Light-induced c-Fos expression in the SCN and behavioural phase shifts of Djungarian hamsters with a delayed activity onset

C-Fos expression in the suprachiasmatic nucleus (SCN) and phase shifts of the activity rhythm following photic stimulation were investigated in Djungarian hamsters (Phodopus sungorus) of two different circadian phenotypes. Wild-type (WT) hamsters display robust daily patterns of locomotor activity according to the light/dark conditions. Hamsters of the DAO (delayed activity onset) phenotype, however, progressively delay the activity onset, whereas activity offset remains coupled to “light-on”. Although the exact reason for the delayed activity onset is not yet clarified, it is connected with a disturbed interaction between the light/dark cycle and the circadian clock. The aim was to test the link between photoreception and the behavioral output of the circadian system in hamsters of both phenotypes, to get further insight in the underlying mechanism of the DAO phenomenon. Animals were exposed to short light pulses at different times during the dark period to analyze phase shifts of the activity rhythm and expression of Fos protein in the SCN. The results indicate that the photosensitive phase in DAO hamsters is shifted like the activity onset. Also, phase shifts were significantly smaller in DAO hamsters. At the same time, levels of Fos expression did not differ between phenotypes regarding the circadian phase. The results provide evidence that the shifted photosensitivity of the circadian system in DAO hamsters does not differ from that of WT animals, and lead us to conclude that processes within the SCN that enable light information to reset the circadian pacemaker might offer an explanation for the DAO phenomenon.     

Re-Entrainment Behavior of Djungarian Hamsters (phodopus sungorus) with Different Rhythmic Phenotype Following Light-Dark Shifts

Djungarian hamsters bred at the authors' institute reveal two distinct circadian phenotypes, the wild-type (WT) and DAO type. The latter is characterized by a delayed activity-onset, probably due to a deficient mechanism for photic entrainment. Experiments with zeitgeber shifts have been performed to gain further insight into the mechanisms underlying this phenomenon. Advancing and delaying phase shifts were produced by a single lengthening or shortening of the dark (D) or light (L) time by 6?h. Motor activity was recorded by passive infrared motion detectors. All WT hamsters re-entrained following various zeitgeber shifts and nearly always in the same direction as the zeitgeber shift. On the other hand, a considerable proportion of the DAO animals failed to re-entrain and showed, instead, diurnal, arrhythmic, or free-running activity patterns. All but one of those hamsters that re-entrained did so by delaying their activity rhythm independently of the direction of the LD shift. Resynchronization occurred faster following a delayed than an advanced shift and also after changes of D rather than L. WT animals tended to re-entrain faster, particularly following a zeitgeber advance (where DAO hamsters re-entrained by an 18-h phase delay instead of a 6-h phase advance). However, the difference between phenotypes was statistically significant only with a shortening of L. To better understand re-entrainment behavior, Type VI phase-response curves (PRCs) were constructed. To do this, both WT and DAO animals were kept under LD conditions, and light pulses (15 min, 100 lux) were applied at different times of the dark span. In WT animals, activity-offset always showed phase advances, whereas activity-onset was phase delayed by light pulses applied during the first half of the dark time and not affected by light pulses applied during the second half. When the light pulse was given at the beginning of D, activity-onset responded more strongly, but light pulses given later in D produced significant changes only in activity-offset. In accord with the delayed activity-onset in DAO hamsters, no or only very weak phase-responses were observed when light pulses were given during the first hours of D. However, the second part of the PRCs was similar to that of WT hamsters, even though it was compressed to an interval of only a few hours and the shifts were smaller. Due to these differences, the first light-on or light-off following an LD shift fell into different phases of the PRC and thus caused different re-entrainment behavior. The results show that it is not only steady-state entrainment that is compromised in DAO hamsters but also their re-entrainment behavior following zeitgeber shifts. (Author correspondence: weinert@zoologie.uni-halle.de)     

Re-entrainment behavior of Djungarian hamsters (Phodopus sungorus) with different rhythmic phenotype following light-dark shifts

Djungarian hamsters bred at the authors' institute reveal two distinct circadian phenotypes, the wild-type (WT) and DAO type. The latter is characterized by a delayed activity-onset, probably due to a deficient mechanism for photic entrainment. Experiments with zeitgeber shifts have been performed to gain further insight into the mechanisms underlying this phenomenon. Advancing and delaying phase shifts were produced by a single lengthening or shortening of the dark (D) or light (L) time by 6?h. Motor activity was recorded by passive infrared motion detectors. All WT hamsters re-entrained following various zeitgeber shifts and nearly always in the same direction as the zeitgeber shift. On the other hand, a considerable proportion of the DAO animals failed to re-entrain and showed, instead, diurnal, arrhythmic, or free-running activity patterns. All but one of those hamsters that re-entrained did so by delaying their activity rhythm independently of the direction of the LD shift. Resynchronization occurred faster following a delayed than an advanced shift and also after changes of D rather than L. WT animals tended to re-entrain faster, particularly following a zeitgeber advance (where DAO hamsters re-entrained by an 18-h phase delay instead of a 6-h phase advance). However, the difference between phenotypes was statistically significant only with a shortening of L. To better understand re-entrainment behavior, Type VI phase-response curves (PRCs) were constructed. To do this, both WT and DAO animals were kept under LD conditions, and light pulses (15 min, 100 lux) were applied at different times of the dark span. In WT animals, activity-offset always showed phase advances, whereas activity-onset was phase delayed by light pulses applied during the first half of the dark time and not affected by light pulses applied during the second half. When the light pulse was given at the beginning of D, activity-onset responded more strongly, but light pulses given later in D produced significant changes only in activity-offset. In accord with the delayed activity-onset in DAO hamsters, no or only very weak phase-responses were observed when light pulses were given during the first hours of D. However, the second part of the PRCs was similar to that of WT hamsters, even though it was compressed to an interval of only a few hours and the shifts were smaller. Due to these differences, the first light-on or light-off following an LD shift fell into different phases of the PRC and thus caused different re-entrainment behavior. The results show that it is not only steady-state entrainment that is compromised in DAO hamsters but also their re-entrainment behavior following zeitgeber shifts.     

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 1h 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.     

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.     

Voluntary exercise stabilizes photic entrainment of djungarian hamsters (Phodopus sungorus) with a delayed activity onset

ABSTRACT

The Djungarian hamsters of our breeding colony show unstable daily activity patterns when kept under standard laboratory conditions. Moreover, part of them develops a delayed activity onset (DAO) or an arrhythmic phenotype. In former studies, we have shown that the system of photic entrainment works at its limits. If the period length (tau) increases, which is the case in DAO hamsters, the light-induced phase advances are too small to compensate the daily delay of the activity rhythm caused by tau being longer than 24 h. Accordingly, under natural conditions, there must be further (environmental) factors to enable a stable entrainment. One of these may be the higher level of motor activity. Animals must cover long distances to search for food, sexual partners and others. In the laboratory, hamsters are kept singly in small cages. This does restrict animals’ options for motor activity. Also, there is less need for moving around as the hamsters are fed ad libitum.

In the present study, a series of experiments was performed to investigate the putative effect of the activity level. To begin with, wild type (WT) and DAO animals were given access to running wheels. 50% of DAO hamsters developed a WT activity pattern. As the main reason for the DAO phenomenon is their long tau together with a too weak photic phase response, the effect of wheel running on these parameters was investigated in further experiments. With higher activity level, tau decreased in WT hamsters but increased in DAO animals even though the increase for the activity onset was only close to significance. Moreover, the photic phase responses were weaker though significant only for the activity offset of DAO hamsters.

Based on the assumptions that running wheel activity will affect the phase response and/or the free running period, the results of the present paper do not provide an explanation for why part of DAO hamsters developed a WT phenotype when they had access to running wheels. Obviously, mechanisms downstream from the suprachiasmatic nuclei must be taken into account when investigating the stabilizing, improving circadian entrainment effect of motor activity.     

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.     

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)     

Novel object recognition of Djungarian hamsters depends on circadian time and rhythmic phenotype

Circadian rhythms have been shown to influence learning and memory. In this study, cognitive functions of Djungarian hamsters revealing different circadian phenotypes were evaluated using a novel object recognition (NOR) task. Wild type (WT) animals show a clear and well-synchronized daily activity rhythm, whereas DAO hamsters are characterized by a delayed activity onset. The phenomenon is caused by a diminished ability of photic synchronization. In arrhythmic (AR) hamsters, the suprachiasmatic nuclei (SCN) do not generate a circadian signal at all. The aim of this study was to investigate consequences of these deteriorations for learning and memory processes. Hamsters were bred and kept under standardized housing conditions with food and water ad libitum and a 14?L/10?D lighting regimen. Experimental animals were assigned to different groups (WT, DAO and AR) according to their activity pattern obtained by means of infrared motion sensors. Activity onset of DAO animals was delayed by 3?±?0.5?h. NOR tests were performed in an open arena and consisted of habituation, training (two identical objects) and test sessions (one of the two objects being replaced). The training–test interval was 60?min. Tests were performed at different Zeitgeber times (ZT 0?=?light-on). Every hamster was tested at all times with an interval of one week between experiments. As activity onset of DAO animals is delaying continuously day by day, they could be tested at only three times (ZT 13, ZT 16 and ZT 19). The times animals did explore the novel and the familiar objects were recorded, and the discrimination index as a measure of cognitive performance was calculated. Behavioral analyzes revealed that, WT hamsters were able to discriminate between familiar and novel objects at ZT 13, ZT 16 and ZT 19, i.e. one hour before and during their activity period. In accordance with their delayed activity onset, DAO hamsters could discriminate between objects only at ZT 16 and ZT 19 what corresponds also to 1?h before and 2?h after their activity onset. In contrast, AR hamsters were not able to perform the NOR task at any time. The results show that the SCN modulate learning and memory in a circadian manner. Moreover, the loss of circadian rhythmicity results in cognitive impairments.     

An inbred lineage of djungarian hamsters with a strongly attenuated ability to synchronize

An inbred lineage of Ph. sungorus was established at our institute showing some unusual characteristics of the circadian system that appear incompatible with an adequate adaptation to the periodic environment. We identified a hamster for which activity onset was delayed under light-dark conditions (L:D=14:10 h) by about 4 h in relation to the light-dark transition. As the activity offset remained synchronized with the time of light-on, the activity period (alpha) became compressed to 6 h. By means of a special breeding program, the percentage of animals showing such a phenomenon increased, indicating that it has a genetic component. Also, it is possible now to breed a larger number of hamsters to further investigate the rhythm deviations and the underlying mechanisms. Activity rhythms were investigated using passive infrared motion sensors. Whereas some of the hamsters showed a rather stable phase delay of activity onset relative to the onset of darkness, some animals progressively delayed their activity onset up to a critical, minimal length of alpha (3.03+/-0.02 h). Thereafter, the rest-activity rhythm started to free-run with a remarkably long period (tau=25.02 h) or became arrhythmic. Some hamsters showed several consecutive cycles alternating between a free-running rhythm and entrainment, with increasing tau and reducing the phases of temporary entrainment. Finally, these hamsters became arrhythmic. The total amount of activity per day was similar in the wild type and delayed activity onset hamsters. The latter did increase the intensity of activity, thereby compensating for the shorter alpha. The period length in constant darkness was significantly longer in the delayed hamsters compared to wild type animals (24.37+/-0.03 h vs. 24.24+/-0.02 h; p<0.001). However, this difference seems too small to cause the later activity onset. The phase response following a light pulse (100 lux, 15' at CT14 where CT12=activity onset) was similar in delayed and wild type hamsters (-1.66+/-0.12 h and -1.82+/-0.16 h). As access to running wheels is known to influence the circadian pacemaker, particularly to strengthen oscillator coupling, a set of further experiments was conducted. The free-running period was significantly shorter when the hamsters were provided with running wheels (24.25+/-0.04 h and 24.07+/-0.04 h in wild type and delayed hamsters, respectively; p<0.005 and p<0.05). However, the effect on the activity onset was not unequivocal. In many hamsters it was still delayed, whereas in others the unlocking of the wheels led to an expansion of alpha. The described inbred lineage appears to be an excellent model to further investigate the properties and the interaction of the two oscillators underlying the daily activity pattern.     

Running activity mediates the phase-advancing effects of dark pulses on hamster circadian rhythms

Summary Pulses of darkness can phase-shift the circadian activity rhythms of hamsters,Mesocricetus auratus, kept in constant light. Dark pulses under these conditions alter photic input to the circadian system, but they also commonly trigger wheel-running activity. This paper investigates the contribution of running activity to the phase-shifting effects of dark pulses. A first experiment showed that running activity by itself can phaseshift rhythms in constant light. Hamsters were induced to run by being confined to a novel wheel for 3–5 h. When this was done at circadian times (CT) 0, 6, and 9, the mean steady-state phase-shifts were 0.6 h, 3.5 h, and 2.3 h, respectively. The latter two values are at least as large as those previously obtained with dark pulses of similar durations and circadian phases. A second experiment showed that restricting the activity of hamsters during 3-h dark pulses at CT 9 reduces the amplitude of the phase-shifts. Unrestrained animals phase-advanced by 1.1 h, but this shift was halved in animals whose wheel was locked, and completely abolished in animals confined to nest boxes during the dark pulse. Activity restriction in itself (without dark pulses) had only minimal phase-delaying effects on free-running rhythms when given between ca. CT 10 and CT 13. These results support the idea that, in hamsters at least, dark pulses affect the circadian system mostly by altering behavioural states rather than by altering photic input to the internal clock.Abbreviations CT circadian time - DD constant darkness - LD light-dark - LL constant light - PRC phase response curve - period of rhythm     

Entrainment of 2 subjective nights by daily light:dark:light:dark cycles in 3 rodent species

Recent work with exotic 24-h light:dark:light:dark (LDLD) cycles indicates surprising flexibility in the entrainment patterns of Syrian hamsters. Following exposure to an LDLD cycle, hamsters may adopt a form of rhythm splitting in which markers of subjective night (e.g., activity, melatonin) are expressed in each of the twice daily scotophases. This pattern contrasts markedly with that of conventionally entrained hamsters in which markers of subjective night are expressed once daily in only 1 of the 2 dark periods. The "split" entrainment pattern was examined further here in Syrian and Siberian hamsters and in mice exposed to LDLD 7:5:7:5, a condition that reliably induces split activity rhythms in all 3 species. The phase angle of entrainment and activity duration were generally similar comparing the 2 daily activity bouts in each species. The stability of this split entrainment state was assessed by deletions of photophases on individual days, by exposure to skeleton photoperiods, and by transfer to constant darkness. As in Syrian hamsters, the one-time substitution of darkness for one 7-h photophase did not grossly alter activity patterns of Siberian hamsters but acutely disrupted the split rhythms of mice. Skeleton light pulses of progressively shorter duration did not significantly alter split entrainment patterns of either Syrian or Siberian hamsters. Both species continued to exhibit stable entrainment with activity expressed in alternate scotophases of an LD 1:5 cycle presented 4 times daily. In contrast, the split activity rhythms of mice were not maintained under skeleton pulses. In constant darkness, rhythms of Siberian hamsters remained distinctly split for a minimum of 2 cycles. Split entrainment to these novel LDLD and 4-pulse skeleton lighting regimes demonstrates a marked degree of plasticity common to the circadian systems of several rodent species and identifies novel entrainment patterns that may be reliably elicited with simple environmental manipulations. Inter- and intraspecific differences in the stability of split activity rhythms likely reflect differences in coupling interactions between the component circadian oscillators, which, adopting separate phase relations to these novel LD cycles, yield a split entrainment pattern.     

Circadian phase shifting induced by clonidine injections in Syrian hamsters

Abstract

The mammalian circadian pacemaker can be phase shifted by photic, pharmacological, and behaviorally‐derived stimuli. The phase‐response curves (PRCs) characterizing these diverse stimuli may comprise two distinct families; a photic PRC typified by the response to brief light pulses, and a non‐photic PRC, typified by the response to dark pulses and to behavioral activation. The present study examined the phase shifting effects of acute systemic treatment with the alpha2‐adrenoceptor agonist, clonidine, in Syrian hamsters. Clonidine injections (0.25 mg/kg, ip) delivered during subjective night mimicked the phase shifting effects of light pulses in animals housed in both constant darkness (DD) and constant red light (RR), but similar effects were not seen in saline‐treated controls. Both clonidine and saline injections resulted in phase advances during subjective day, but only in RR‐housed animals. Clonidine‐induced phase shifting was dose‐dependent, but rather high doses were required to induce phase shifts. Pretreatment with the selective noradrenergic neurotoxin, DSP‐4, blocked clonidine‐induced phase shifting. These results suggest that clonidine acts at presynaptic alpha2‐adrenergic autoreceptors to disinhibit spontaneous and/or evoked activity in the photic entrainment pathway.     

Evidence for independence of circadian characters and extent of photoresponsiveness in the Djungarian hamster, Phodopus sungorus.

Djungarian hamsters (Phodopus sungorus) exposed to a short-day photoperiod generally respond with a syndrome of physiological and behavioral changes, such as body weight loss and molt to a white pelage. The extent of the short-day-induced responses differs among individuals. Furthermore, some hamsters show no photoresponse. In this study, we sought to determine whether variation in the photoresponse would be associated with circadian function: whether phase angle or free-running period (tau) would differ between responsive and nonresponsive hamsters; and whether changes in these circadian characters would correlate with the extent of weight loss and molt (and the timing of molt onset) in photoresponsive hamsters. Adult hamsters were kept in a short-day photoperiod (9 hr light, 15 hr dark) for 14 weeks, during which time body weight and molt were measured biweekly. Hamsters were then transferred to cages equipped with running wheels; we measured the phase angle of activity onset under a short-day photoperiod and tau in constant dark. Hamsters exhibiting a short-day-induced molt had a significantly shorter tau and a less negative phase angle than nonmolting animals. Hamsters that exhibited weight loss also had a significantly less negative phase angle, but no difference in tau. No significant Pearson's or Spearman's correlation coefficients were found between extent (or timing) of the photoresponse and the circadian characters in responsive hamsters. Although these results indicate that threshold for photoresponsiveness is related to circadian function, the extent (and timing) of the photoresponse may not be.     

Circadian phase response curves for dark pulses in the hamster

Summary Hamsters maintained under constant illumination were exposed to 2- or 6-h pulses of darkness at various phases of their circadian activity rhythms. When presented around the time of activity onset, the pulses resulted in phase advances, and when presented toward the end of daily activity, they resulted in phase delays. Since others have shown that light pulses presented at the same phases in constant darkness cause phase shifts in the opposite directions, these results indicate that phase response curves for light and dark pulses are mirror images.Dark pulses also caused phase-dependent changes, both transient and long-lasting, in the period of the free-running rhythms, and a few pulses were immediately followed by splitting of the activity rhythms into two components. Such effects may reflect a differential responsiveness of two coupled oscillators to dark pulses.Abbreviations CT circadian time - DD constant dark - LD lightdark - LL constant light - PRC phase response curve - SD subjective day - SN subjective night - period of a circadian rhythm Supported by grants from the NSERC of Canada to B. Rusak and to G.V. Goddard. We are grateful to Dr. Goddard for his support and encouragement     

An Inbred Lineage of Djungarian Hamsters with a Strongly Attenuated Ability to Synchronize

An inbred lineage of Ph. sungorus was established at our institute showing some unusual characteristics of the circadian system that appear incompatible with an adequate adaptation to the periodic environment. We identified a hamster for which activity onset was delayed under light‐dark conditions (L:D=14∶10 h) by about 4 h in relation to the light‐dark transition. As the activity offset remained synchronized with the time of light‐on, the activity period (α) became compressed to 6 h. By means of a special breeding program, the percentage of animals showing such a phenomenon increased, indicating that it has a genetic component. Also, it is possible now to breed a larger number of hamsters to further investigate the rhythm deviations and the underlying mechanisms. Activity rhythms were investigated using passive infrared motion sensors. Whereas some of the hamsters showed a rather stable phase delay of activity onset relative to the onset of darkness, some animals progressively delayed their activity onset up to a critical, minimal length of α (3.03±0.02 h). Thereafter, the rest‐activity rhythm started to free‐run with a remarkably long period (τ=25.02 h) or became arrhythmic. Some hamsters showed several consecutive cycles alternating between a free‐running rhythm and entrainment, with increasing τ and reducing the phases of temporary entrainment. Finally, these hamsters became arrhythmic. The total amount of activity per day was similar in the wild type and delayed activity onset hamsters. The latter did increase the intensity of activity, thereby compensating for the shorter α. The period length in constant darkness was significantly longer in the delayed hamsters compared to wild type animals (24.37±0.03 h vs. 24.24±0.02 h; p<0.001). However, this difference seems too small to cause the later activity onset. The phase response following a light pulse (100 lux, 15′ at CT14 where CT12=activity onset) was similar in delayed and wild type hamsters (?1.66±0.12 h and ?1.82±0.16 h). As access to running wheels is known to influence the circadian pacemaker, particularly to strengthen oscillator coupling, a set of further experiments was conducted. The free‐running period was significantly shorter when the hamsters were provided with running wheels (24.25±0.04 h and 24.07±0.04 h in wild type and delayed hamsters, respectively; p<0.005 and p<0.05). However, the effect on the activity onset was not unequivocal. In many hamsters it was still delayed, whereas in others the unlocking of the wheels led to an expansion of α. The described inbred lineage appears to be an excellent model to further investigate the properties and the interaction of the two oscillators underlying the daily activity pattern.     

Nonphotic entrainment in a diurnal mammal, the European ground squirrel (Spermophilus citellus).

Entrainment by nonphotic, activity-inducing stimuli has been investigated in detail in nocturnal rodents, but little is known about nonphotic entrainment in diurnal animals. Comparative studies would offer the opportunity to distinguish between two possibilities. (1) If nonphotic phase shifts depend on the phase of the activity cycle, the phase response curve (PRC) should be about 180 degrees out of phase in nocturnal and diurnal mammals. (2) If nonphotic phase shifts depend on the phase of the pacemaker, the two PRCs should be in phase. We used the diurnal European ground squirrel (Spermophilus citellus) in a nonphotic entrainment experiment to distinguish between the two possibilities. Ten European ground squirrels were kept under dim red light (<1 lux) and 20 +/- 1 degrees C. During the entrainment phase of the experiment, the animals were confined every 23.5 h (T) to a running wheel for 3 h. The circadian rhythms of 6 squirrels entrained, 2 continued to free run, and 2 possibly entrained but displayed arrhythmicity during the experiment. In a second experiment, a photic pulse was used in a similar protocol. Five out of 9 squirrels entrained, 1 did not entrain, and 3 yielded ambiguous results. During stable entrainment, the phase-advancing nonphotic pulses coincided with the end of the subjective day, while phase-advancing light pulses coincided with the start of the subjective day: mean psi(nonphotic) = 11.4 h; mean psi(photic) = 0.9 h (psi defined as the difference between the onset of activity and the start of the pulse). The data for nonphotic entrainment correspond well with those from similar experiments with nocturnal Syrian hamsters where psi(nonphotic) varied from 8.09 to 11.34 h. This indicates that the circadian phase response to a nonphotic activity-inducing stimulus depends on the phase of the pacemaker rather than on the phase of the activity cycle.     

Circadian rhythm of locomotor activity in the Japanese honeybee, Apis cerana japonica

Abstract.  To reveal circadian characteristics and entrainment mechanisms in the Japanese honeybee Apis cerana japonica , the locomotor-activity rhythm of foragers is investigated under programmed light and temperature conditions. After entrainment to an LD 12 : 12 h photoperiodic regime, free-running rhythms are released in constant dark (DD) or light (LL) conditions with different free-running periods. Under the LD 12 : 12 h regime, activity offset occurs approximately 0.4 h after lights-off transition, assigned to circadian time (Ct) 12.4 h. The phase of activity onset, peak and offset, and activity duration depends on the photoperiodic regimes. The circadian rhythm can be entrained to a 24-h period by exposure to submultiple cycles of LD 6 : 6 h, as if the locomotive rhythm is entrained to LD 18 : 6 h. Phase shifts of delay and advance are observed when perturbing single light pulses are presented during free-running under DD conditions. Temperature compensation of the free-running period is demonstrated under DD and LL conditions. Steady-state entrainment of the locomotor rhythm is achieved with square-wave temperature cycles of 10 °C amplitude, but a 5 °C amplitude fails to entrain.     

Effects of the duper mutation on circadian responses to light

The circadian mutation duper in Syrian hamsters shortens the free-running circadian period (τ(DD)) by 2 hours when expressed on a tau mutant (τ(ss)) background and by 1 hour on a wild-type background. We have examined the effects of this mutation on phase response curves and entrainment. In contrast to wild types, duper hamsters entrained to 14L:10D with a positive phase angle. Super duper hamsters (expressing duper on a τ(ss) background) showed weak entrainment, while τ(ss) animals either completely failed to entrain or showed sporadic entrainment with episodes of relative coordination. As previously reported, wild-type and τ(ss) hamsters show low amplitude resetting in response to 15-minute light pulses after short-term (10 days) exposure to DD. In contrast, super duper hamsters show high amplitude resetting. This effect is attributable to the duper allele, as hamsters carrying duper on a wild-type background also show large phase shifts. Duper mutants that were born and raised in DD also showed high amplitude resetting in response to 15-minute light pulses, indicating that the effect of the mutation on PRC amplitude is not an aftereffect of entrainment to 14L:10D. Hamsters that are heterozygous for duper do not show amplified resetting curves, indicating that for this property, as for determination of free-running period, the mutant allele is recessive. In a modified Aschoff type II protocol, super duper and duper hamsters show large phase shifts as soon as the second day of DD. Despite the amplification of the PRC in super duper hamsters, the induction of Period1 gene expression in the SCN by light is no greater in these mutants than in wild-type animals. Period2 expression in the SCN did not differ between super duper and wild-type hamsters exposed to light at CT15, but albumin site D-binding protein (Dbp) mRNA showed higher basal levels and greater light induction in the SCN of super duper compared to wild-type animals. These results indicate that the duper mutation alters the amplitude of the circadian oscillator and further distinguish it from the tau mutation.