A Noradrenergic Mechanism Influences the Circadian Timing System in Rats and Hamsters

Brainstem monoaminergic projections to the suprachiasmatic nucleus (SCN), and to the intergeniculate leaflet (IGL), appear to modulate both photic and non-photic effects on the circadian system. Recent work in this laboratory has concentrated on the role of noradrenaline in the regulation of circadian period and phase. Previously, this lab has shown that chronic administration of the alpha₂ adrenergic agonist, clonidine, to rats maintained in constant light (LL) shortens free-running circadian period and promotes dissociation of rhythmicity, while acute clonidine administration to hamsters produces phase shifts similar to those observed with photic stimuli. These results suggest an interaction between clonidine and photic input on circadian rhythmicity, and so the present study was designed to examine systematically the relationship between chronic clonidine administration and photic input in both rats and hamsters. In DD and low intensity LL, clonidine did not alter free-running circadian wheel-running rhythms of rats, but under moderate to high intensity LL, clonidine significantly reduced the period-lengthening effects of LL. Chronic clonidine administration also altered several aspects of circadian phase in hamsters; phase shifts in response to light pulses of varying intensity at CT 19 were reduced; steady-state entrainment phase under a 24-h light-dark cycle (LD 14:10)was delayed; and synchronization to a 23-h light-dark cycle (LD 13:10) was impaired. Clonidine appeared to have little effect on free-running period of hamsters, but a trend towards dissociation of rhythmicity under LL was observed. These effects may reflect an action of clonidine at the photic input pathways to the circadian system, or directly at the circadian pacemaker, since alpha 2 adrenoceptors have been localized both in the suprachiasmatic nucleus (SCN) and in several of its projection areas. As both clinical and experimental studies suggest that clonidine may have depressogenic properties, chronic administration of clonidine to rodents may provide an animal model of the alterations in circadian rhythmicity seen in human depression.     

Species Differences Between Hamsters and Rats with Regard to the Putative Role of Serotonin in the Circadian System

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). Among various other afferents, the SCN receives a dense serotonergic input from the mesencephalic raphe complex. Experimental evidence obtained so far in Syrian hamsters suggests that serotonin (5-HT) mimics the effect of nonphotic stimuli during subjective day and modulates photic input to the SCN during subjective night. These findings are consistent with a putative role of serotonergic pathways in the transmission of the state of arousal to the SCN. In this paper, we review recent evidence for different modes of 5-HT action and/or the involvement of different 5-HT receptor subtypes in hamsters and rats. In intact rats, 5-HT agonists induce photic-like phase shifts of locomotor activity and melatonin rhythms as well as c-Fos expression in the ventral SCN. These results suggest a role for 5-HT in the transmission of photic rather than nonphotic information to the rat SCN. Such a function of 5-HT would also explain why the circadian system of rats is less sensitive or even insensitive to nonphotic stimuli.     

Species Differences Between Hamsters and Rats with Regard to the Putative Role of Serotonin in the Circadian System

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). Among various other afferents, the SCN receives a dense serotonergic input from the mesencephalic raphe complex. Experimental evidence obtained so far in Syrian hamsters suggests that serotonin (5-HT) mimics the effect of nonphotic stimuli during subjective day and modulates photic input to the SCN during subjective night. These findings are consistent with a putative role of serotonergic pathways in the transmission of the state of arousal to the SCN. In this paper, we review recent evidence for different modes of 5-HT action and/or the involvement of different 5-HT receptor subtypes in hamsters and rats. In intact rats, 5-HT agonists induce photic-like phase shifts of locomotor activity and melatonin rhythms as well as c-Fos expression in the ventral SCN. These results suggest a role for 5-HT in the transmission of photic rather than nonphotic information to the rat SCN. Such a function of 5-HT would also explain why the circadian system of rats is less sensitive or even insensitive to nonphotic stimuli.     

Glaucoma Alters the Circadian Timing System

Glaucoma is a widespread ocular disease and major cause of blindness characterized by progressive, irreversible damage of the optic nerve. Although the degenerative loss of retinal ganglion cells (RGC) and visual deficits associated with glaucoma have been extensively studied, we hypothesize that glaucoma will also lead to alteration of the circadian timing system. Circadian and non-visual responses to light are mediated by a specialized subset of melanopsin expressing RGCs that provide photic input to mammalian endogenous clock in the suprachiasmatic nucleus (SCN). In order to explore the molecular, anatomical and functional consequences of glaucoma we used a rodent model of chronic ocular hypertension, a primary causal factor of the pathology. Quantitative analysis of retinal projections using sensitive anterograde tracing demonstrates a significant reduction (∼50–70%) of RGC axon terminals in all visual and non-visual structures and notably in the SCN. The capacity of glaucomatous rats to entrain to light was challenged by exposure to successive shifts of the light dark (LD) cycle associated with step-wise decreases in light intensity. Although glaucomatous rats are able to entrain their locomotor activity to the LD cycle at all light levels, they require more time to re-adjust to a shifted LD cycle and show significantly greater variability in activity onsets in comparison with normal rats. Quantitative PCR reveals the novel finding that melanopsin as well as rod and cone opsin mRNAs are significantly reduced in glaucomatous retinas. Our findings demonstrate that glaucoma impacts on all these aspects of the circadian timing system. In light of these results, the classical view of glaucoma as pathology unique to the visual system should be extended to include anatomical and functional alterations of the circadian timing system.     

Inactivation of Glucocorticoid Receptor in Noradrenergic System Influences Anxiety- and Depressive-Like Behavior in Mice

The aim of this study was to investigate whether conditional inactivation of the glucocorticoid receptors (GRs) in noradrenergic neurons affects animal behavior in mice. Selective ablation of GRs in the noradrenergic system was achieved using the Cre/loxP approach. We crossed transgenic mice expressing the Cre recombinase under the dopamine beta-hydroxylase (DBH) promoter with animals harboring the floxed GR gene. The resulting GR^DBHCre mutant mice exhibited no alterations in terms of normal cage behavior, weight gain, spatial memory or spontaneous locomotor activity, regardless of gender. To assess depressive- and anxiety-like behaviors we performed the Tail Suspension Test and the Light-Dark Box Test. While male mutant animals did not show any alternations in both tests, female GR^DBHCre mutants displayed depressive- and anxiety-like behavior. Additionally, male GR^DBHCre mice were exposed to chronic restraint stress but still exhibited immobility times and anxiety statuses similar to those of non-stressed animals while stressed control mice clearly revealed depressive- and anxiety-like phenotype. Thus, in males the effects of the mutation were precipitated only after chronic restraint stress procedure. Our data reveal a possible gender-dependent role of GRs in the noradrenergic system in anxiety- and depressive-like behavior in mice.     

Short-Day Response in Djungarian Hamsters of Different Circadian Phenotypes

In Djungarian hamsters (Phodopus sungorus) bred at the authors' institute, a certain number of animals show activity patterns incompatible with proper entrainment of their endogenous circadian pacemaker to the environmental light-dark (LD) cycle. Even though the activity-offset in these animals is stably coupled to “light-on,” activity-onset is increasingly delayed, leading to a compression of the activity time (α). If α falls below a critical value, the circadian rhythm in these so called delayed activity-onset (DAO) hamsters starts to free-run and finally breaks down. Animals then show an arrhythmic activity pattern (AR hamsters). Previous studies revealed the mechanisms of photic entrainment have deteriorated (DAO) or the suprachiasmatic nucleus (SCN) does not generate a rhythmic signal (AR). The aim of the present study was to investigate the consequences that these deteriorations have upon photoperiodic time measurement. Animals were bred and kept under standardized housing conditions with food and water ad libitum and a 14L/10D (long day, LD) regimen. Locomotor activity was recorded continuously using passive infrared motion detectors. Body mass, testes size, and fur coloration were measured weekly or biweekly to further quantify the photoperiodic reaction. In a first experiment, adult male wild-type (WT), DAO, and AR hamsters were transferred initially to a 16L/8D cycle. After 3–4 wks, the light period was shortened symmetrically by 8?h. After 14 wks, none of the DAO and AR hamsters, and only 1 of 8 WT hamsters showed short-day (SD) traits. Therefore, in a second experiment, hamsters were transferred to SD conditions (8L/16D cycle) for 8 wks directly from standard LD conditions. In 6 of 7 WT hamsters, activity time expanded, body mass and testes size decreased, and fur coloration changed from summer to winter pelage. In contrast, none of the DAO and AR hamsters displayed an SD response. In a third experiment, DAO and AR hamsters were kept in constant darkness (DD) for 8 and 14 wks. After 8 wks, DAO hamsters showed a similar photoperiodic reaction to WT hamsters that had been kept for 8 wks under SD conditions. However, the level of adaptation was still less compared to WT hamsters, but this difference was not apparent after 14 wks. In contrast, AR animals did not display any photoperiodic reaction, even after 14 wks in DD. Type VI phase response curves (PRCs) were constructed to better understand the mechanism behind the SD response. In WT hamsters, the photosensitive phase, where light pulses induce phase shifts, was lengthened in SD condition. In DAO hamsters, in contrast, the PRCs were similar under LD and SD conditions with a compressed photosensitive phase corresponding to α. Also, “light-on” induced only weak phase advances of activity-onset, insufficient to compensate for the long endogenous period. The results show that physiological mechanisms necessary for seasonal adaptation are working in DAO hamsters and that it is the inadequate interaction of the LD cycle with the SCN that prevents the photoperiodic reaction. AR hamsters, on the other hand, are incapable of measuring photoperiodic time due to a complete disruption of circadian rhythmicity.     

Potent Circadian Effects of Dim Illumination at Night in Hamsters

Conventional wisdom holds that the circadian pacemaker of rodents and humans is minimally responsive to light of the intensity provided by dim moonlight and starlight. However, dim illumination (<0.005 lux) provided during the daily dark periods markedly alters entrainment in hamsters. Under dimly lit scotophases, compared to completely dark ones phases, the upper range of entrainment is increased by ～4 h, and re‐entrainment is accelerated following transfer from long to short day lengths. Moreover, the incidence of bimodal entrainment to 24 h light:dark:light:dark cycles is increased fourfold. Notably, the nocturnal illumination inducing these pronounced effects is equivalent in photic energy to that of a 2 sec, 100 lux light pulse. These effects may be parsimoniously interpreted as an action of dim light on the phase relations between multiple oscillators comprising the circadian pacemaker. An action of dim light distinct from that underlying bright‐light phase‐resetting may promote more effective entrainment. Together, the present results refute the view that scotopic illumination is environmental “noise” and indicate that clock function is conspicuously altered by nighttime illumination like that experienced under dim moonlight and starlight. We interpret our results as evidence for a novel action of dim light on the coupling of multiple circadian oscillators.     

The Daily Melatonin Pattern in Djungarian Hamsters Depends on the Circadian Phenotype

Djungarian hamsters (Phodopus sungorus) bred at the Institute of Halle reveal three different circadian phenotypes. The wild type (WT) shows normal locomotor activity patterns, whereas in hamsters of the DAO (delayed activity onset) type, the activity onset is continuously delayed. Since the activity offset in those hamsters remains coupled to “light-on,” the activity time becomes compressed. Hamsters of the AR (arrhythmic) type are episodically active throughout the 24?h. Previous studies showed that a disturbed interaction of the circadian system with the light-dark (LD) cycle contributes to the phenomenon observed in DAO hamsters. To gain better insight into the underlying mechanisms, the authors investigated the daily melatonin rhythm, as it is a reliable marker of the circadian clock. Hamsters were kept individually under standardized laboratory conditions (LD 14:10, T?=?22°C?±?2°C, food and water ad libitum). WT, DAO (with exactly 5?h delay of activity onset), and AR hamsters were used for pineal melatonin and urinary 6-sulfatoxymelatonin (aMT6s) measurement. Pineal melatonin content was determined at 3 time points: 4?h after “light-off” [D?+?4], 1?h before “light-on” [L???1], and 1?h after “light-on” [L?+?1]). The 24-h profile of melatonin secretion was investigated by transferring the animals to metabolic cages for 27?h to collect urine at 3-h intervals for aMT6s analysis. WT hamsters showed high pineal melatonin content during the dark time (D?+?4, L???1), which significantly decreased at the beginning of the light period (L?+?1). In contrast, DAO hamsters displayed low melatonin levels during the part of the dark period when animals were still resting (D?+?4). At the end of the dark period (L???1), melatonin content increased significantly and declined again when light was switched on (L?+?1). AR hamsters showed low melatonin levels, comparable to daytime values, at all 3 time points. The results were confirmed by aMT6s data. WT hamsters showed a marked circadian pattern of aMT6s excretion. The concentration started to increase 3?h after “light-off” and reached daytime values 5?h after “light-on.” In DAO hamsters, in contrast, aMT6s excretion started about 6?h later and reached significantly lower levels compared to WT hamsters. In AR animals, aMT6s excretion was low at all times. The results clearly indicate the rhythm of melatonin secretion in DAO hamsters is delayed in accord with their delayed activity onset, whereas AR hamsters display no melatonin rhythm at all. Since the regulatory pathways for the rhythms of locomotor activity and melatonin synthesis (which are downstream from the suprachiasmatic nucleus [SCN]) are different but obviously convey the same signal, we conclude that the origin of the phenomenon observed in DAO hamsters must be located upstream of the SCN, or in the SCN itself. (Author correspondence: weinert@zoologie.uni-halle.de)     

Synchronization of the Circadian Activity Rhythm in Hamsters Following Intermittent Schedule Shifts

The time course of resynchronization of the circadian activity rhythm of hamsters was observed following a 10-hr advance or delay in the light-dark cycle (LD 12:12). Twenty-six shift patterns of the lighting schedule were studied; they consisted of continuous (daily), three-step, two-step and one-step shifting. So long as the daily shift of the lighting schedule was 1 hr or less, the locomotor rhythm followed the continuous shift perfectly. As the amount of daily shift increased, the time course of activity onset deviated more from the time of lights off; the tendency was more marked in advancing than in delaying shifts. Responses of the activity rhythm to stepwise shifting were essentially the same as those to a continuous shift. They were, however, characterized by larger individual variations, and it took additional days before entrainment was achieved. By fitting the time course of entrainment to an exponential model with a constant term, estimates of time constant and shift error were derived. The time constant became shorter with increasing amounts of daily shifts up to 2 hr per day, increasing the number of shift steps, and/or reducing the amount of the initial shift of the seies. The shift error estimated was 0.51 ± 0.12 hr, indicating precise resynchronization. Accordingly, a quicker resynchronization may be expected when a multiple step shift with a moderate initial shift are employed. In the case of a 10-hr shift, for example, a shift of 3 hr followed by another 7 hr may be recommended.     

Synchronization of the Circadian Activity Rhythm in Hamsters Following Intermittent Schedule Shifts

The time course of resynchronization of the circadian activity rhythm of hamsters was observed following a 10-hr advance or delay in the light-dark cycle (LD 12:12). Twenty-six shift patterns of the lighting schedule were studied; they consisted of continuous (daily), three-step, two-step and one-step shifting. So long as the daily shift of the lighting schedule was 1 hr or less, the locomotor rhythm followed the continuous shift perfectly. As the amount of daily shift increased, the time course of activity onset deviated more from the time of lights off; the tendency was more marked in advancing than in delaying shifts. Responses of the activity rhythm to stepwise shifting were essentially the same as those to a continuous shift. They were, however, characterized by larger individual variations, and it took additional days before entrainment was achieved. By fitting the time course of entrainment to an exponential model with a constant term, estimates of time constant and shift error were derived. The time constant became shorter with increasing amounts of daily shifts up to 2 hr per day, increasing the number of shift steps, and/or reducing the amount of the initial shift of the seies. The shift error estimated was 0.51 ± 0.12 hr, indicating precise resynchronization. Accordingly, a quicker resynchronization may be expected when a multiple step shift with a moderate initial shift are employed. In the case of a 10-hr shift, for example, a shift of 3 hr followed by another 7 hr may be recommended.     

Serotoninergic System in Scrapie-Infected Hamsters

Hamsters inoculated with scrapie virus show a dramatic hypersensitivity to serotoninergic drugs, developing a behavioral syndrome not unlike that obtained with pharmacologically induced lesions of the raphe nuclei. In an attempt to explain the state of hypersensitivity and to determine whether or not serotoninergic neurons were targets of the scrapie virus, pre- and postsynaptic serotoninergic sites were studied in the cerebral cortices of scrapie-infected and sham-inoculated hamsters. [3H]Imipramine binding and the uptake of endogenous 5-hydroxytryptamine (5-HT, serotonin) in synaptosomes prepared from scrapie-inoculated animals were not different from those of controls. This suggests integrity of the serotoninergic neurons in scrapie-infected hamsters. In contrast, affinity for the 5-HT1 receptor (which modulates inhibitory response) was diminished whereas that for the 5-HT2 receptor (which modulates excitatory response) was increased. This "imbalance" between the two receptors which is amplified in in vivo responses may account for the 5-HT hypersensitivity. The alteration in the affinity of the two postsynaptic 5-HT receptors supports the observation that scrapie virus alters cell plasma membranes.     

A Mechanistic Model of the Regulation of Division Timing by the Circadian Clock in Cyanobacteria

The cyanobacterium Synechococcus elongatus possesses a circadian clock in the form of a group of proteins whose concentrations and phosphorylation states oscillate with daily periodicity under constant conditions. The circadian clock regulates the cell cycle such that the timing of the cell divisions is biased toward certain times during the circadian period, but the mechanism underlying this phenomenon remains unclear. Here, we propose a mechanism in which a protein limiting for division accumulates at a rate proportional to the cell volume growth and is modulated by the clock. This “modulated rate” model, in which the clock signal is integrated over time to affect division timing, differs fundamentally from the previously proposed “gating” concept, in which the clock is assumed to suppress divisions during a specific time window. We found that although both models can capture the single-cell statistics of division timing in S. elongatus, only the modulated rate model robustly places divisions away from darkness during changes in the environment. Moreover, within the framework of the modulated rate model, existing experiments on S. elongatus are consistent with the simple mechanism that division timing is regulated by the accumulation of a division limiting protein in a phase with genes whose activity peaks at dusk.     

Modifications of Reactions of the Hypothalamo-Hypophyseal-Adrenocortical System to Noradrenergic and Corticotropic Stimulations in Rats Subjected to Prenatal Hydrocortisone Treatment

We studied the reactions of the adrenal cortex to corticotropic and central noradrenergic stimulations in mature adult male and female rats which, in the final week of the prenatal period, developed under conditions of an artificial increase in the level of glucocorticoids in the maternal organism (everyday injections of 50 µg/kg of hydrocortisone acetate suspension to pregnant females). Experiments were carried out on unanesthetized offsprings of both sexes under conditions of free behavior; the level of corticosterone was repeatedly measured in the blood plasma with 30-min-long intervals within a 90 to 120 min period after injection of a stimulating agent. There was practically no adrenocortical reaction to infusion of adrenaline into the cerebral ventricle III in males whose mothers were injected with hydrocorticosterone acetate in the pregnancy period. At the same time, males born by intact mothers demonstrated a significant increase in the corticosterone level 30 min after the above-mentioned infusion. Noradrenergic stimulation increased the corticosterone concentration in the blood plasma in female offspring of both control and experimental groups, but the dynamics of reactions in females prenatally treated by hydrocortisone acetate demonstrated certain specificity (the reaction was longer, and the corticosterone level in the blood was higher even at the 90th min after noradrenaline infusion). At the same time, there were no changes in the sensitivity of the adrenal cortex to β-1-24-corticotropin either in males or in females of all observed groups. These results show that an artificial increase in the level of glucocorticoid hormones in the blood of a pregnant female and fetus modifies the noradrenergic reaction of the hypothalamo-hypophyseal-adrenocortical system, but the direction of the respective changes in offspring males and females is opposite to that observed in prenatally stressed animals.Neirofiziologiya/Neurophysiology, Vol. 37, No. 1, pp. 21–25, January–February, 2005.     

Rubidium Chloride Fuses Split Circadian Activity Rhythms in Hamsters Housed in Bright Constant Light

Chronotypic effects of rubidium (Rb) were examined in hamsters whose circadian activity rhythms had split into two components while they were housed in bright constant light. Seven of 12 hamsters receiving RbCl in drinking water for 10 weeks showed fusing of the components into an intact rhythm compared with none of 7 control hamsters (p = 0.016). Rb may modify coupling between circadian oscillators via reduced photic input to the suprachiasmatic nuclei. Alternative mechanisms include changes in potassium metabolism or endocrine function or behavioral changes that in turn alter circadian function. This normalization of a circadian anomaly by a putative antidepressant suggests that Rb may be valuable in strengthening coupling between oscillators in cases of human chronopathology, including those implicated in the etiology of some affective disorders.     

Rubidium Chloride Fuses Split Circadian Activity Rhythms in Hamsters Housed in Bright Constant Light

Chronotypic effects of rubidium (Rb) were examined in hamsters whose circadian activity rhythms had split into two components while they were housed in bright constant light. Seven of 12 hamsters receiving RbCl in drinking water for 10 weeks showed fusing of the components into an intact rhythm compared with none of 7 control hamsters (p = 0.016). Rb may modify coupling between circadian oscillators via reduced photic input to the suprachiasmatic nuclei. Alternative mechanisms include changes in potassium metabolism or endocrine function or behavioral changes that in turn alter circadian function. This normalization of a circadian anomaly by a putative antidepressant suggests that Rb may be valuable in strengthening coupling between oscillators in cases of human chronopathology, including those implicated in the etiology of some affective disorders.     

Circadian Changes in Anxiolysis-Related Behavior of Syrian Hamsters. Correlation with Hypothalamic GABA Release

The recently developed method of studying fungi inside a soil by fluorescence microscopy was used to examine the periodism of trap formation by predacious fungi. Therefore, this method was supplemented by a technique, which also permitted serial sections of the soil and the counting of capture organs of Arthrobotrys oligospora in the soil. The combination of these methods also allowed spotting of the traps at their exact location, thus giving evidence about their spatial, three dimensional pattern and demonstrating the relative predatory success in soil. It was shown that, in spite of a virtually homogeneous induction by eelworms, the distribution is distinctly periodical. A period of 29.65 ± 4.14 h was calculated. Additional EDX-measurements of the mycelia showed maxima of mineral content, the distance of which agreed with the period length observed. These mineral accumulations obviously marked the sites of potential trap formation.