Sleep Deprivation and Caffeine Treatment Potentiate Photic Resetting of the Master Circadian Clock in a Diurnal Rodent.

Circadian rhythms in nocturnal and diurnal mammals are primarily synchronized to local time by the light/dark cycle. However, nonphotic factors, such as behavioral arousal and metabolic cues, can also phase shift the master clock in the suprachiasmatic nuclei (SCNs) and/or reduce the synchronizing effects of light in nocturnal rodents. In diurnal rodents, the role of arousal or insufficient sleep in these functions is still poorly understood. In the present study, diurnal Sudanian grass rats, Arvicanthis ansorgei, were aroused at night by sleep deprivation (gentle handling) or caffeine treatment that both prevented sleep. Phase shifts of locomotor activity were analyzed in grass rats transferred from a light/dark cycle to constant darkness and aroused in early night or late night. Early night, but not late night, sleep deprivation induced a significant phase shift. Caffeine on its own induced no phase shifts. Both sleep deprivation and caffeine treatment potentiated light-induced phase delays and phase advances in response to a 30 min light pulse, respectively. Sleep deprivation in early night, but not late night, potentiated light-induced c-Fos expression in the ventral SCN. Caffeine treatment in midnight triggered c-Fos expression in dorsal SCN. Both sleep deprivation and caffeine treatment potentiated light-induced c-Fos expression in calbindin-containing cells of the ventral SCN in early and late night. These findings indicate that, in contrast to nocturnal rodents, behavioral arousal induced either by sleep deprivation or caffeine during the sleeping period potentiates light resetting of the master circadian clock in diurnal rodents, and activation of calbindin-containing suprachiasmatic cells may be involved in this effect.SIGNIFICANCE STATEMENT Arousing stimuli have the ability to regulate circadian rhythms in mammals. Behavioral arousal in the sleeping period phase shifts the master clock in the suprachiasmatic nuclei and/or slows down the photic entrainment in nocturnal animals. How these stimuli act in diurnal species remains to be established. Our study in a diurnal rodent, the Grass rat, indicates that sleep deprivation in the early rest period induces phase delays of circadian locomotor activity rhythm. Contrary to nocturnal rodents, both sleep deprivation and caffeine-induced arousal potentiate the photic entrainment in a diurnal rodent. Such enhanced light-induced circadian responses could be relevant for developing chronotherapeutic strategies.

Circadian desynchronization triggers premature cellular aging in a diurnal rodent.

Chronic jet lag or shift work is deleterious to human metabolic health, in that such circadian desynchronization is associated with being overweight and the prevalence of altered glucose metabolism. Similar metabolic changes are observed with age, suggesting that chronic jet lag and accelerated cell aging are intimately related, but the association remains to be determined. We addressed whether jet lag induces metabolic and cell aging impairments in young grass rats (2-3 mo old), using control old grass rats (12-18 mo old) as an aging reference. Desynchronized young and control old subjects had impaired glucose tolerance (+60 and +280%) when compared with control young animals. Despite no significant variation in liver DNA damage, shorter telomeres were characterized, not only in old animal liver cells (-18%), but also at an intermediate level in desynchronized young rats (-9%). The same pattern was found for deacetylase sirtuin (SIRT)-1 (-57 and -29%), confirming that jet-lagged young rats have an intermediate aging profile. Our data indicate that an experimental circadian desynchronization in young animals is associated with a precocious aging profile based on 3 well-known markers, as well as a prediabetic phenotype. Such chronic jet lag-induced alterations observed in a diurnal species constitute proof of principle of the need to develop preventive treatments in jet-lagged persons and shift workers.

The role of PPARß/δ in the regulation of glutamatergic signaling in the hamster suprachiasmatic nucleus.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as transcription factors that regulate gene expression in numerous biological processes. Although the PPARß/δ subtype is highly expressed in the brain, its physiological roles in neuronal function remain to be elucidated. In this study, we examined the presence of PPARß/δ in the master circadian clock of the Syrian hamster and investigated its putative functional role in this structure. In mammals, the central circadian clock, located in the suprachiasmatic nucleus (SCN), is entrained by the light-dark (LD) cycle via photic6 signals conveyed by a direct pathway whose terminals release glutamate. Using immunocytochemical and qRT-PCR analysis, we demonstrated that the rhythmic expression of PPAR ß/δ within the SCN of hamsters raised under an LD cycle was detectable only at the transcriptional level when the hamsters were maintained under constant darkness (DD). The increase in the number of immunoreactive PPARß/δ cells observed under DD after light stimulation during the early subjective night (CT14), but not during the subjective day (CT06), demonstrated that the expression of PPARß/δ can be up-regulated according to the photosensitive phase of the circadian clock. All of the PPARß/δ-positive cells in the SCN also expressed the glutamate receptor NMDAR1. Moreover, we demonstrated that at the photosensitive point (CT14), the administration of L-16504, a specific agonist of PPARß/δ, amplified the phase delay of the locomotor response induced by a light pulse. Taken together, these data suggest that PPARß/δ activation modulates glutamate release that mediates entrainment of the circadian clock by light.

Setting the main circadian clock of a diurnal mammal by hypocaloric feeding.

Caloric restriction attenuates the onset of a number of pathologies related to ageing. In mammals, circadian rhythms, controlled by the hypothalamic suprachiasmatic (SCN) clock, are altered with ageing. Although light is the main synchronizer for the clock, a daily hypocaloric feeding (HF) may also modulate the SCN activity in nocturnal rodents. Here we report that a HF also affects behavioural, physiological and molecular circadian rhythms of the diurnal rodent Arvicanthis ansorgei. Under constant darkness HF, but not normocaloric feeding (NF), entrains circadian behaviour. Under a light­dark cycle, HF at midnight led to phase delays of the rhythms of locomotor activity and plasma corticosterone. Furthermore, Per2 and vasopressin gene oscillations in the SCN were phase delayed in HF Arvicanthis compared with animals fed ad libitum. Moreover, light-induced expression of Per genes in the SCN was modified in HF Arvicanthis, despite a non-significant effect on light-induced behavioural phase delays. Together, our data show that HF affects the circadian system of the diurnal rodent Arvicanthis ansorgei differentially from nocturnal rodents. The Arvicanthis model has relevance for the potential use of HF to manipulate circadian rhythms in diurnal species including humans.

Short day-length increases sucrose consumption and adiposity in rats fed a high-fat diet.

BACKGROUND: Photoperiod, i.e., the relative day-length per 24h, may modulate the metabolic responses to high-fat diet (HFD) and sucrose consumption. METHODS: To test this hypothesis, hormonal changes, fat accretion and sucrose intake were measured in rats exposed to short- or long-day for 4 weeks and fed with a standard high-carbohydrate low-fat pelleted diet (high-carbohydrate diet (HCD)) or a high-fat, medium-carbohydrate pelleted diet (HFD), with or without free access to 10% sucrose solution in addition to water available ad libitum. RESULTS: Plasma leptin and adiposity index, defined as epididymal white fat expressed as percentage of body mass, were markedly increased only in HFD-fed animals drinking sucrose under short, but not long, photoperiods. Voluntary ingestion of sucrose under short days was greater in HFD rats compared with HCD animals over the experiment, while a trend for the opposite effect was visible under long days. Total energy intake was not changed overall, as rats proportionally decreased chow intake when they drank sucrose. A noteworthy exception was the HFD group with sucrose access under short days that significantly increased their total calorie intake. Fasting blood glucose was generally unaltered, except for an increase in HFD-fed animals drinking sucrose under long days compared to control animals, suggesting a decrease in glucose tolerance. Insulin resistance was not yet affected by nutritional or photoperiodic conditions after 4 experimental weeks. CONCLUSIONS: Even if photoperiod cannot be considered as an obesogenic environmental factor per se, the metabolic effects resulting from the combination of high-fat feeding and voluntary intake of sucrose were dependent on day-length. Exposure to short days triggers a larger increase of sucrose ingestion and hyperleptinemia in rats fed with HFD compared to the control diet. Considering that the cardinal symptoms of winter depression include carbohydrate craving and increased adiposity, the present data provide an experimental basis for developing new animal models of seasonal affective disorder.

Reciprocal relationships between general (Propofol) anesthesia and circadian time in rats.

Several common postdischarge symptoms, such as sleep disorders, headache, drowsiness or general malaise, evoke disturbances of circadian rhythms due to jet lag (ie crossing time zones) or shift work rotation. Considering that general anesthesia is associated with numerous effects on the central nervous system, we hypothesized that it may also act on the circadian timing system. We first determined the effects of the circadian timing on general anesthesia. We observed that identical doses of propofol showed marked circadian fluctuations in duration of effects, with a peak at the middle of the resting period (ie 7 h after lights on). Then, we examined the effects of general anesthesia on circadian timing, by analysing stable free-running circadian rhythms (ie in constant environmental conditions), an experimental approach used widely in circadian biology. Free-running rats were housed in constant darkness and temperature to assess possible phase-shifting effects of propofol anesthesia according to the time of the day. When administered around (+/-2 h) the daily rest/activity transition point, a 30-min propofol anesthesia induced a 1-h phase advance in the free-running rest-activity rhythm, while anesthesia had no significant resetting effect at other times of the day. Anesthesia-induced hypothermia was not correlated with the phase-shifting effects of propofol anesthesia. From our results, anesthesia itself can reset circadian timing, and acts as a synchronizing cue for the circadian clock.

Increasing the fat-to-carbohydrate ratio in a high-fat diet prevents the development of obesity but not a prediabetic state in rats.

Metabolic disorders induced by high-fat feeding in rodents evoke some, if not all, of the features of human metabolic syndrome. The occurrence and severity of metabolic disorders, however, varies according to rodent species, and even strain, as well as the diet. Therefore, in the present study, we investigated the long-term obesogenic and diabetogenic effects of three high-fat diets differing by their fat/carbohydrate ratios. Sprague-Dawley rats were fed a control high-carbohydrate and low-fat diet [HCD; 3:16:6 ratio of fat/carbohydrate/protein; 15.48 kJ/g (3.7 kcal/g)], a high-fat and medium-carbohydrate diet [HFD1; 53:30:17 ratio of fat/carbohydrate/protein; 19.66 kJ/g (4.7 kcal/g)], a very-high-fat and low-carbohydrate diet [HFD2; 67:9:24 ratio of fat/carbohydrate/protein; 21.76 kJ/g (5.2 kcal/g)] or a very-high-fat and carbohydrate-free diet [HFD3; 75:0:25 ratio of fat/carbohydrate/protein; 24.69 kJ/g (5.9 kcal/g)] for 10 weeks. Compared with the control diet (HCD), rats fed with high-fat combined with more (HFD1) or less (HFD2) carbohydrate exhibited higher BMI (body mass index; +13 and +10% respectively; P<0.05) and abdominal fat (+70% in both HFD1 and HFD2; P<0.05), higher plasma leptin (+130 and +135% respectively; P<0.05), lower plasma adiponectin levels (-23 and -30% respectively; P<0.05) and impaired glucose tolerance. Only the HFD1 group had insulin resistance. By contrast, a very-high-fat diet devoid of carbohydrate (HFD3) led to impaired glucose tolerance, insulin resistance and hypoadiponectinaemia (-50%; P<0.05), whereas BMI, adiposity and plasma leptin did not differ from respective values in animals fed the control diet. We conclude that increasing the fat-to-carbohydrate ratio to the uppermost (i.e. carbohydrate-free) in a high-fat diet prevents the development of obesity, but not the prediabetic state (i.e. altered glucose tolerance and insulin sensitivity).

Leptin normalizes photic synchronization in male ob/ob mice, via indirect effects on the suprachiasmatic nucleus.

Mounting evidence indicates a strong link between metabolic diseases and circadian dysfunctions. The metabolic hormone leptin, substantially increased in dietary obesity, displays chronobiotic properties. Here we investigated whether leptin is involved in the alteration of timing associated with obesity, via direct or indirect effects on the suprachiasmatic nucleus (SCN), the site of the master clock. Photic synchronization was studied in obese ob/ob mice (deficient in leptin), either injected or not with high doses of recombinant murine leptin (5 mg/kg). This was performed first at a behavioral level, by shifting the light-dark cycle and inducing phase shifts by 30-minute light pulses and then at molecular levels (c-FOS and P-ERK1/2). Moreover, to characterize the targets mediating the chronomodulatory effects of leptin, we studied the induction of phosphorylated signal transducer and activator of transcription 3 (P-STAT3) in the SCN and in different structures projecting to the SCN, including the medial hypothalamus. Ob/ob mice showed altered photic synchronization, including augmented light-induced phase delays. Acute leptin treatment normalized the photic responses of the SCN at both the behavioral and molecular levels (decrease of light-induced c-FOS). Leptin-induced P-STAT3 was modulated by light in the arcuate nucleus and both the ventromedial and dorsomedial hypothalamic nuclei, whereas its expression was independent of the presence of leptin in the SCN. These results suggest an indirect action of leptin on the SCN, possibly mediated by the medial hypothalamus. Taken together, these results highlight a central role of leptin in the relationship between metabolic disturbances and circadian disruptions.

Food-anticipatory activity in Syrian hamsters: behavioral and molecular responses in the hypothalamus according to photoperiodic conditions.

When food availability is restricted, animals adjust their behavior according to the timing of food access. Most rodents, such as rats and mice, and a wide number of other animals express before timed food access a bout of activity, defined as food-anticipatory activity (FAA). One notable exception amongst rodents is the Syrian hamster, a photoperiodic species that is not prone to express FAA. The present study was designed to understand the reasons for the low FAA in that species. First, we used both wheel-running activity and general cage activity to assess locomotor behavior. Second, the possible effects of photoperiod was tested by challenging hamsters with restricted feeding under long (LP) or short (SP) photoperiods. Third, because daytime light may inhibit voluntary activity, hamsters were also exposed to successive steps of full and skeleton photoperiods (two 1-h light pulses simulating dawn and dusk). When hamsters were exposed to skeleton photoperiods, not full photoperiod, they expressed FAA in the wheel independently of daylength, indicating that FAA in the wheel is masked by daytime light under full photoperiods. During FAA under skeleton photoperiods, c-Fos expression was increased in the arcuate nuclei independently of the photoperiod, but differentially increased in the ventromedial and dorsomedial hypothalamic nuclei according to the photoperiod. FAA in general activity was hardly modulated by daytime light, but was reduced under SP. Together, these findings show that food-restricted Syrian hamsters are not prone to display FAA under common laboratory conditions, because of the presence of light during daytime that suppresses FAA expression in the wheel.

Aging-like circadian disturbances in folate-deficient mice.

The elderly population shows various circadian disturbances, including dampened amplitude of rhythmicity and decreased responsiveness to light. The common poor folate status in the elderly might account for these aging-related circadian disturbances. To test this hypothesis, we investigated whether folate deficiency in mice affects circadian oscillations of the master clock in the suprachiasmatic nuclei, and the shifting responses to light. Mice fed a diet without folate for 6 weeks displayed markedly reduced (4.5-fold) erythrocyte folate concentration and increased (2.3-fold) homocysteinemia compared with control mice. Folate deficiency decreased the circadian amplitude of vasopressin and the clock protein PERIOD 2 (PER2) in the master clock, slowed the rate of re-entrainment of behavioral rhythms after delayed light-dark cycle and reduced light-induced phase-delays, without detectable morphologic changes in the retina, such as the number of melanopsinergic ganglion cells, that might have impaired photodetection. In conclusion, folate deficiency and consecutive hyperhomocysteinemia led to dampened PER2 and vasopressin oscillations in the master clock and reduced responsiveness to photic resetting, which constitute hallmarks of aging effects on circadian rhythmicity.

Photic and pineal modulation of food anticipatory circadian activity rhythms in rodents.

Restricted daily feeding schedules entrain circadian oscillators that generate food anticipatory activity (FAA) rhythms in nocturnal rodents. The location of food-entrainable oscillators (FEOs) necessary for FAA remains uncertain. The most common procedure for inducing circadian FAA is to limit food access to a few hours in the middle of the light period, when activity levels are normally low. Although light at night suppresses activity (negative masking) in nocturnal rodents, it does not prevent the expression of daytime FAA. Nonetheless, light could reduce the duration or magnitude of FAA. If so, then neural or genetic ablations designed to identify components of the food-entrainable circadian system could alter the expression of FAA by affecting behavioral responses to light. To assess the plausibility of light as a potential mediating variable in studies of FAA mechanisms, we quantified FAA in rats and mice alternately maintained in a standard full photoperiod (12h of light/day) and in a skeleton photoperiod (two 60 min light pulses simulating dawn and dusk). In both species, FAA was significantly and reversibly enhanced in the skeleton photoperiod compared to the full photoperiod. In a third experiment, FAA was found to be significantly attenuated in rats by pinealectomy, a procedure that has been reported to enhance some effects of light on behavioral circadian rhythms. These results indicate that procedures affecting behavioral responses to light can significantly alter the magnitude of food anticipatory rhythms in rodents.

Timed hypocaloric feeding and melatonin synchronize the suprachiasmatic clockwork in rats, but with opposite timing of behavioral output.

Temporal organization of the molecular clockwork and behavioral output were investigated in nocturnal rats housed in constant darkness and synchronized to nonphotic cues (daily normocaloric or hypocaloric feeding and melatonin infusion) or light (light-dark cycle and daily 1-h light exposure). Clock gene (Per1, Per2 and Bmal1) and clock-controlled gene (Vasopressin) expression in the suprachiasmatic nuclei was assessed over 24 h. Light and exogenous melatonin synchronized the molecular clock, signaling, respectively, 'daytime' and 'nighttime', without affecting temporal organization of behavioral output (rest/activity rhythm). By contrast, synchronization to hypocaloric feeding led to a striking temporal change between gene expression in the suprachiasmatic clock and waveform of locomotor activity rhythm, rats then becoming active during the subjective day (diurnal-like temporal organization). When the time of feeding coincided with activity offset, normocaloric feeding also synchronized the locomotor activity rhythm with no apparent switch in temporal organization. Peak of Per2 expression in the piriform cortex occurred between the beginning and the middle of the activity/feeding period, depending on the synchronizer. These data demonstrate that even though the suprachiasmatic clockwork can be synchronized to nonphotic cues, hypocaloric feeding likely acts downstream from clock gene oscillations in the suprachiasmatic nuclei to yield a stable yet opposite organization of the rest/activity cycle.