments, the effect of access to novel running wheels on the rate of re-entrainment was studied after a 6 h phase advance/delay in 24 h (12:12 h) light/dark (LD) cycles. Experimental animals were given access to novel running wheels for 3-h, 1 h after the 'lights-off' only on the first day of the 'new LD cycles'. Experimental animals took fewer cycles to re-entrain to 6-h phase advanced LD cycles compared to the control animals. After a phase delay in the LD cycles by 6h, the experimental animals took more number of cycles to re-entrain compared to the control animals. These results thus suggest that access to novel running wheel can act as a Zeitgeber for the circadian clocks of the nocturnal mouse M. booduga, and can also modify the rates of re-entrainment to phase shifted LD cycles, in a time-dependent manner.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Phase Shifting by Dark Pulses (III)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought to be responsible for melatonin circadian rhythms. The enzyme has circadian properties--its rhythm persists in constant conditions, and it is precisely controlled by light and dark. Experiments are reported in which 4-h light or dark pulses were imposed on chicks (Gallus domesticus) over a 24-h period. Pineal NAT profiles were measured during and subsequent to the pulses. The phase of the NAT cycle following pulses was plotted to obtain phase-response curves. Light pulses produced a maximum phase shift (advance of 5 h) 8 h after the expected time of lights-out; dark pulses produced a maximum phase shift (advance of 4 h) 3 h after the expected time of lights-out. Maximum phase delays (-2 h) occurred 1-2 h after the expected lights-out for light pulses and 8 h after expected lights-on for dark pulses.     

Photic and nonphotic inputs to the diurnal circadian clock

Diurnal animals occupy a different temporal niche from nocturnal animals and are consequently exposed to different amounts of light as well as different dangers. Accordingly, some variation exists in the way that diurnal animals synchronize their internal circadian clock to match the external 24-hour daily cycle. First, though the brain mechanisms underlying photic entrainment are very similar among species with different daily activity patterns, there is evidence that diurnal animals are less sensitive to photic stimuli compared to nocturnal animals. Second, stimuli other than light that synchronize rhythms (i.e. nonphotic stimuli) can also entrain and phase shift daily rhythms. Some of the rules that govern nonphotic entrainment in nocturnal animals as well as the brain mechanisms that control nonphotic influences on rhythms do not appear to apply to diurnal animals, however. Some evidence supports the idea that arousal or activity plays an important role in entraining rhythms in diurnal animals, either during the light (active) or dark (inactive) phases, though no consistent pattern is seen. GABAergic stimulation induces phase shifts during the subjective day in both diurnal and nocturnal animals. In diurnal Arvicanthis niloticus (Nile grass rats), SCN GABA_A receptor activation at this time results in phase delays while in nocturnal animals phase advances are induced. It appears that the effect of GABA at this circadian phase results from the inhibition of period gene expression in both diurnal and nocturnal animals. Nonetheless, the resulting phase shifts are in opposite directions. It is not known what stimuli or behaviours ultimately induce changes in GABA activity in the SCN that result in alterations of circadian phase in diurnal grass rats. Taken together, studies such as these suggest that it may be problematic to apply the principles governing nocturnal nonphotic entrainment and its underlying mechanisms to diurnal species including humans.     

Pubertal maturation and time of day differentially affect behavioral and neuroendocrine responses following an acute stressor

Puberty markedly influences stress responsiveness such that prepubertal animals show a more protracted corticosterone (CORT) and progesterone response following acute stress compared to adults. In both adult and juvenile rats, circadian time modulates adrenocortical steroids with basal CORT and progesterone levels rising prior to the onset of the dark phase of the light-dark cycle (i.e., active period). How time of day affects the pubertal difference in stress responsiveness and if the behaviors of prepubertal and adult animals are differentially affected by stress and time of testing remain unknown. Thus, we exposed group housed (3 per cage) prepubertal (28d) and adult (77d) male rats to 30 min of restraint in either the early portion of the behaviorally inactive, light (circadian nadir of CORT and progesterone) or behaviorally active, dark (circadian peak) phase of their light-dark cycle and measured ACTH, CORT, progesterone, and home cage behavior before and after the stressor. We found that the extended hormonal stress response demonstrated by prepubertal males occurred at both times of day. However, differences in post-stress behavior were dependent on time of testing. Specifically, although pre- and post-stress behaviors were similarly affected by the stressor in the light phase in prepubertal and adult males, during the dark phase, stress suppressed play behavior in the prepubertal males, and increased their time spent resting together (huddling), while these behaviors were unaffected by stress in the adults. These data indicate that pubertal development and time of day interact to modulate post-stress behavior and demonstrate a dissociation between post-stress hormonal and behavioral responses.     

Circadian rhythm and response to an acute stressor of urinary corticosterone, testosterone, and creatinine in adult male mice

In small laboratory species, steroid measures can be obtained more frequently and less invasively from urine than blood. Insofar as urinary levels reflect systemic levels, they could provide advantages particularly for measurement of glucocorticoids, whose blood levels react rapidly to handling and stress. In Experiment 1, urinary samples were collected from male mice every second hour over a 14:10 h light:dark cycle. Samples were analyzed via enzyme immunoassay for corticosterone, testosterone, and creatinine. Corticosterone had peak concentrations 1 h after light offset and a trough 1 h after light onset. Testosterone showed peak concentrations 5-7 h after light onset and lowest concentrations during the dark phase of the cycle. Creatinine showed some variation over the light-dark cycle, but steroid measures showed similar trends with and without adjustment for creatinine. In Experiment 2, mice were stressed via an injection at times close to the determined peak and trough levels of corticosterone. In urinary samples taken 90 min after injection, corticosterone was significantly higher in injected animals at both times relative to levels in control animals, but testosterone was unaffected by injection stress. In Experiment 3, serum and urine samples were collected from mice every sixth hour across the diurnal cycle. Corticosterone peaked in urine and serum immediately after light offset, and urinary measures predicted those in serum. These data indicate that urinary corticosterone reflects systemic levels in mice, document circadian variation in urinary testosterone, and indicate that circadian variation in creatinine is minimal, but potentially relevant in stressed animals.     

Circadian rhythms in heart rate, motility, and body temperature of wild-type C57 and eNOS knock-out mice under light-dark, free-run, and after time zone transition

The nitric oxide (NO) system is involved in the regulation of the cardiovascular system in controlling central and peripheral vascular tone and cardiac functions. It was the aim of this study to investigate in wild-type C57BL/6 and endothelial nitric oxide synthase (eNOS) knock-out mice (eNOS-/-) the contribution of NO on the circadian rhythms in heart rate (HR), motility (motor activity [MA]), and body temperature (BT) under various environmental conditions. Experiments were performed in 12:12 h of a light:dark cycle (LD), under free-run in total darkness (DD), and after a phase delay shift of the LD cycle by -6 h (i.e., under simulation of a westward time zone transition). All parameters were monitored by radiotelemetry in freely moving mice. In LD, no significant differences in the rhythms of HR and MA were observed between the two strains of mice. BT, however, was significantly lower during the light phase in eNOS-/- mice, resulting in a significantly greater amplitude. The period of the free-running rhythm in DD was slightly shorter for all variables, though not significant. In general, rhythmicity was greater in eNOS-/- than in C57 mice both in LD and DD. After a delay shift of the LD cycle, HR and BT were resynchronized to the new LD schedule within 5-6 days, and resynchronization of MA occurred within 2-3 days. The results in telemetrically instrumented mice show that complete knock-out of the endothelial NO system—though expressed in the suprachiasmatic nuclei and in peripheral tissues—did not affect the circadian organization of heart rate and motility. The circadian regulation of the body temperature was slightly affected in eNOS-/- mice.     

Resetting of the hamster circadian system by dark pulses

Circadian rhythms of animals are reset by exposure to light as well as dark; however, although the parameters of photic entrainment are well characterized, the phase-shifting actions of dark pulses are poorly understood. Here, we determined the tonic and phasic effects of short (0.25 h), moderate (3 h), and long (6-9 h) duration dark pulses on the wheel-running rhythms of hamsters in constant light. Moderate- and long-duration dark pulses phase dependently reset behavioral rhythms, and the magnitude of these phase shifts increased as a function of the duration of the dark pulse. In contrast, the 0.25-h dark pulses failed to evoke consistent effects at any circadian phase tested. Interestingly, moderate- and long-dark pulses elevated locomotor activity (wheel-running) on the day of treatment. This induced wheel-running was highly correlated with phase shift magnitude when the pulse was given during the subjective day. This, together with the finding that animals pulsed during the subjective day are behaviorally active throughout the pulse, suggests that both locomotor activity and behavioral activation play an important role in the phase-resetting actions of dark pulses. We also found that the robustness of the wheel-running rhythm was weakened, and the amount of wheel-running decreased on the days after exposure to dark pulses; these effects were dependent on pulse duration. In summary, similarly to light, the resetting actions of dark pulses are dependent on both circadian phase and stimulus duration. However, dark pulses appear more complex stimuli, with both photic and nonphotic resetting properties.     

Circadian locomotor analysis of male mice lacking the gene for neuronal nitric oxide synthase (nNOS-/-)

Nitric oxide (NO) is an endogenous gas that functions as a neurotransmitter. Because NO is very labile with a half-life of less than 5 sec, most functional studies of NO have manipulated its synthetic enzyme, NO synthase (NOS). Three isoforms of NOS have been identified: (1) in the endothelial lining of blood vessels (eNOS), (2) an inducible form found in macrophages (iNOS), and (3) in neurons (nNOS). Most pharmacological studies to date have blocked all three isoforms of NOS. Previous studies using such agents have revealed that NO might be necessary for photic entrainment of circadian rhythms; general NOS inhibitors attenuate phase shifts of free-running behavior, light-induced c-fos expression in the suprachiasmatic nucleus (SCN), and phase shifts of neural firing activity in SCN maintained in vitro. To assess the specific role of nNOS in mediating entrainment of circadian rhythms, mice with targeted deletion of the gene encoding the neuronal isoform of NOS (nNOS-/-) were used. Wild-type (WT) and nNOS-/- mice initially were entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was either phase advanced or phase delayed. After an additional 3 weeks, animals were held in either constant dim light or constant dark. WT and nNOS-/- animals did not differ in their ability to entrain to the LD cycle, phase shift locomotor activity, or free run in constant conditions. Animals held in constant dark were killed after light exposure during either the subjective day or subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos expression in the SCN of both WT and nNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that NO from neurons might not be necessary for photic entrainment.     

Circadian clock functioning is linked to acute stress reactivity in rats

At least two major physiological systems are involved in the adaptation of the organism to environmental challenges: the circadian system and the stress reaction. This study addressed the possibility that interindividual differences in stress sensitivity and in the functioning of the circadian system are related. At 2 months of age, corticosterone secretion in response to a 20-min restraint stress was assessed in 9 Sprague-Dawley rats for which running wheel activity was recorded as a rhythmic behavioral marker of the circadian clock. Two weeks later, the adaptive response of the circadian system to an abrupt shift in the light:dark (LD) cycle was assessed in those rats using a jet-lag paradigm. Finally, after resynchronization to the new LD cycle, rats were transferred to constant darkness to assess the free-running period of their circadian rhythm of running-wheel activity. Results indicate that stress-induced corticosterone secretion was (1) positively correlated with the number of days to resynchronize the circadian activity rhythm to the new LD cycle, and with the value of its free-running period, and (2) negatively correlated with the intensity of daily locomotor activity. Those data, emphasizing the interactions between the stress response of an organism and the functioning of its circadian system, could explain interindividual differences in humans' susceptibility to shift work or other circadian-related disorders.