Early Subjective Night Research Articles

Effects of light intensity and restraint on dark-pulse-induced circadian phase shifting during subjective night in Syrian hamsters.

Dark pulses presented on a background of constant light (LL) result in phase advances during midsubjective day and early subjective night, and phase delays during late subjective night, as shown in the dark-pulse phase response curve. In hamsters, the phase-shifting effects of dark pulses are thought to be mediated by increased activity, as previous studies have shown that restraining animals during dark pulses blocks the phase shifts observed in midsubjective day and late subjective night. This study focuses on dark-pulse-induced phase shifting during early subjective night, examining the influence of both LL intensity and restraint on the magnitude of these phase shifts. Syrian hamsters were maintained in LL of four different illumination levels (1, 10, 100, or 600 lux) and periodically presented with 6-h pulses (dark pulse alone, restraint alone, or dark pulse plus restraint) beginning at circadian time 11. Phase advances were observed in response to dark pulses alone, and the magnitude of these shifts was dependent on background illumination, with significantly larger advances seen under higher intensities. No relationship was found between the amount of activity displayed during dark pulses and phase shift magnitude. Six-hour periods of restraint resulted in phase delays, the magnitude of which was also dependent on background illumination. Restraining hamsters during dark pulses reduced the magnitude of phase advances, but the extent of this reduction could be predicted from the additive effects of the dark-pulse-alone and restraint-alone conditions. These results indicate that the phase-shifting effects of dark pulses during early subjective night are not mediated by behavioral activation and may instead reflect a mirror image of the phase-delaying effects of light pulses at this phase.

Correlative association between N-methyl-D-aspartate receptor-mediated expression of period genes in the suprachiasmatic nucleus and phase shifts in behavior with photic entrainment of clock in hamsters.

Because the rapid induction of Period (Per) genes is associated with the photic entrainment of the biological clock, we examined whether N-methyl-D-aspartate (NMDA) receptors were involved in the photic induction of Per genes in the hamster suprachiasmatic nucleus (SCN). In situ hybridization observation revealed that light during the early subjective night [circadian time (CT) 13.5] or the late subjective night (CT20) caused an induction of Per1 and Per2 but not Per3 mRNA in the SCN. Photic induction of Per mRNA at CT13.5 was observed especially in the ventrolateral SCN, whereas that at CT20 was more widespread from the ventrolateral to the dorsal SCN. A noncompetitive NMDA receptor antagonist, +MK801, dose-dependently (0. 1-5.0 mg/kg) suppressed only the ventrolateral part of Per1 and Per2 mRNA induction by light at CT13.5 or CT20 in the SCN. The suppressive effects of +MK801 on Per mRNA strongly correlated with the attenuating action of this compound on phase shifts by light at both CT13.5 and CT20. A competitive NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV), also exhibited inhibitory actions on light (CT20)-induced Per1 and Per2 mRNA expression in the ventrolateral SCN. Furthermore, local injection of NMDA into the SCN resulted in the induction of Per1 and Per2 mRNA in the SCN. Among NMDA receptors, NR2B and NR2C mRNA were expressed in the ventrolateral and dorsal SCN, respectively. These results suggest that the activation of NMDA receptor is a critical step for photic induction of Per1 and Per2 transcripts in the SCN, which are linked to a photic behavioral entrainment.

Characterization of the Chicken SerotoninN-Acetyltransferase Gene: ACTIVATION VIA CLOCK GENE HETERODIMER/E BOX INTERACTION

The abundance of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) mRNA in the chicken pineal gland exhibits a circadian rhythm, which is translated into a circadian rhythm in melatonin production. Here we have started to elucidate the molecular basis of the circadian rhythm in chicken AANAT (cAANAT). The 5'-flanking region of the cAANAT gene was isolated and found to contain an E box DNA element that confers strong luciferase reporter activity. In transfection experiments using chicken pineal cells, an E box mutation dramatically decreased reporter activity. Northern blot analysis indicated that several putative clock genes (bmal1, Clock, and MOP4) are co-expressed in the chicken pineal gland. bmal1 mRNA is expressed in a rhythmic manner in the chicken pineal gland, with peak levels at early subjective night, coincident with the increase in cAANAT expression. Co-transfection experiments in COS cells demonstrated that chicken BMAL1/CLOCK and human BMAL1/MOP4 heterodimers bound the AANAT E box element and enhanced transcription. These observations suggest that binding of clock gene heterodimers to the cAANAT E box is a critical element in the expression of the cAANAT gene in vitro.

Nonphotic Entrainment by 5-HT1A/7Receptor Agonists Accompanied by ReducedPer1andPer2mRNA Levels in the Suprachiasmatic Nuclei

In mammals, the environmental light/dark cycle strongly synchronizes the circadian clock within the suprachiasmatic nuclei (SCN) to 24 hr. It is well known that not only photic but also nonphotic stimuli can entrain the SCN clock. Actually, many studies have shown that a daytime injection of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT), a serotonin 1A/7 receptor agonist, as a nonphotic stimulus induces phase advances in hamster behavioral circadian rhythms in vivo, as well as the neuron activity rhythm of the SCN in vitro. Recent reports suggest that mammalian homologs of the Drosophila clock gene, Period (Per), are involved in photic entrainment. Therefore, we examined whether phase advances elicited by 8-OH DPAT were associated with a change of Period mRNA levels in the SCN. In this experiment, we cloned partial cDNAs encoding hamster Per1, Per2, and Per3 and observed both circadian oscillation and the light responsiveness of Period. Furthermore, we found that the inhibitory effect of 8-OH DPAT on hamster Per1 and Per2 mRNA levels in the SCN occurred only during the hamster's mid-subjective day, but not during the early subjective day or subjective night. The present findings demonstrate that the acute and circadian time-dependent reduction of Per1 and/or Per2 mRNA in the hamster SCN by 8-OH DPAT is strongly correlated with the phase resetting in response to 8-OH DPAT.

Photic phase response curve in Octodon degus: assessment as a function of activity phase preference.

Light exposure during the early and late subjective night generally phase delays and advances circadian rhythms, respectively. However, this generality was recently questioned in a photic entrainment study in Octodon degus. Because degus can invert their activity phase preference from diurnal to nocturnal as a function of activity level, assessment of phase preference is critical for computations of phase reference [circadian time (CT) 0] toward the development of a photic phase response curve. After determining activity phase preference in a 24-h light-dark cycle (LD 12:12), degus were released in constant darkness. In this study, diurnal (n = 5) and nocturnal (n = 7) degus were randomly subjected to 1-h light pulses (30-35 lx) at many circadian phases (CT 1-6: n = 7; CT 7-12: n = 8; CT 13-18: n = 8; and CT 19-24: n = 7). The circadian phase of body temperature (Tb) onset was defined as CT 12 in nocturnal animals. In diurnal animals, CT 0 was determined as Tb onset + 1 h. Light phase delayed and advanced circadian rhythms when delivered during the early (CT 13-16) and late (CT 20-23) subjective night, respectively. No significant phase shifts were observed during the middle of the subjective day (CT 3-10). Thus, regardless of activity phase preference, photic entrainment of the circadian pacemaker in Octodon degus is similar to most other diurnal and nocturnal species, suggesting that entrainment mechanisms do not determine overt diurnal and nocturnal behavior.

Protein synthesis is a required process for the optic lobe circadian clock in the cricket Gryllus bimaculatus

The effects of a translation inhibitor, cycloheximide (CHX), on the circadian neuronal activity rhythm of the optic lamina–medulla compound eye complex cultured in vitro were investigated in the cricket Gryllus bimaculatus. When the complex was treated with 10 −5 M CHX for 6 h, the rhythm exhibited a marked phase shift. The magnitude and direction of the phase shift were dependent on the phase at which the complex was treated with CHX; phase delays occurred during the late subjective day to early subjective night, whereas phase advances occurred around the late subjective night. Continuous application of CHX abolished circadian rhythms of both the spontaneous neuronal activity and the visually evoked response. However, it abolished neither the spontaneous activity nor the visually evoked response. As washed with fresh medium after CHX treatment, the rhythm soon reappeared and the subsequent phase was clearly correlated to the termination time of the treatment. These results suggest that protein synthesis is also involved in the cricket optic lobe circadian clock, and that the clock-related protein synthesis may be active during the late subjective day to subjective night.

PHOTIC ENTRAINMENT OF CIRCADIAN ACTIVITY PATTERNS IN THE TROPICAL LABRID FISH HALICHOERES CHRYSUS

Yellow wrasses (Halichoeres chrysus) show clear daily activity patterns. The fish hide in the substrate at (subjective) night, during the distinct rest phase. Initial entrainment in a 12h:12h light-dark (12:12 LD) cycle (mean period 24.02h, SD 0.27h, n = 16 was followed by a free run (mean period 24.42h, SD 1.33h) after transition into constant dim light conditions. Light pulses of a comparable intensity as used in the light part of the LD cycles did not result in significant phase shifts of the free-running rhythm in constant darkness. Application of much brighter 3h light pulses resulted in a phase-response curve (PRC) for a fish species, with pronounced phase advances during late subjective night. The PRCs differed from those mainly obtained in other vertebrate taxa by the absence of significant phase delays in the early subjective night. At that circadian phase, significant tonic effects of the light pulses caused a shortening of the circadian period length. Entrainment to skeleton photoperiods of 1:11 LD was observed in five of six wrasses exposed, also after a 3h phase advance of this LD cycle. Subsequently, a 1:11.25 LD cycle resulted in entrainment in four of the six fish. It is suggested that the expression of the circadian system in fish can be interpreted as a functional response to a weak natural zeitgeber, as present in the marine environment. This response allows photic entrainment as described here in the yellow wrasse. (Chronobiology International, 17(5), 613–622, 2000)

Light and serotonin phase-shift the circadian clock in the cricket optic lobe in vitro

Light and serotonin were found to cause phase shifts of the circadian neural activity rhythm in the optic lobe of the cricket Gryllus bimaculatus cultured in vitro. The two phase-shifting agents yielded phase-response curves different in shape. Light induced phase delay and advance in the early and late subjective night, respectively, and almost no shifts in the subjective day, whereas serotonin phase-advances the clock during the subjective day and induced delay shifts during the subjective night. The largest phase advance and delay occurred at circadian time 21 and 12, respectively, for light, and circadian time 3 and 18, respectively, for serotonin. Quipazine, a nonspecific serotonin agonist, induced phase advance and phase delay at circadian time 3 and 18, respectively, like serotonin. (±)8-OH-DPAT, a specific 5-HT1A agonist, phase delayed by 2 h at the subjective night, but produced no significant phase shifts at the subjective day. When NAN-190, a specific 5-HT1A antagonist, was applied together with quipazine, it completely blocked the phase delay at circadian time 18, whereas it had no effect on the advance shifts induced by quipazine. The results suggest that the phase dependency of serotonin-induced phase shifts of the clock may be partly attributable to the daily change in receptor type.

Circadian Rhythms in the Suprachiasmatic Nucleus are Temperature-Compensated and Phase-Shifted by Heat PulsesIn Vitro

Temperature compensation and the effects of heat pulses on rhythm phase were assessed in the suprachiasmatic nucleus (SCN). Circadian neuronal rhythms were recorded from the rat SCN at 37 and 31 degrees C in vitro. Rhythm period was 23.9 +/- 0.1 and 23.7 +/- 0.1 hr at 37 and 31 degrees C, respectively; the Q(10) for tau was 0.99. Heat pulses were administered at various circadian times (CTs) by increasing SCN temperature from 34 to 37 degrees C for 2 hr. Phase delays and advances were observed during early and late subjective night, respectively, and no phase shifts were obtained during midsubjective day. Maximum phase delays of 2.2 +/- 0.3 hr were obtained at CT 14, and maximum phase advances of 3.5 +/- 0.2 hr were obtained at CT 20. Phase delays were not blocked by a combination of NMDA [AP-5 (100 microM)] and non-NMDA [CNQX (10 microM)] receptor antagonists or by tetrodotoxin (TTX) at concentrations of 1 or 3 microM. The phase response curve for heat pulses is similar to ones obtained with light pulses for behavioral rhythms. These data demonstrate that circadian pacemaker period in the rat SCN is temperature-compensated over a physiological range of temperatures. Phase delays were not caused by activation of ionotropic glutamate receptors, release of other neurotransmitters, or temperature-dependent increases in metabolism associated with action potentials. Heat pulses may have phase-shifted rhythms by directly altering transcriptional or translational events in SCN pacemaker cells.

Interactions between light and melatonin on the circadian clock of mice.

Melatonin and light synchronize the biological clock and are used to treat sleep/wake disturbances in humans. However, the two treatments affect circadian rhythms differently when they are combined than when they are administered individually. To elucidate the nature of the interaction between melatonin and light, the present study assessed the effect of melatonin on circadian timing and immediate-early gene expression in the suprachiasmatic nucleus (SCN) when administered in the presence of light. Male C3H/HeN mice, housed in constant dark in cages equipped with running wheels, were treated with either melatonin (90 microg, s.c.) or vehicle (3% ethanol-saline) 5 min prior to exposure to light (15 min, 300 lux) at various times in the circadian cycle. Combined treatment resulted in lower magnitude phase delays of circadian activity rhythms than those obtained with light alone during the early subjective night and advances in phase when melatonin and light were administered during the subjective day (p < .001). The reduction in phase delays with combined treatment at Circadian Time (CT) 14 was significant when light exposure measured 300 lux but not at lower light levels (p < .05). When light preceded melatonin administration, the inhibition of phase delays attained significance only when the light exposure reached 1000 lux (p < .05). Neither basal nor light-induced expression of c-fos mRNA in the SCN was modified by melatonin administration at CT 14 or CT 22. Together, these results suggest that combined administration of melatonin and light affect circadian timing in a manner not predicted by summing the two treatments given individually. Furthermore, the interaction is not likely to be due to inhibition of photic input to the clock by melatonin but might arise from a photically induced enhancement of melatonin's actions on circadian timing.

Electrical stimulation of the median or dorsal raphe nuclei reduces light-induced FOS protein in the suprachiasmatic nucleus and causes circadian activity rhythm phase shifts

Several pharmacological studies have suggested that the large median raphe serotonergic projection to the circadian clock in the suprachiasmatic nucleus may modulate circadian rhythm phase. The present experiments studied the role of dorsal and median raphe nuclei as regulators of circadian rhythmicity by evaluating the ability of electrical stimulation to shift rhythm phase or to alter photic induction of FOS protein synthesis. Male hamsters implanted with bipolar electrodes in either the median or dorsal raphe nucleus were stimulated during the early subjective night coincident with exposure to a saturating light pulse. About 90 min later, animals were anesthetized, perfused and the brains processed for FOS protein immunoreactivity. As previously demonstrated, light alone induces FOS immunoreactivity in nuclei of suprachiasmatic nucleus neurons. This was significantly attenuated by stimulation of either the median or dorsal raphe nucleus, with the extent of attenuation proportional to the intensity of stimulation. Electrical stimulation without light exposure had no effect on FOS expression. The effect of light on FOS expression in the suprachiasmatic nucleus was not modified by pre-treatment with the 5-HT 1/2 serotonin receptor antagonist, metergoline, although it greatly reduced electrical stimulation-induced FOS expression in the hippocampus. In a second experiment, hamsters housed with running wheels in constant light were electrically stimulated in the median or dorsal raphe nucleus 6 h prior to (CT6) or 2 h after (CT14) expected activity onset. Regardless of which raphe nucleus was electrically stimulated, approximately 22 min phase advances were elicited at CT6 and 36 min phase delays were elicited at CT14. Despite the fact that the sole direct projection from the raphe complex to the suprachiasmatic nucleus is from the median nucleus, the present data do not distinguish between the median and dorsal raphe with respect to their impact on circadian rhythm regulation. Instead, two possible roles for each raphe nucleus are demonstrated. One main effect is that both raphe nuclei modulate rhythm phase. The second is an interaction between raphe efferent activity and light which, in the present studies, is demonstrated by the ability of raphe stimulation to modulate the action of light on the circadian system. While serotonin is a likely neurotransmitter mediating one or both effects, alternatives such as GABA, must be considered.

Functional diversities of two activity components of circadian rhythm in genetical splitting mice (CS strain).

CS mice, an inbred strain, showed two distinctive characteristics in the circadian rhythm of locomotor activity: (1) large variation in the freerunning period, and (2) spontaneous rhythm splitting under continuous darkness. In the splitting rhythm there was a positive correlation between the freerunning period of the evening component and the activity time of the morning component. The phase-shifting effect of a 15-min light pulse was examined on the two activity components of the splitting rhythm. There were significant differences in the amount of light-induced phase response between the two components. A light pulse during the late subjective night induced a phase advance shift only in the morning component, while a light pulse during the early subjective night induced a phase delay shift only in the evening component. These results indicate functional diversities of the two activity components in the circadian locomotor rhythm of CS mice, and suggest that the circadian system in CS mice consists of two mutually coupled oscillators which have different circadian periods and different responsiveness to light. The CS mouse is a useful model to explore a genetic background of oscillator coupling in the circadian system of nocturnal rodents.

Reduced glucose availability attenuates circadian responses to light in mice.

To test whether circadian responses to light are modulated by decreased glucose availability, we analyzed photic phase resetting of the circadian rhythm of locomotor activity in mice exposed to four metabolic challenges: 1) blockade of glucose utilization induced by 2-deoxy-D-glucose (2-DG), 2) fasting (food was removed for 30 h), 3) insulin administration, and 4) insulin treatment after fasting. In mice housed in constant darkness, light pulses applied during early subjective night induced phase delays of the rhythm of locomotor activity, whereas light pulses applied during late subjective night caused phase advances. There was an overall reduction of light-induced phase shifts, with a more pronounced effect for delays, in mice pretreated with 500 mg/kg ip 2-DG compared with mice injected with saline. Administration of glucose with 2-DG prevented the reduction of light-induced phase delays. Furthermore, phase delays were reduced in fed mice pretreated with 5 IU/kg sc insulin and in fasted mice injected with saline or insulin compared with control fed mice. These results show that circadian responses to light are reduced when brain glucose availability is decreased, suggesting a metabolic modulation of light-induced phase shifts.

Retinal GABA(A) receptors participate in the regulation of circadian responses to light.

A role for retinal gamma-aminobutyric acid Type A (GABA(A)) receptors in the regulation of circadian responses to light was examined. Intraocular injections of the GABA(A) antagonist, bicuculline, were performed during the early (Circadian Time [CT] 13.5) and late subjective night (CT 20), followed by a light pulse. Bicuculline significantly decreased the magnitude of phase delays induced by light to 65%, whereas it had no effect on phase advances. To explore the nature of the inhibition elicited by bicuculline, an intensity-response curve was performed. Intraocular injections of bicuculline inhibited phase delays only when induced by high-saturating light illuminances (20 and 100 lux). No effect was observed at light intensities < or = 5 lux. These results suggest that retinal GABA(A) receptors modulate the responsivity of the circadian system to light.

An NK 1 receptor antagonist affects the circadian regulation of locomotor activity in golden hamsters

Substance P (SP) is a neuromodulator which may participate in the photic regulation of the circadian timing system in mammals. The biological effects of SP are mediated by interaction with specific receptors, designated as NK 1, NK 2, and NK 3. The NK 1 subtype receptor is expressed in the circadian system. Experiment 1 was designed to test whether an NK 1 antagonist mimics the effects of dark pulses. Hamsters were housed in constant lighting conditions, either constant darkness or constant light (around 250 lx), and they received an i.p. injection of either the specific NK 1 receptor antagonist, L-760,735 (5 mg/kg), or saline during the mid-subjective day, a time when dark pulses cause a phase-advance in circadian rhythm of locomotor activity. After treatment with the NK 1 antagonist, significant phase-advances of wheel-running activity rhythm were found in constant light, but not in constant darkness. Experiment 2 was designed to test the ability of the NK 1 antagonist to block the phase-delaying and/or the phase-advancing effects of light in animals kept in constant darkness. Phase-advances of locomotor activity rhythm that can normally be induced by light pulses given during the late subjective night were markedly reduced by pre-treatment with the NK 1 antagonist. By contrast, phase-delays that can be induced by lights pulses given during the early subjective night were unaffected by the NK 1 antagonist. These data support the hypothesis that SP within the circadian system may, by interacting with NK 1 receptors, modulate photic responses of the SCN pacemaker.

Circadian Period Modulation and Masking Effects Induced by Repetitive Light Pulses in Locomotor Rhythms of the Cricket, Gryllus bimaculatus

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 +/- 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.

Methylcobalamin amplifies melatonin-induced circadian phase shifts by facilitation of melatonin synthesis in the rat pineal gland

Effects of methylcobalamin (methyl-B 12), a putative drug for treating human circadian rhythm disorders, on the melatonin-induced circadian phase shifts were examined in the rat. An intraperitoneal injection of 1–100 μg/kg melatonin 2-h before the activity onset time (CT 10) induced phase advances of free-running activity rhythms in a dose-dependent manner (ED 50=1.3 μg/kg). Injection of methyl-B 12 (500 μg/kg) prior to melatonin (1 μg/kg) injection induced larger phase advances than saline preinjected controls, while the injection of methyl-B 12 in combination with saline did not induce a phase advance. These results indicate amplification of melatonin-induced phase advances by methyl-B 12. Pinealectomy abolished the phase alternating effect of methyl-B 12, suggesting a site of action within the pineal gland. In fact, methyl-B 12 significantly increased the content of melatonin in the pineal collected 2-h after activity onset (CT 14). In contrast, no difference in melatonin content was found at CT 10, indicating that the effect of methyl-B 12 may be gated after the activity onset time when endogenous melatonin synthesis is known to increase. These results suggest that methyl-B 12 amplifies melatonin-induced phase advances via an increase in melatonin synthesis during the early subjective night at a point downstream from the clock regulation.

Three period Homologs in Mammals: Differential Light Responses in the Suprachiasmatic Circadian Clock and Oscillating Transcripts Outside of Brain

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows ∼37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

Entrainment in calorie-restricted mice: conflicting zeitgebers and free-running conditions.

Phase-shifting effects of timed calorie restriction were investigated in mice during exposure to a 12:12-h light-dark cycle. Food-anticipatory activity (FAA), the output of a food-entrainable pacemaker, was expressed before the time of feeding whether mice received daily hypocaloric food (3.3 g of chow/day) or normocaloric food (5 g of chow/day) at zeitgeber time (ZT) 2 (ZT12 = lights off). Subsequently, mice were placed in constant darkness and fed ad libitum. The onset of the nocturnal period of locomotor activity was phase advanced by 1 h in calorie-restricted mice compared with normocalorie-fed controls. The phase advance still occurred when FAA was prevented by restraining calorie-restricted mice. Giving hypocaloric food at ZT2, ZT10, ZT14, or ZT22 phase advanced the nocturnal pattern of activity by 1, 3, 1, and 1 h, respectively. After transfer to constant darkness, FAA free ran in parallel with the normal nocturnal period of locomotor activity. A light pulse during the early subjective night phase delayed both components. These results indicate that 1) timed calorie restriction under a light-dark cycle can phase advance the light-entrainable pacemaker with a phase-dependent magnitude, 2) FAA feedback is not crucial for the observed phase advance, and 3) the light-entrainable pacemaker may control the period of the food-entrainable pacemaker in mice fed ad libitum.

Neonatal clomipramine treatment of Syrian hamsters: effect on the circadian system

The circadian behavior of male Syrian hamsters injected with the serotonin/norepinephrine reuptake inhibitor clomipramine (15 mg/kg from postnatal days 8 to 21) was examined. Clomipramine treatment significantly augmented mean activity values of wheel running rhythm, as well as delayed its acrophase. After a 6-h phase advance of the light–dark cycle, reentrainment of clomipramine-treated hamsters took significantly longer than controls. Clomipramine-treated hamsters exhibited a shorter circadian period than controls in constant light conditions, but no differences were found in constant darkness. Light pulses applied at late subjective night to clomipramine-treated hamsters caused significantly reduced phase advances as compared to controls, while no differences were found in phase delay magnitudes when light pulses were applied during early subjective night. Administration of the 5-HT 1A receptor agonist 8-hydroxy-2-(di- n-propylamino) tetralin (8-OH-DPAT) at circadian time 8 significantly advanced the onset of activity to a greater extent in clomipramine-treated hamsters than in controls. The results indicate that neonatal clomipramine treatment of hamsters causes long-lasting changes in the circadian system, by increasing activity levels and by partially inhibiting light-evoked responses. An enhancement of a non-photic, serotonergic-induced response was also unveiled.