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.     

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)     

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.     

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.     

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.     

Intensive voluntary wheel running may restore circadian activity rhythms and improves the impaired cognitive performance of arrhythmic Djungarian hamsters

Circadian rhythms are highly important not only for the synchronization of animals and humans with their periodic environment but also for their fitness. Accordingly, the disruption of the circadian system may have adverse consequences. A certain number of animals in our breeding stock of Djungarian hamsters are episodically active throughout the day. Also body temperature and melatonin lack 24-h rhythms. Obviously in these animals, the suprachiasmatic nuclei (SCN) as the central pacemaker do not generate a circadian signal. Moreover, these so-called arrhythmic (AR) hamsters have cognitive deficits. Since motor activity is believed to stabilize circadian rhythms, we investigated the effect of voluntary wheel running. Hamsters were bred and kept under standardized housing conditions with food and water ad libitum and a 14 L/10 D lighting regimen. AR animals were selected according to their activity pattern obtained by means of passive infrared motion detectors. In a first step, the daily activity behavior was investigated for 3 weeks each without and with running wheels. To estimate putative photic masking effects, hamsters were exposed to light (LPs) and DPs and also released into constant darkness for a minimum of 3 weeks. A novel object recognition (NOR) test was performed to evaluate cognitive abilities both before and after 3 weeks of wheel availability. The activity patterns of hamsters with low wheel activity were still AR. With more intense running, daily patterns with higher values in the dark time were obtained. Obviously, this was due to masking as LPs did suppress and DPs induced motor activity. When transferred to constant darkness, in some animals the daily rhythm disappeared. In other hamsters, namely those which used the wheels most actively, the rhythm was preserved and free-ran, what can be taken as indication of a reconstitution of circadian rhythmicity. Also, animals showing a 24-h activity pattern after 3 weeks of extensive wheel running were able to recognize the novel object in the NOR test but not so before. The results show that voluntary exercise may reestablish circadian rhythmicity and improve cognitive performance.     

Non-parametric photic entrainment of Djungarian hamsters with different rhythmic phenotypes

To investigate the role of non-parametric light effects in entrainment, Djungarian hamsters of two different circadian phenotypes were exposed to skeleton photoperiods, or to light pulses at different circadian times, to compile phase response curves (PRCs). Wild-type (WT) hamsters show daily rhythms of locomotor activity in accord with the ambient light/dark conditions, with activity onset and offset strongly coupled to light-off and light-on, respectively. Hamsters of the delayed activity onset (DAO) phenotype, in contrast, progressively delay their activity onset, whereas activity offset remains coupled to light-on. The present study was performed to better understand the underlying mechanisms of this phenomenon. Hamsters of DAO and WT phenotypes were kept first under standard housing conditions with a 14:10 h light–dark cycle, and then exposed to skeleton photoperiods (one or two 15-min light pulses of 100 lx at the times of the former light–dark and/or dark–light transitions). In a second experiment, hamsters of both phenotypes were transferred to constant darkness and allowed to free-run until the lengths of the active (α) and resting (ρ) periods were equal (α:ρ = 1). At this point, animals were then exposed to light pulses (100 lx, 15 min) at different circadian times (CTs). Phase and period changes were estimated separately for activity onset and offset. When exposed to skeleton-photoperiods with one or two light pulses, the daily activity patterns of DAO and WT hamsters were similar to those obtained under conditions of a complete 14:10 h light–dark cycle. However, in the case of giving only one light pulse at the time of the former light–dark transition, animals temporarily free-ran until activity offset coincided with the light pulse. These results show that photic entrainment of the circadian activity rhythm is attained primarily via non-parametric mechanisms, with the “morning” light pulse being the essential cue. In the second experiment, typical photic PRCs were obtained with phase delays in the first half of the subjective night, phase advances in the second half, and a dead zone during the subjective day. ANOVA indicated no significant differences between WT and DAO animals despite a significantly longer free-running period (tau) in DAO hamsters. Considering the phase shifts induced around CT0 and the different period lengths, it was possible to model the entrainment patterns of both phenotypes. It was shown that light-induced phase shifts of activity offset were sufficient to compensate for the long tau in WT and DAO hamsters, thus enabling a stable entrainment of their activity offsets to be achieved. With respect to activity onsets, phase shifts were sufficient only in WT animals; in DAO hamsters, activity onset showed increasing delays. The results of the present paper clearly demonstrate that, under laboratory conditions, the non-parametric component of light and dark leads to circadian entrainment in Djungarian hamsters. However, a stable entrainment of activity onset can be achieved only if the free-running period does not exceed a certain value. With longer tau values, hamsters reveal a DAO phenotype. Under field conditions, therefore, non-photic cues/zeitgebers must obviously be involved to enable a proper circadian entrainment.     

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.     

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.     

Spatial Memory and Long-Term Object Recognition Are Impaired by Circadian Arrhythmia and Restored by the GABAAAntagonist Pentylenetetrazole

Performance on many memory tests varies across the day and is severely impaired by disruptions in circadian timing. We developed a noninvasive method to permanently eliminate circadian rhythms in Siberian hamsters (Phodopussungorus) so that we could investigate the contribution of the circadian system to learning and memory in animals that are neurologically and genetically intact. Male and female adult hamsters were rendered arrhythmic by a disruptive phase shift protocol that eliminates cycling of clock genes within the suprachiasmatic nucleus (SCN), but preserves sleep architecture. These arrhythmic animals have deficits in spatial working memory and in long-term object recognition memory. In a T-maze, rhythmic control hamsters exhibited spontaneous alternation behavior late in the day and at night, but made random arm choices early in the day. By contrast, arrhythmic animals made only random arm choices at all time points. Control animals readily discriminated novel objects from familiar ones, whereas arrhythmic hamsters could not. Since the SCN is primarily a GABAergic nucleus, we hypothesized that an arrhythmic SCN could interfere with memory by increasing inhibition in hippocampal circuits. To evaluate this possibility, we administered the GABA_A antagonist pentylenetetrazole (PTZ; 0.3 or 1.0 mg/kg/day) to arrhythmic hamsters for 10 days, which is a regimen previously shown to produce long-term improvements in hippocampal physiology and behavior in Ts65Dn (Down syndrome) mice. PTZ restored long-term object recognition and spatial working memory for at least 30 days after drug treatment without restoring circadian rhythms. PTZ did not augment memory in control (entrained) animals, but did increase their activity during the memory tests. Our findings support the hypothesis that circadian arrhythmia impairs declarative memory by increasing the relative influence of GABAergic inhibition in the hippocampus.     

Memory for Time of Day (Time Memory) Is Encoded by a Circadian Oscillator and Is Distinct From Other Context Memories

We report that the neural representation of the time of day (time memory) in golden hamsters involves the setting of a 24-h oscillator that is functionally and anatomically distinct from the circadian clock in the suprachiasmatic nucleus (SCN), but is entrained by the SCN acting as a weak zeitgeber. In hamsters, peak conditioned place avoidance (CPA) was expressed only near the time of day of the learning experience (±2?h) for the first days after conditioning. On a 14:10 light:dark cycle, with conditioning at the end of the light period (zeitgeber time 11 [ZT11]), CPA behavior, including time of day memory, was retained for more than 18 d. With conditioning in the early day (zeitgeber time 03 [ZT03]), CPA was completely lost after 5 d but reemerged after an additional 6 d, with the peak avoidance time shifted to ZT11. When the entraining light cycle was shifted immediately following learning at either ZT11 or ZT03, with no additional experience in the training apparatus, peak CPA 18 d later was always found at ZT11 on the shifted light cycles. When conditioned at ZT03, then placed into constant dark for 18 cycles, the peak shifted to subjective circadian time 11 (CT11). In all experiments, the peak CPA time was set initially to the time of experience, and was reset subsequently to the end of the subjective day, without memory loss for other context associations. In the absence of an SCN, peak avoidance was not reset. Therefore, time memory is distinct from other context memories, and involves the setting of a non-SCN circadian oscillator. We suggest that circadian oscillators underlying time memory work in concert with the SCN to enable anticipation of critical conditions according to both immediate- and long-term probabilities of where and when important conditions could be encountered again. (Author correspondence: ralph@psych.utoronto.ca)     

Novel wheel running blocks the preovulatory luteinizing hormone surge and advances the hamster circadian pacemaker

In rodents, the preovulatory luteinizing hormone (LH) surge is timed by a circadian rhythm. We recently reported that a phenobarbital-induced delay of the estrous cycle in Syrian hamsters is associated with an approximately 2-h phase advance in both the circadian locomotor activity rhythm and the timing of the LH surge. The following study tests the hypothesis that a >2-h nonpharmacological phase advance in the circadian pacemaker that delays the estrous cycle by a day will also phase advance the LH surge by approximately 2 h. Activity rhythms were continuously monitored in regularly cycling hamsters using running wheels or infrared detectors for about 10 days prior to jugular cannulation. The next day, on proestrus, hamsters were transferred to the laboratory for 1 of 3 treatments: transfer to a "new cage" (and wheel) from zeitgeber time (ZT) 4 to 8 (with ZT12 defined as time of lights-off), or exposure to a "novel wheel" at ZT5 or ZT1. All animals were then placed in constant dark (DD). Blood samples were obtained just before onset of DD and hourly for the next 6 h, on that day and the next day for determination of plasma LH concentrations. Running activity was monitored in DD for about 10 more days. Transfer to a novel wheel at either ZT5 or ZT1 delayed the LH surge to day 2 in most hamsters, whereas exposure to a new cage did not. Only the delayed LH surges were phase advanced at least 2.5 h on average in all 3 groups. However, wheel-running activity was similarly phase advanced in all 3 groups regardless of the timing of the LH surge; thus, the phase advances in circadian activity rhythms were not associated with the 1-day delay of the LH surge. Interestingly, the number of wheel revolutions was closely associated with the 1-day delay of LH surges following exposure to a novel wheel at either ZT1 or ZT5. These results suggest that the intensity of wheel running (or an associated stimulus) plays an important role in the circadian timing mechanism for the LH surge.     

Effect of melatonin on changes in locomotor activity rhythm of Syrian hamsters injected with beta amyloid peptide 25-35 in the suprachiasmatic nuclei

1. Alzheimer's disease is associated with circadian rhythm disturbances, probably because of beta amyloid-induced neuronal damage of hypothalamic suprachiasmatic nuclei (SCN).2. Since there is no published study on the circadian consequences of injecting beta amyloid peptide in experimental animals, one objective of the present study was to examine circadian locomotor activity in Syrian hamsters injected with beta amyloid peptide 25–35 into both SCN.3. Because one of the proposed therapies for circadian alterations in dementia is the administration of melatonin, a chronobiotic agent with antioxidant properties, the preventive effect of melatonin on the circadian changes produced by beta amyloid microinjection into SCN was also assessed.4. Wheel running activity was recorded by using the Dataquest III system in male golden hamsters kept under 14:10 light–dark photoperiods. Animals received microinjections of beta amyloid peptide 25–35 (100 M solution, 1 L) or saline in each SCN. Only those animals with neuronal lesions larger than 10% of SCN after beta amyloid injection were considered for further analysis.5. To assess the effect of melatonin on beta-amyloid peptide activity, melatonin was given in the drinking water (25 g/mL) starting 15 days in advance to the microinjection of beta amyloid peptide into SCN.6. Beta amyloid-treated hamsters exhibited a significant phase advance of onset of running activity of about 22 min as compared to saline-injected animals. They also showed a significantly greater variability in onset time of wheel running activity, mainly evident from 6 to 15 days of treatment.7. Melatonin administration in the drinking water prevented the phase advance of onset time and the increased variability of onset time brought about by beta amyloid peptide.8. The results support the existence of a neuroprotective effect of melatonin on beta amyloid-induced circadian changes in hamsters.     

A change in the pattern in circadian running‐wheel activity after lesions of the intergeniculate leaflet and ventral lateral geniculate nucleus in the Syrian hamster

Abstract

The suprachiasmatic nuclei (SCN) contain the endogenous mammalian circadian pacemaker, which generates the circadian rhythm in locomotor activity. In Syrian hamsters with free‐running rhythms, the onset of running‐wheel activity is very precise and predictable while the end (offset) is more variable. From the thalamic intergeniculate leaflet (IGL) and the ventral lateral geniculate nucleus (vLGN) a projection to the SCN originates. Animals with a lesion aimed at the IGL/vLGN and sham‐and unoperated controls were kept in continuous darkness. With linear regression, lines were fitted through 10 successive onsets and offsets of activity and the mean deviation of the onsets and offsets from the fitted lines was determined. Animals with a complete or partial lesion of the IGL/vLGN had a smaller mean deviation of the circadian activity offset from the fitted regression line (0.313 h) compared with the grouped control animals (0.678 h). To test the difference statistically, we compared the sum of the square residuals of the circadian offsets between the groups. This difference was highly significant (F(69,64)=4.16, p<0.0001), which indicates that animals with a lesion of the IGL/ vLGN have a less variable circadian offset of running‐wheel activity. No differences were observed in the variability in the circadian onset of locomotor activity between experimental and control animals. It is concluded that the IGL/vLGN influence the variability of the offset of the circadian running‐wheel activity.     

Daily novel wheel running reorganizes and splits hamster circadian activity rhythms.

The phenomenon of splitting of locomotor activity rhythms in constant light has implied that the mammalian circadian pacemaker is composed of multiple interacting circadian oscillators. Exposure of male Syrian hamsters to novel running wheels also induces splitting in some reports, although novel wheel running (NWR) is better known for its effects on altering circadian phase and the length of the free-running period. In three experiments, the authors confirm and extend earlier reports of split rhythms induced by NWR. Male Syrian hamsters, entrained to LD 14:10, were transferred for 6 to 11 consecutive days to darkened novel Wahmann wheels at ZT 4 and were returned to their home cages at ZT 9. All hamsters ran robustly in the novel wheels. NWR caused a marked reorganization of home cage wheel-running behavior: Activity onsets delayed progressively with each additional day of NWR. After 11 days, activity onset in the nighttime scotophase was delayed by 7 h and disappeared completely in 2 hamsters (Experiment 1). After 6 to 7 days of NWR (Experiment 2), activity onset delayed by 5 h. Transfer of hamsters to constant darkness (DD) after 7 days of NWR revealed clearly split activity rhythms: The delayed nighttime activity bout was clearly identifiable and characterized by a short duration. A second bout associated with the former time of NWR was equally distinct and exhibited a similarly short duration. These components rejoined after 3 to 5 days in DD accomplished via delays and advances of the nighttime and afternoon components, respectively. The final experiment established that rejoining of activity components could be prevented by perpetuating the light-dark:light-dark cycle used to induce split rhythms. The data suggest that NWR causes selective phase shifting of some circadian oscillators and that component oscillators interact strongly in constant darkness.     

Daily torpor alters multiple gene expression in the suprachiasmatic nucleus and pineal gland of the Djungarian hamster (Phodopus sungorus)

Circadian rhythms are still expressed in animals that display daily torpor, implying a temperature compensation of the pacemaker. Nevertheless, it remains unclear how the clock works in hypothermic states and whether torpor itself, as a temperature pulse, affects the circadian system. To reveal changes in the clockwork during torpor, we compared clock gene and neuropeptide expression by in situ hybridization in the suprachiasmatic nucleus (SCN) and pineal gland of normothermic and torpid Djungarian hamsters (Phodopus sungorus). Animals from light-dark (LD) 8ratio16 were sacrificed at 8 time points throughout 24 h. To investigate the effect of a previous torpor episode on the clock, we sacrificed a group of normothermic hamsters 1 day after torpor. In normothermic animals, Per1 peaked at zeitgeber time (ZT)4; whereas, Bmal1 reached maximal expression between ZT16 and ZT19. AVP mRNA in the SCN showed highest levels at ZT7. On the day of torpor, the levels of all mRNAs investigated, except for AVP mRNA, were increased during the torpor bout. Moreover, the Bmal1 rhythm was advanced. On the day after the hypothermia, Bmal1 and AVP rhythms showed severely depressed amplitude. Those distinct amplitude changes of Bmal1 and AVP on the day after a torpor episode expression suggests that torpor affects the circadian system, probably by altered translational processes that might lead to a modified protein feedback on gene expression. In the pineal gland, an important clock output, Aanat expression, peaked between ZT16 and ZT22 in normothermic animals. Aanat levels were significantly advanced on the day of hypothermia, an effect which was still visible 1 day afterward. In summary, this study showed that daily torpor affects the phase and amplitude of rhythmic clock gene and clock-controlled gene expression in the SCN. Furthermore, the rhythmic gene expression in a peripheral oscillator, the pineal gland, is also affected.     

大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性

本研究旨在观察和比较视交叉上核（suprachiasmatic nucleus,SCN）与松果体（pineal gland,pG）中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗（constant darkness,DD）和12h光照：12h黑暗交替（12hourlight：12hour-darkcycle,LD）光制下分别被饲养8周（n=36）和4周n=36）后,在一昼夜内每隔4h采集一组SCN和PG组织（n=6）,提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点（circadian times．CT or zeitgeber times．ZT）各样品中Clock基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律性转录变化。结果如下：（1）SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化（P〈0．05）,PG中Clock基因的转录也显示相似的内源性节律外观,即峰值出现于主观夜晚（SCN为CTl5,PG为CT18）,谷值位于主观白天（SCN为CT3,PG为CT6）（P〉0．05）。（2）LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡（P〈0．05）,但与其DD光制下节律外观相比,呈现反时相节律变化（P〈0．05）,且其表达的振幅及峰值的mRNA水平均增加（P〈0．05）,而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反（P〈0．05）。（3）在LD光制下,光照使PG中Clock基因转录的节律外观反时相于SCN（P〈0．05）,即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17,谷值分别位于黑暗期ZT22和光照期ZT5。结果表明,Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质,而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。     

Fos immunoreactivity in the suprachiasmatic nuclei of golden hamsters bearing an ectopic pituitary graft

Young male golden hamsters, made hyperprolactinemic by a pituitary graft under the kidney capsule, were exposed to a light pulse (1,000 lx/30 min) at Zeitgeber time (ZT) 18. Controls included hamsters receiving a sham graft (muscle). Fos immunoreactive cells were counted in both suprachiasmatic nuclei (SCN) of each animal, using an image analyzer system. The Fos immunoreactivity (Fos-ir) of the ventrolateral and dorsomedial SCN regions was greater in the pituitary-grafted hamsters. Indeed, light induced the greatest response in grafted animals in both SCN regions. However, the SCN of pituitary-grafted hamsters in the absence of light showed the lowest Fos-ir in both regions. The results support the occurrence of a dual effect of hyperprolactinemia on Fos-ir in the SCN of hamsters at ZT 18, with inhibition of Fos expression in the absence of light and potentiation of early gene expression when animals were exposed to a light pulse.     

Circadian rhythm phenotype of 5-HT7 receptor knockout mice: 5-HT and 8-OH-DPAT-induced phase advances of SCN neuronal firing

In vitro neuronal recordings in the SCN have clearly documented shifts in the peak of unit activity following the application of serotonergic agents, and yet selectivity issues with these very tools have limited progress in establishing the precise receptor mechanisms. As an alternative strategy, mice were bred (C57BL/6J) lacking 1 serotonin receptor, the 5-HT(7), to serve as a null background for this subtype; earlier work had documented the involvement of 5-HT(7) receptors in the phase advances elicited by 8-OH-DPAT, a mixed 5-HT(1A/7) agonist, in SCN slices prepared from rat donors. Single-unit recordings in sequential electrode passes revealed peaks of activity that occurred at nearly the same time in the knockout (KO; ZT4.2 +/- 0.6) and wild-type animals (WT; ZT4.3 +/- 0.1), where ZT0 marks the beginning of the light phase in a 12:12 LD cycle. Bath application of 8-OH-DPAT produced a phase advance in neuronal firing (2.1 +/- 0.5 h) when applied 1 circadian cycle earlier at ZT6 (10 microM, 10 min), but surprisingly, the mean phase advance in slices prepared from KO mice (2.3 +/- 0.1 h) was no different. Coapplication of 8-OH-DPAT with WAY-100,635 (10 microM), a highly selective 5-HT(1A) antagonist, significantly reduced the phase advance, both in experiments with WT and KO mice, suggesting the greater importance of this serotonin sub-type independent of genetic modification. 5-HT itself (0.5 +/-M, 10 min) at ZT6 also yielded phase advances that were indistinguishable in slices prepared from WT and KO mice (1.8 +/- 0.4 h and 2.1 +/- 0.2 h, respectively) and that were also sensitive to WAY-100,635. Unlike the pattern with 8-OH-DPAT, however, 5-HT-induced phase advances, in both WT and KO mice, were blocked by ritanserin, in this paradigm useful as a 5-HT(5A/7) antagonist (in addition to its more typical role as a 5-HT2A/2C antagonist). Serotonin antagonists when administered alone were without effect in slices from WT mice but produced significant phase shifts when administered to those from KO animals. Taken together, these results highlight the importance of the species used in establishing receptor mechanism. More provocatively, they support the involvement of multiple serotonin receptors in shifting the phase of circadian rhythms at ZT6.