Rat Circadian System Research Articles

Melatonin is a redundant entraining signal in the rat circadian system

The role of melatonin in maintaining proper function of the circadian system has been proposed but very little evidence for such an effect has been provided. To ascertain the role, the aim of the study was to investigate impact of long-term melatonin absence on regulation of circadian system. The parameters of behavior and circadian clocks of rats which were devoid of the melatonin signal due to pinealectomy (PINX) for more than one year were compared with those of intact age-matched controls. PINX led to a decrease in spontaneous locomotor activity and a shortening of the free-running period of the activity rhythm driven by the central clock in the suprachiasmatic nuclei (SCN) in constant darkness. However, the SCN-driven rhythms in activity and feeding were not affected and remained well entrained in the light/dark cycle. In contrast, in these conditions PINX had a significant effect on amplitudes of the clock gene expression rhythms in the duodenum and also partially in the liver. These results demonstrate the significant impact of long-term melatonin absence on period of the central clock in the SCN and the amplitudes of the peripheral clocks in duodenum and liver and suggest that melatonin might be a redundant but effective endocrine signal for these clocks.

New light on the serotonergic paradox in the rat circadian system

The main mammalian circadian clock, localized in the suprachiasmatic nuclei can be synchronized not only with light, but also with serotonergic activation. Serotonergic agonists and serotonin reuptake inhibitors (e.g., fluoxetine) have a non-photic influence (shifting effects during daytime and attenuation of photic resetting during nighttime) on hamsters' and mice' main clock. Surprisingly, in rats serotonergic modulation of the clock shows essentially photic-like features in vivo (shifting effects during nighttime). To delineate this apparent paradox, we analyzed the effects of fluoxetine and serotonin agonists on rats' clock. First, fluoxetine induced behavioral phase-advances associated with down-regulated expression of the clock genes Per1 and Rorbeta and up-regulated expression of Rev-erbalpha during daytime. Moreover, fluoxetine produced an attenuation of light-induced phase-advances in association with altered expression of Per1, Per2 and Rorbeta during nighttime. Second, we showed that 5-HT(1A) receptors -maybe with co-activation of 5-HT(7) receptors- were implicated in non-photic effects on the main clock. By contrast, 5-HT(3) and 5-HT(2C) receptors were involved in photic-like effects and, for 5-HT(2C) subtype only, in potentiation of photic resetting. Thus this study demonstrates that as for other nocturnal rodents, a global activation of the serotonergic system induces non-photic effects in the rats' clock during daytime and nighttime.

Quantitative changes in neuronal and glial cells in the suprachiasmatic nucleus as a function of the lighting conditions during weaning

To examine whether lighting conditions during the development of the rat circadian system affect the morphology of the suprachiasmatic nucleus (SCN), three groups of rats were born and maintained until they were 24 days old under constant light (LL), constant darkness (DD) or 24-h light–dark cycles (LD, 12-h light and 12-h darkness). We applied a stereological method to study whether these conditions lead to alterations in the volume of the SCN and changes in the total number of neurons and glial cells. While lighting conditions did not induce differences in the SCN volume, the number of both neurons and glial cells did differ between groups. The DD rats showed the lowest number of neurons. Glial cells were also lower in this group than in the other two groups; however the number of glial cells in LL rats was lower than in LD rats. Moreover, females had more glial cells than males but males and females showed a similar number of neurons. These findings indicate the plasticity of the SCN in response to lighting conditions during the developmental stage.

Circadian change in tryptophan hydroxylase protein levels within the rat intergeniculate leaflets and raphe nuclei

Serotonin (5-HT) is involved in the synchronisation of the mammalian circadian clock located in the suprachiasmatic nuclei of the hypothalamus (SCN). This clock is synchronised by light (photic cues) and by non-photic cues. Non-photic cues are notably conveyed to the SCN by a direct 5-HT pathway arising from the mesencephalic median raphe nucleus (MRN). Furthermore, an indirect projection conveys non-photic inputs by 5-HT fibres from the mesencephalic dorsal raphe nucleus (DRN) to the intergeniculate leaflets of the thalamus (IGL) which project to the SCN. In the rat, the quantitative distribution of tryptophan hydroxylase (TpH), used as an index of 5-HT synthesis, was studied by in situ immunoautoradiography in both the serotoninergic cell bodies area of the raphe nuclei and the serotoninergic terminal field of the IGL. Under a 12 h light: 12 h dark (LD 12:12), TpH protein amount exhibited a rhythmic variation within the IGL. The maximum levels were reached at the day/night transition. In both MRN and the lateral groups of the DRN, TpH variations were opposite to those observed in the IGL. Such phase opposition was reported previously in the MRN/SCN pathway and was correlated with a rhythmic release of 5-HT within the SCN [Eur J Neurosci 15 (2002) 833]. Thus, the daily rhythmicity of TpH levels observed in DRN-IGL pathway may be correlated with a rhythmic release of 5-HT in the IGL at the beginning of the night. Under constant darkness, TpH rhythmic variations in the two serotoninergic pathways were maintained and similar to those observed under light/dark cycle. These results demonstrate the existence of a circadian endogenous functioning in the 5-HT neurones projecting to the rat circadian system.

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.

Requirements for Conditioning of the Rat Circadian System Remain Elusive

Requirements for Conditioning of the Rat Circadian System Remain Elusive

Expression profiles of JunB and c-Fos proteins in the rat circadian system

The immediate-early genes c-Fos and JunB are implicated in light signaling within the suprachiasmatic nucleus (SCN), the mammalian circadian clock. Light induces phase-dependent expression of c-Fos and JunB within the retinorecipient SCN. In the absence of light, rhythmic expression of these genes has been observed in the dorsal region of the SCN with peak expression observed near dawn. The present study examined the pattern of induction of c-Fos and JunB in the SCN and intergeniculate leaflet (IGL) of rats housed in constant conditions, under light–dark cycles, or in dark-adapted light-stimulated animals. In contrast with previous studies, no evidence of expression of c-Fos and JunB was observed within the dorsal SCN, regardless of circadian time. Strain differences could account for the absence of rhythmic JunB expression, whereas the use of a monoclonal anti-c-Fos antibody in the present study may account for the absence of dorsal SCN c-Fos staining. The profile of light-induced c-Fos and JunB expression was consistent with previous observations. In the SCN, light-induced expression of c-Fos and JunB was phase dependent, whereas in the IGL light-induced both c-Fos and JunB regardless of circadian time.

Local effects of the serotonin agonist quipazine on the suprachiasmatic nucleus of rats

In mammals, circadian rhythms of locomotor activity and many other behavioral and physiological functions are controlled by an endogenous pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). One of the SCN's afferents is a dense serotonergic input from the mesencephalic raphe complex. Previous work from this laboratory demonstrated that systemic administrations of the serotonin agonist quipazine mimic the effects of light on the circadian system of rats, i.e. they induce photic-like phase shifts of the circadian activity rhythm as well as c-Fos expression in the SCN. In contrast, no such effect has been demonstrated so far in the isolated rat SCN slice preparation. In this study we demonstrate that local injections of quipazine (0.5 microg/kg) into the region of the SCN induce photic-like effects similar to those induced by systemic injections. These findings suggest a role for 5-HT in the transmission of photic information to the rat circadian system through a direct action at the level of the SCN.

Photic entrainment and induction of immediate-early genes within the rat circadian system

Immediate-early genes (IEGs) are transiently expressed within the rodent circadian system in response to nocturnal light. The two most studied light-induced IEGs within this system are Fos and Jun-B. Molecular expression of these two genes within the hypothalamic suprachiasmatic nucleus (SCN) correlates with light-induced behavioral phase shifts. Previous studies of the role of Fos and Jun-B in circadian clock resetting have used light stimuli that induce strong phase shifts. However, the relationship of Fos and Jun-B expression in the SCN and light-induced phase shifts in an entrainment context is undocumented in rats. In this study, male rats for which the free running period was determined were entrained to a 0.5 h:23.5 h LD cycle. On the fifteenth day of stable entrainment, the entraining light pulse was reduced to 10 min. Animals were killed 50 min later and brains were processed for IEG immunocytochemistry. Strong Fos induction was observed in the SCN and the intergeniculate leaflet (IGL). Strong Jun-B immunoreactivity was observed only in the SCN whereas Jun-B labeling in the IGL was weak. Significant correlations were obtained between the magnitude of light-induced IEGs in the SCN and the magnitude of the daily phase shift required for stable entrainment to the 0.5 h:23.5 h LD cycle. Further, a significant correlation was observed between the number of Fos and Jun-B immunoreactive cells in the SCN and IGL. These data suggest that the magnitude of Fos and Jun-B induction within the SCN is related to the magnitude of the daily phase shift required for stable entrainment.

Conditioned stimulus control in the rat circadian system depends on clock resetting during conditioning.

Conditioned stimulus control in the rat circadian system depends on clock resetting during conditioning.

Conditioned stimulus control in the rat circadian system depends on clock resetting during conditioning.

The authors examined the ability of a conditioned stimulus (CS; mild air disturbance) previously paired with an entraining light pulse to reset the circadian pacemaker in rats. Rats were entrained to a single 30-min light stimulus delivered every 25 hr or 24 hr (T cycle). Each daily light presentation was paired with the CS. After at least 20 days of stable entrainment to each of the T cycles, the rats were allowed to free run and were then presented with the CS at circadian time 15. CS-induced phase shifts in wheel-running activity rhythms were taken as evidence for conditioning. For the most part, conditioning occurred after CS-light pairings on the 25-hr but not 24-hr T cycle. The results suggest that CS control of the circadian clock phase depends on the effect that the entraining light pulse has on the clock during conditioning.

Feeding behavior and entrainment limits in the circadian system of the rat.

The entrainment limits of the circadian rhythms of feeding activity were studied in Wistar rats exposed to gradually increasing and decreasing or to static light-dark cycles. In the former, the entrainment limits of feeding behavior were 22 h 10 min and 26 h 40 min. In the latter, the upper limit was higher, because rats under zeitgeber period (t) length = 27 h (t27) and t28 met the criteria of entrainment. The lower limit, on the other hand, was not modified because none of the t22 animals showed entrained rhythms and one-half of the t23 rats exhibited two components in their circadian feeding rhythms, one with a period of 23 h and the other free running. This 23-h component reflected not only the masking effect of light-dark cycles but also seemed a true light-entrained component. In well-synchronized animals, food intake seemed to depend more on the number of cycles that the animal experienced than on actual time lived; however, other feeding parameters, such as meal frequency and feeding duration, remained constant when expressed per 24 h, irrespective of the t cycle. These results concerning feeding duration, meal frequency, and food intake revealed that the homeostatic and circadian controls interacted to a degree that depended on the type of variable considered. In conclusion, the entrainment limits appeared much more imprecise than they were previously thought to be, because the circadian system can only be partially synchronized near its entrainment limits. The hypothesis that the rat's circadian system is composed of multiple oscillators with different intrinsic frequencies and varying capacities for light synchronization would explain the partial desynchronization observed near the entrainment limits.

Circadian Modification of Uterine Responses by Day-Night Dexamethasone Treatment during Decidualization in Rats

The effect of treatment (1 mg/rat for 5 days, post-implantation) with the synthetic glucocorticoid, dexamethasone (Dex) on the biochemical components in uterine proliferation during decidualization in rats was assessed. Dex was administered subcutaneously either at only one time (two hours after lights off) of the scotophase, or again at one time (two hours after lights on) during the photophase of a fixed 24 hours (12L: 12D) photoperiod. The uterine parameters included the wet weight, protein and DNA concentrations, nuclear estrogen receptors, enzymatic activities of alkaline phosphatase, isocitrate dehydrogenase and the matrix metalloproteinases, and serum levels of estradiol and progesterone. Certainly the rat circadian system, under the influence of the hypothalamic suprachiasmatic nucleus is fully entrained by the alternation of the experiment. Hence, the results suggest that between the two injections times, there may be a circadian difference in the uterine responses to Dex. The findings have meaningful implications for Dex and the optimal effectiveness of its application times for implantation problems.

Aggressive and Sexual Social Stimuli Do not Phase Shift the Orcadian Temperature Rhythm in Rats

The objective of the present study was to determine whether the rat circadian system is sensitive to social stimuli. Male rats were subjected to a sociosexual interaction with an estrous female or to an aggressive interaction with a dominant male conspecific. The interactions lasted for lh and took place in the middle of the circadian resting phase. Control animals were picked up and handled for a few minutes, but were otherwise left undisturbed. Animals were housed under constant dim red light during the whole period of the experiment. To assess the effects of the interactions on free-running circadian rhythmicity, body temperature was measured by means of radio telemetry. Neither the sociosexual interaction with a female nor the aggressive interaction with another male induced phase shifts or changes in the free-running period. The rat circadian system does not seem to be sensitive to social stimuli directly. Moreover, the finding that aggressive interactions do not phase shift circadian rhythms indicates that the endogenous pacemaker in rats is not sensitive to stressors.

Light-induced suppression of the rat circadian system.

In a constant environment, circadian rhythms persist with slightly altered period lengths. Results of studies with continuous light exposure are less clear, because of short exposure durations and single-variable monitoring. This study sought to characterize properties of the oscillator(s) controlling the rat's circadian system by monitoring both body temperature and locomotor activity. We observed that prolonged exposure of male Sprague-Dawley rats to continuous light (LL) systematically induced complete suppression of body temperature and locomotor activity circadian rhythms and their replacement by ultradian rhythms. This was preceded by a transient loss of coupling between both functions. Continuous darkness (DD) restored circadian synchronization of temperature and activity circadian rhythms within 1 wk. The absence of circadian rhythms in LL coincided with a mean sixfold decrease in plasma melatonin and a marked dampening but no abolition of its circadian rhythmicity. Restoration of temperature and activity circadian rhythms in DD was associated with normalization of melatonin rhythm. These results demonstrated a transient internal desynchronization of two simultaneously monitored functions in the rat and suggested the existence of two or more circadian oscillators. Such a hypothesis was further strengthened by the observation of a circadian rhythm in melatonin, despite complete suppression of body temperature and locomotor activity rhythms. This rat model should be useful for investigating the physiology of the circadian timing system as well as to identify agents and schedules having specific pharmacological actions on this system.

Dose dependent effects of S-20098, a melatonin agonist, on direction of re-entrainment of rat circadian activity rhythms

The chronobiotic properties of melatonin are well documented. For example, following an 8-h phase advance of the light-dark cycle daily injections of melatonin administered at the pre-shift dark onset alter the direction of re-entrainment of rat activity rhythms. Using this 8-h phase advance paradigm, the effects of the melatonin agonist S-20098 (1 mg/kg and 3 mg/kg) on the rat circadian system were compared with those of melatonin. S-20098 altered the direction of re-entrainment in the same manner as melatonin. A study using lower doses of S-20098 showed that the effect on direction of re-entrainment was dose-dependent, with 100% of rats responding at a dose of 100 micrograms/kg. S-20098 may, therefore, have therapeutic potential as a chronobiotic in the treatment of circadian disorders in humans.

Pinealectomized rats entrain and phase-shift to melatonin injections in a dose-dependent manner.

Previous work has shown that daily injections of the pineal hormone melatonin (N-acetyl, 5-methoxytryptamine) entrain the free-running locomotor rhythms of rats held in constant darkness (with a median effective dose [ED50] of 5.45 +/- 1.33 micrograms/kg) and in constant bright light. The present experiments determined the dose-response characteristics of entrainment and phase shifting to daily and single melatonin injections in both sham-operated (SHAM) and pinealectomized (PINX) rats. The data indicated an ED50 of 332 +/- 53 ng/kg and 121 +/- 22 ng/kg for SHAM and PINX rats, respectively, during the entrainment experiment. The ED50's for the entrainment experiment were considerably lower than doses previously employed, and much lower than doses employed in reproductive and metabolic studies in rats and hamsters. The data indicated that no partial entrainment occurred; nor were there differences in phase angle, length of activity, or period among all effective doses. Next, a single injection of 1 mg/kg melatonin has previously been shown to cause a phase advance of approximately 45 min when administered at about circadian time (CT) 10. We found that both SHAM and PINX animals phase-advanced, in a dose-dependent manner to a single melatonin injection given at CT 10. The data for the phase-shifting experiment indicated an ED50 of 8.19 +/- 0.572 micrograms/kg and 2.16 +/- 0.326 micrograms/kg for SHAM and PINX animals, respectively, with an average phase advance of 40 min for both groups. Together, the data suggest that the presence of the pineal gland is not necessary for the effects of melatonin on the rat circadian system, and that PINX animals are marginally more sensitive to melatonin than their SHAM controls.

Dose-dependent entrainment of rat circadian rhythms by daily injection of melatonin.

Previous work in our laboratory has shown that daily injection of large doses of the pineal hormone melatonin entrains the free-running locomotor rhythms of rats held in constant darkness and synchronizes the disrupted patterns of rats maintained in constant bright light. The present experiments determined the dose-response characteristics of entrainment to daily melatonin injections and made preliminary biochemical estimates of blood melatonin levels and half-lives after two critical doses of the hormone. The data indicated that the median effective dose for melatonin as an entraining agent in free-running rats was 5.45 +/- 1.33 micrograms/kg, considerably lower than doses previously employed and lower than doses employed in reproductive and metabolic studies in rats and hamsters. The data further indicated that the response to melatonin was quantal; rats either entrained to melatonin or they did not. No "partial entrainment" was evident, nor were there differences in phase angle, activity, or period among all effective doses. Biochemical estimates of blood melatonin after either 1 mg/kg or 1 microgram/kg of melatonin indicated that all effective doses resulted in supraphysiological levels of blood melatonin, although doses of 1 microgram/kg resulted in blood levels that were within one order of magnitude of normal nighttime values. Together, the data suggest that the rat circadian system is sensitive to the pineal hormone melatonin at or below doses required to effect rodent reproduction. Whether this sensitivity reflects a role for the pineal gland in rat circadian organization, however, still remains to be determined.

Daily variation in 5-hydroxytryptamine in rat spleen and the effect of stimuli on the rhythm.

AbstractFluorometric measurements were made of 5‐hydroxytryptamine levels in spleens obtained from separate subgroups of adult male rats killed at two‐hourly intervals over four separate 24‐hour time spans. For three weeks prior to obtaining tissue, all rats were standardized carefully and were subjected to 12 hours of light (0600–1800) followed by 12 hours of darkness. Analyses showed in each study very significant fluctuation over the 24‐hour time scale; the range of change over this period was as great as 131%. The phasing of the rhythms, in spite of the rigid environmental synchronization, was not similar in any of the four studies. Possible explanations for this unexpected desynchronization are discussed. Spleen weights also fluctuated with a significant 24‐hour circadian frequency.In addition, the rhythm characterizing this amine was described in female rats as well as the effect of different stimuli on the same rhythm. These stimuli were ether, immobilization and a ‘novelty’ situation; and all three significantly increased the levels of 5‐HT in spleen when the overall 24‐hour mean values of each experimental group were compared with controls. However, in all cases there were discrete time points when significant differences in 5‐HT levels could not be obtained with any of the stimuli. This study demonstrates that the effect of the stimuli used depends on the phase of the rat's circadian system during which it was applied. The necessity of considering the natural rhythmic fluctuation in any investigation evaluating this biogenic amine in spleen is demonstrated.