Oestrus cycle of the Desert hamster (Phodopus roborovskii, Satunin, 1903).

The Desert hamster, Phodopus roborovskii, is one of the lesser-known laboratory animal models and therefore knowledge of its reproductive system and physiology is limited. This study investigated the time course of vaginal and behavioural oestrus cycles by means of vaginal smear cytology, serum luteinizing hormone (LH) levels, wheel-running activity patterns, and pairing tests. The oestrus cycle lasts between four and six days and follows a pattern rather similar to that described for other rodent species. An important finding of this study is the existence of a fifth cycle stage between pre-oestrus and oestrus, the so-called early oestrus. Early oestrus differs from pre-oestrus by a complete lack of leucocytes in the vaginal cytology and a dramatic increase of serum LH levels just before activity onset. The early oestrus stage lasts 4-6 h, but was not observed in every female. When present it affects the length of the whole oestrus cycle. With early oestrus the pre-oestrus stage lasts only 14-18 h and the total length of the oestrus cycle length is four days. Without early oestrus, the pre-oestrus is prolonged to 18-36 h and the oestrus cycle length varies between four and six days. Desert hamsters showed only subtle oestrus-correlated changes in wheel-running activity, i.e. they failed to show the characteristic scalloping of activity onset, but showed prolonged activity during early oestrus. Pairing tests revealed characteristic changes in the relative frequencies of socio-positive, neutral, aggressive and sexual behaviour during the course of the oestrus cycle, with an elevated level of sexual behaviour during oestrus.

Involvement of the retinohypothalamic tract in the photic-like effects of the serotonin agonist quipazine in the rat.

Light is the major synchronizer of the mammalian circadian pacemaker located in the suprachiasmatic nucleus. Photic information is perceived by the retina and conveyed to the suprachiasmatic nucleus either directly by the retinohypothalamic tract or indirectly by the intergeniculate leaflet and the geniculohypothalamic tract. In addition, serotonin has been shown to affect the suprachiasmatic nucleus by both direct and indirect serotonin projections from the raphe nuclei. Indeed, systemic as well as local administrations of the serotonin agonist quipazine in the region of the suprachiasmatic nucleus mimic the effects of light on the circadian system of rats, i.e. they induce phase-advances of the locomotor activity rhythm as well as c-FOS expression in the suprachiasmatic nucleus during late subjective night. The aim of this study was to localize the site(s) of action mediating those effects. Phase shifts of the locomotor activity rhythm as well as c-FOS expression in the suprachiasmatic nucleus after s.c. injection of quipazine (10 mg/kg) were assessed in Lewis rats, which had received either radio-frequency lesions of the intergeniculate leaflet or infusions of the serotonin neurotoxin 5,7-dihydroxytryptamine into the suprachiasmatic nucleus (25 microg) or bilateral enucleation. Lesions of intergeniculate leaflet and serotonin afferents to the suprachiasmatic nucleus did not reduce the photic-like effects of quipazine, whereas bilateral enucleation and the subsequent degeneration of the retinohypothalamic tract abolished both the phase-shifting and the FOS-inducing effects of quipazine. The results indicate that photic-like effects of quipazine are mediated via the retinohypothalamic tract.

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

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

Interstrain differences in activity pattern, pineal function, and SCN melatonin receptor density of rats.

We investigated the possibility that strain-dependent differences in the diurnal pattern of wheel running activity rhythms are also reflected in the melatonin profiles. The inbred rat strains ACI/Ztm, BH/Ztm, and LEW/Ztm. LEW were examined for diurnal [12:12-h light-dark (LD)] wheel running activity, urinary 6-sulphatoxymelatonin (aMT6s) excretion, melatonin concentrations of plasma and pineal glands, and melatonin receptor density in the suprachiasmatic nuclei (SCN). ACI rats displayed unimodal activity patterns with a high level of activity, whereas BH and LEW rats showed multimodal activity patterns with ultradian components and reduced activity levels. In contrast, the individual daily profiles of aMT6s excretion and mean melatonin synthesis followed a unimodal time pattern in all three strains, suggesting that different output pathways of the SCN are responsible for the temporal organization of locomotor activity and pineal melatonin synthesis. In addition, melatonin synthesis at night and SCN melatonin receptor density at day were significantly higher in BH and LEW rats than in ACI rats. These results support the hypothesis of a long-term stimulating effect of melatonin on its own receptor density in the SCN.

Serotonin agonist quipazine induces photic-like phase shifts of the circadian activity rhythm and c-Fos expression in the rat suprachiasmatic nucleus.

Nonphotic stimuli can reset and entrain circadian activity rhythms in hamsters and mice, and serotonin is thought to be involved in the phase-resetting effects of these stimuli. In the present study, the authors examined the effect of the serotonin agonist quipazine on circadian activity rhythms in three inbred strains of rats (ACI, BH, and LEW). Furthermore, they investigated the effect of quipazine on the expression of c-Fos in the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN). Quipazine reduced the amount of running wheel activity for 3 h after treatment, however, no long-term changes in tau and in the activity level were observed. More important, quipazine induced significant phase advances of the activity rhythm and c-Fos production in the SCN at the end of the subjective night (Circadian Time [CT] 22), whereas neither phase shifts nor c-Fos induction were observed during the subjective day. Quipazine injections also resulted in moderate phase delays at the beginning of the subjective night (CT 14). A similar phase-response characteristic typically can be observed for photic stimuli. By contrast, nonphotic stimuli normally produce phase advances during the subjective day. The present results suggest species differences between the hamster and the rat with respect to the serotonergic action on circadian timekeeping and indicate that serotonergic pathways play a role in the transmission of photic information to the SCN of rats.

Locking and unlocking of running wheel affects circadian period stability differently in three inbred strains of rats.

Running-wheel access has been shown to shorten the circadian period length (tau) of various mammalian species. Due to the close correlation between tau and the level of activity, running wheel-induced changes of the activity level are thought to be responsible for the observed changes in tau. In the present study, the influence of the running wheel on tau and the activity level was examined in three inbred strains of rats (ACI, BH, LEW). Four animals of each strain had free access to their running wheels, while the wheels of the other 4 animals of each strain were mechanically locked. These conditions were changed twice, so that each animal encountered both kinds of changes, that is, from a locked to an unlocked running wheel and vice versa. During the whole study, overall activity was measured by infrared detectors. Running-wheel access resulted in a significant increase of overall activity in strains LEW and ACI. However, significant changes of tau were observed only in LEW rats. These rats showed a significant shortening of tau after the second change of the housing conditions regardless of whether the wheel was locked or unlocked. Consequently, no causal relationship was found between changes of tau and running wheel-induced changes of overall activity. Instead, the results suggest that subtle environmental influences like locking or unlocking the running wheel affect tau in a strain-dependent manner, whereas changes in the activity level are neither necessary nor sufficient to induce changes of tau.

Effects of an activity-correlated feeding regime on circadian locomotor activity rhythms in LEW/Ztm rats.

The aim of the present study was to examine the possible feedback effect of activity on the circadian system in rats. The animals were housed in running wheel cages equipped with food dispensers and were forced to run for their food during parts of the study. Overall level and free-running period tau of wheel-running activity were recorded continuously to quantify the relationship between tau and the level of activity. Surprisingly, the activity-dependent feeding regime failed to increase the level of wheel-running activity and did not affect the activity pattern. Nevertheless, the period of wheel-running activity was shortened in 50% of the animals subjected to food dispensers and in 38% of the animals fed only once a day at irregular times, indicating that subtle environmental differences can affect the circadian pacemaker system without changing the level or the pattern of activity. In addition, 26% of the animals showed a shortening of tau after 3 weeks, i.e., before any change in the experimental setup. This shortening was probably caused by the access to the running wheel at the beginning of the experiment. The present results suggest that the effect on the circadian pacemaker common to both of these experimental manipulations is a change in the physiological or emotional state of the animal rather than an increase of the overall level of activity.

Timing of torpor bouts during hibernation in European hamsters (Cricetus cricetus L.).

Body temperature (Tb) of seven European hamsters maintained at constant ambient temperature (Ta = 8 degrees C) and constant photoperiod (LD 8:16) was recorded throughout the hibernating season using intraperitoneal temperature-sensitive HF transmitters. The animals spent about 30% of the hibernation season in hypothermia and 70% in inter-bout normothermy. Three types of hypothermia, namely deep hibernation bouts (DHBs), short hibernation bouts (SHBs), and short and shallow hibernation bouts (SSHBs), were distinguished by differences in bout duration and minimal body temperature (Tm). A gradual development of SSHBs from the diel minimum of Tb during normothermy could be seen in individual hamsters, suggesting a stepwise decrease of the homeostatic setpoint of Tb regulation during the early hibernation season. Entry into hibernation followed a 24-h rhythm occurring at preferred times of the day in all three types of hypothermia. DHBs and SHBs were initiated approximately 4 h before SSHBs, indicating a general difference in the physiological initiation of SSHBs on the one hand and DHBs and SHBs on the other. Arousals from SHBs and SSHBs also followed a 24-h rhythm, whereas spontaneous arousals from DHBs were widely scattered across day and night. Statistical analyses of bout length and the interval between arousals revealed evidence for a free-running circadian rhythm underlying the timing of arousals. The results clearly demonstrate that entries into hypothermia are linked to the light/dark-cycle. However, the role of the circadian system in the timing of arousals from DHBs remains unclear.

Creatinine is an appropriate reference for urinary sulphatoxymelatonin of laboratory animals and humans.

In our studies on diurnal 6-sulphatoxymelatonin (aMT6s) rhythms in various species, we have sometimes obtained fluctuating patterns. In most of these, the volume of individual urine fractions was not accurately measured because of methodological problems. Here, we report a simple method to overcome these problems by using urinary creatinine to estimate urine volume. The benefit of this method is demonstrated in two representative examples of the diurnal aMT6s rhythms of rats, domestic pigs and humans. Because the human urine fractions were collected accurately, the qualitative pattern of the aMT6s rhythm was not altered by using urinary creatinine as a substitute for urine volume. The total creatinine excretion (urine volume x creatinine concentration) was constant within a small range and showed no diurnal rhythm. In rats and pigs, the highly variable aMT6s concentrations relative to urine volume throughout the 24-hr period were changed drastically by referring to creatinine. All aMT6s patterns became stable and qualitatively similar to those of the rest of the group. From these results it can be concluded that creatinine is an adequate substitute for urine volume and a beneficial parameter with which to overcome technical problems with urine collection from laboratory animals or unknown urine volumes in human studies.

Strain differences in the distribution of arginine-vasopressin- and neuropeptide Y-immunoreactive neurons in the suprachiasmatic nucleus of laboratory rats.

We have studied the number of arginine-vasopressin (AVP)-immunoreactive (ir) somata and the area size of AVP- and neuropeptide Y (NPY)-ir fibers in the suprachiasmatic nuclei (SCN) of three strains of laboratory rats exhibiting a strong unimodal (ACI), a bimodal (BH), and a weak multimodal pattern (LEW) of wheel running activity. In all three strains, AVP-ir somata and fibers were located predominantly in the dorsomedial SCN. Significant strain-differences were found for the area size of AVP-ir fibers as well as for the number and density of AVP-ir somata. The total number of AVP-ir somata was significantly higher in strain ACI (2238 +/- 164) than in strains BH (1552 +/- 137) and LEW (1426 +/- 110), whereas the mean area of AVP-ir fibers was significantly larger in strain LEW (50779 +/- 2202 microns2) than in strains ACI (39034 +/- 2095 microns2) and BH (28052 +/- 1728 microns2). Consequently, the density of AVP-ir somata was significantly lower in LEW rats, which have a weak multimodal activity pattern, than in BH and ACI rats, which have a bimodal and unimodal activity pattern, respectively. These data suggest that AVP neurons may be part of SCN output pathways controlling circadian activity rhythms. NPY-ir fibers have been identified mainly in the ventral part of the SCN. The mean area of NPY-ir fibers was smallest in BH rats (26100 +/- 1822 microns2), which show a rather scattered activity onset, and larger in ACI (29934 +/- 2468 microns2) and LEW rats (31889 +/- 2728 microns2), which have rather precise activity onsets. The inbred strains ACI, BH, and LEW may prove to be suitable models to further study distinct neuronal substrates of the SCN functionally correlated with characteristic parameters of circadian rhythms.

Block of c-Fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock.

Light-induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c-Jun, JunB, c-Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c-Fos and JunB, the predominantly expressed AP-1 proteins for the phase-shifting effects of light, we blocked the expression of c-Fos and JunB in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 microliters of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c-fos and junB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induced a significant phase shift (by 125 +/- 15 min) of the circadian locomotor activity rhythm, whereas application of ASO 6 h before the light pulse completely prevented this phase shift. Application of control nonsense oligodeoxynucleotides had no effect. ASO strongly reduced the light-induced expression of c-Fos and JunB proteins. In contrast, light pulses with or without the control nonsense oligodeoxynucleotides evoked strong nuclear c-Fos and JunB immunoreactivity in the rat suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription factors such as c-Fos and JunB are an essential part of fundamental biological processes in the adult mammalian nervous system, e.g. of light-induced phase shifts of the circadian pacemaker.

Seasonal and daily rhythms of body temperature in the European hamster (Cricetus cricetus) under semi-natural conditions.

Body temperature of five European hamsters exposed to semi-natural environmental conditions at 47 degrees N in Southern Germany was recorded over a 1.5-year period using intraperitoneal temperature-sensitive radio transmitters. The animals showed pronounced seasonal changes in body weight and reproductive status. Euthermic body temperature changed significantly throughout the year reaching its maximum of 37.9 +/- 0.2 degrees C in April and its minimum of 36.1 +/- 0.4 degrees C in December. Between November and March the hamsters showed regular bouts of hibernation and a few bouts of shallow torpor. During hibernation body temperature correlated with ambient temperature. Monthly means of body temperature during hibernation were highest in November (7.9 +/- 0.8 degrees C) and March (8.2 +/- 0.5 degrees C) and lowest in January (4.4 +/- 0.7 degrees C). Using periodogram analysis methods, a clear diurnal rhythm of euthermic body temperature could be detected between March and August, whereas no such rhythm could be found during fall and winter. During hibernation bouts, no circadian rhythmicity was evident for body temperature apart from body temperature following ambient temperature with a time lag of 3-5 h. On average, hibernation bouts lasted 104.2 +/- 23.8 h with body temperature falling to 6.0 +/- 1.7 degrees C. When entering hibernation the animals cooled at a rate of -0.8 +/- 0.2 degrees C.h-1; when arousing from hibernation they warmed at a rate of 9.9 +/- 2.4 degrees C.h-1. Warming rates were significantly lower in November and December than in January and February, and correlated with ambient temperature (r = -0.46, P < 0.01) and hibernating body temperature (r = -0.47, P < 0.01). Entry into hibernation occurred mostly in the middle of the night (mean time of day 0148 hours +/- 3.4 h), while spontaneous arousals were widely scattered across day and night. For all animals regression analysis revealed free-running circadian rhythms for the timing of arousal. These results suggest that entry into hibernation is either induced by environmental effects or by a circadian clock with a period of 24 h, whereas arousal from hibernation is controlled by an endogenous rhythm with a period different from 24 h.

Effect of lithium carbonate on activity level and circadian period in different strains of rats.

Lithium, an important pharmacological agent for the treatment of manic-depressive illness in humans, is known to lengthen the circadian period in a number of different species. Recent experiments, on the other hand, suggest that pharmacological agents may affect the circadian system indirectly through an increase or decrease of activity. To explore the interaction between pharmacological and activity effects on the circadian system, lithium was administered chronically to three different strains of rats (ACI, BH, and LEW) while wheel-running activity was studied quantitatively. Two of these inbred strains (BH and LEW) show profound abnormalities in their circadian activity rhythms, namely, a reduced overall level of activity and bimodal or multimodal activity patterns. Wheel-running activity was monitored for 4 weeks under baseline conditions, followed by 3 weeks with lithium treatment (0.3% Li2CO3 administered with food) and 4 weeks with normal food. Treatment with lithium (average intake per day = 3.6 +/- 0.2 mg) consistently decreased both the overall level and the circadian amplitude of the activity rhythm. The free-running period tau was slightly lengthened during lithium treatment, while the most dramatic effect on period was observed after lithium withdrawal. Correlation analysis, however, revealed only a small negative correlation between activity level and period length, which proved significantly only for animals of the ACI strain. Our data support the traditional interpretation that lithium lengthens circadian period by a direct pharmacological effect on the circadian pacemaker rather than through indirect effects of activity feedback.

The role of Jun transcription factor expression and phosphorylation in neuronal differentiation, neuronal cell death, and plastic adaptations in vivo.

1. To investigate the role of the Jun transcription factors in neuronal differentiation, programmed neuronal cell death, and neuronal plasticity, we used phosphorothioate oligodeoxynucleotides (S-ODN) to inhibit selectively the expression of c-Jun, JunB, and JunD. 2. We have shown previously that in contrast to c-Jun, the JunB and JunD transcription factors are negative regulators of cell growth in various cell lines. Here we confirm this finding in primary human fibroblasts. 3. c-Jun and JunB are counterplayers not only with respect to proliferation, but also in cell differentiation. Since JunB expression is essential for neuronal differentiation, we analyzed possible posttranslational modifications of JunB after induction of PC-12 cell differentiation by nerve growth factor (NGF). 4. JunB was strongly phosphorylated after induction of PC-12 cell differentiation with NGF but not after stimulation of cell proliferation with serum. Thus, while cell proliferation is associated with c-Jun phosphorylation, cell differentiation is correlated with JunB phosphorylation. This supports the finding that c-Jun and JunB play antagonistic roles in both proliferation and differentiation. 5. The JunB transcription factor together with the c-Fos transcription factor is also induced in vivo in the suprachiasmatic nucleus (SCN) of rat brain after a light stimulus that induces resetting of the circadian clock. 6. Using antisense oligonucleotides injected into the third ventricle, we selectively cosuppressed the two transcription factors in vivo as shown by immunohistochemistry. Expression of c-Jun, JunD, and FosB was not affected. Inhibition of JunB and c-Fos expression prevented the light-induced phase shift of the circadian rhythm. In contrast, rats injected with a randomized control oligonucleotide showed the same phase shift as untreated animals. 7. In primary rat hippocampal cultures, anti-c-jun S-ODN selectively inhibited neuronal cell death and promoted neuronal survival. This indicates a causal role of c-Jun in programmed neuronal cell death. 8. These findings demonstrate the essential role of inducible transcription factors in the reprogramming of cells to a different functional state. Jun transcription factors play an essential role not only in fundamental processes such as cell proliferation, differentiation, and programmed neuronal cell death, but also in such complex processes as plastic adaptations in the mature brain. The inhibition of neuronal cell death by anti-c-jun S-ODN shows the great therapeutic potential of selective antisense oligonucleotides.

Effect of photoperiod on the development of wheel-running activity rhythms in LEW/Ztm rats.

Wheel-running activity of LEW/Ztm rats is characterized by a multimodal pattern consisting of two activity bouts about 3-5 h apart. In this study we investigated the development of activity rhythms in LEW rats born and raised under three different photoperiods (LD 18:6, LD 12:12, and LD 6:18). Wheel-running activity was measured for 6 weeks in LD and for another 6 weeks in constant darkness (DD). The length of the photoperiod influenced the phase relationship between the two activity bouts only during the first week after weaning. Then, the characteristic activity pattern was established independently of the length of the photoperiod. However, development under long photoperiods (LD 18:6) resulted in a temporary increase in the level of activity and a significant shortening of the free-running period under DD. These results indicate that the multimodal activity pattern displayed by LEW rats is controlled by separate activity oscillators that establish their unique phase relationship early during development.

Effects of chronic administration and withdrawal of antidepressant agents on circadian activity rhythms in rats.

Experimental and clinical studies indicate that clinical depression may be associated with disturbances of circadian rhythms. To explore the interaction between circadian rhythmicity, behavioral state, and monoaminergic systems, the present study investigated the effects of chronic administration and withdrawal of the following antidepressant agents on circadian wheel-running rhythms of laboratory rats: a) moclobemide, a reversible and selective monoamine oxidase (MAO) type A inhibitor; b) Ro 19-6327, a selective MAO type B inhibitor; c) desipramine, a preferential norepinephrine reuptake inhibitor; d) clomipramine and e) fluoxetine, both serotonin reuptake inhibitors; and f) levoprotiline, an atypical antidepressant whose biochemical mechanism is still unknown. Wheel-running activity rhythms were studied in three inbred strains of laboratory rats (ACI, BH, LEW) under constant darkness (DD). Two of these inbred strains (BH and LEW) show profound abnormalities in their circadian activity rhythms, namely, a reduced overall level of activity and bimodal or multimodal activity patterns. Chronic treatment with moclobemide and desipramine consistently increased the overall level, as well as the circadian amplitude, of the activity rhythm. Furthermore, the abnormal activity pattern of the LEW strain was changed into a unimodal activity pattern like that of other laboratory rats. The free-running period tau was slightly shortened by moclobemide and dramatically shortened by desipramine. Effects of moclobemide and desipramine treatment on overall activity level and duration were reversed shortly after termination of treatment, whereas long aftereffects were observed for the free-running period. All other substances tested had no systematic effects on the activity rhythms of any of the strains. The fact that moclobemide and desipramine altered the period, amplitude, and pattern of circadian activity rhythms is consistent with the hypothesis that monoaminergic transmitters play a significant role in the neuronal control of behavioral state and circadian rhythmicity. Although the present study found that some antidepressives affect parameters of circadian rhythmicity, it could not demonstrate a common effect of all classes of antidepressives.

Wheel-running activity rhythms in two inbred strains of laboratory rats under different photoperiods.

Wheel-running activity patterns were studied in two inbred rat strains (ACI/Ztm and LEW/Ztm) under 24-h light-dark cycles with various photoperiods. The ACI strain was characterized by a unimodal activity pattern, whereas the LEW strain exhibited a multimodal activity pattern consisting of two activity bouts about 3-5 h apart. Harmonic spectral analyses and chi square periodograms revealed strain-specific differences in the characteristic rhythmic components of the activity pattern. The ACI strain showed only a strong 24-h rhythm, whereas the LEW strain showed additional rhythmic components with periods of 6, 4.8, and 4 h. Except in very young rats, these strain specific patterns were not affected by an experimental lengthening of the dark period. However, differences between the two strains were found in the free-running period and in the mode of entrainment. Our results indicate that the multimodal activity pattern of the LEW strain is not due to an ultradian bout oscillator, instead it may be the result of a unique coupling of multiple circadian oscillators.

Strain differences in the pattern and intensity of wheel running activity in laboratory rats.

Wheel running activity rhythms of three inbred rat strains, ACI/Ztm, BH/Ztm, and LEW/Ztm, were compared in order to evaluate the effect of genetic differences on circadian rhythm parameters. Significant strain differences were found in the general pattern of the activity rhythms and their characteristic periodicities as well as in the amount and duration of wheel running activity and the timing of activity onsets and offsets. The results suggest that genetic differences exist in the coupling of the multiple circadian oscillators that generate the overall pattern of wheel running activity.

Phase shifting the circadian clock with cycloheximide: response of hamsters with an intact or a split rhythm of locomotor activity.

Systemic administration of the protein synthesis inhibitor, cycloheximide, induced both phase advances and phase delays in the circadian rhythm of wheel-running activity in hamsters (Mesocricetus auratus) maintained in constant darkness or constant light. The magnitude and direction of the phase shifts were dependent on the circadian time (CT) of drug treatment. The phase response curves in constant darkness and constant light were of similar general shape, but they differed in the overall mean amplitude of the phase shifts. Maximal phase advances were observed after injections around CT 6-8, maximal delays at CT 0-2. Injections of various doses of cycloheximide at CT 0 induced a dose-dependent phase delay in the rhythm with a maximum delay induced by 10 mg cycloheximide. Injections of cycloheximide in animals with a split activity rhythm caused phase shifts of both components in the same direction (20/39) and in different directions (10/39). The results support the hypothesis that 80S ribosomal protein synthesis plays an important role in the biochemical mechanisms of circadian systems.