A refined method to monitor arousal from hibernation in the European hamster.

BACKGROUND: Hibernation is a physiological and behavioural adaptation that permits survival during periods of reduced food availability and extreme environmental temperatures. This is achieved through cycles of metabolic depression and reduced body temperature (torpor) and rewarming (arousal). Rewarming from torpor is achieved through the activation of brown adipose tissue (BAT) associated with a rapid increase in ventilation frequency. Here, we studied the rate of rewarming in the European hamster (Cricetus cricetus) by measuring both BAT temperature, core body temperature and ventilation frequency. RESULTS: Temperature was monitored in parallel in the BAT (IPTT tags) and peritoneal cavity (iButtons) during hibernation torpor-arousal cycling. We found that increases in brown fat temperature preceded core body temperature rises by approximately 48 min, with a maximum re-warming rate of 20.9℃*h-1. Re-warming was accompanied by a significant increase in ventilation frequency. The rate of rewarming was slowed by the presence of a spontaneous thoracic mass in one of our animals. Core body temperature re-warming was reduced by 6.2℃*h-1 and BAT rewarming by 12℃*h-1. Ventilation frequency was increased by 77% during re-warming in the affected animal compared to a healthy animal. Inspection of the position and size of the mass indicated it was obstructing the lungs and heart. CONCLUSIONS: We have used a minimally invasive method to monitor BAT temperature during arousal from hibernation illustrating BAT re-warming significantly precedes core body temperature re-warming, informing future study design on arousal from hibernation. We also showed compromised re-warming from hibernation in an animal with a mass obstructing the lungs and heart, likely leading to inefficient ventilation and circulation.

Entrainment of circadian rhythms of locomotor activity by ambient temperature cycles in the dromedary camel.

In the dromedary camel, a well-adapted desert mammal, daily ambient temperature (Ta)-cycles have been shown to synchronize the central circadian clock. Such entrainment has been demonstrated by examining two circadian outputs, body temperature and melatonin rhythms. Locomotor activity (LA), another circadian output not yet investigated in the camel, may provide further information on such specific entrainment. To verify if daily LA is an endogenous rhythm and whether the desert Ta-cycle can entrain it, six dromedaries were first kept under total darkness and constant-Ta. Results showed that the LA rhythm free runs with a period of 24.8-24.9 h. After having verified that the light-dark cycle synchronizes LA, camels were subjected to a Ta-cycle with warmer temperatures during subjective days and cooler temperatures during subjective nights. Results showed that the free-running LA rhythm was entrained by the Ta-cycle with a period of exactly 24.0 h, while a 12 h Ta-cycle phase advance induced an inversion of the LA rhythm and advanced the acrophase by 9 h. Similarly, activity onset and offset were significantly advanced. All together, these results demonstrate that the Ta-cycle is a strong zeitgeber, able to entrain the camel LA rhythm, hence corroborating previous results concerning the Ta non-photic synchronization of the circadian master clock.

Melatonin rhythm and other outputs of the master circadian clock in the desert goat (Capra hircus) are entrained by daily cycles of ambient temperature.

In desert areas, mammals such as camel and goat are exposed to harsh environmental conditions. The ambient temperature (Ta) cycles have been shown to entrain the circadian clock in the camel. In the present work, we assumed that, in the goat living in a desert biotope, Ta cycles would have the same synchronizing effect on the central clock. Therefore, the effects of Ta cycles on body temperature (Tb), locomotor activity (LA) and melatonin (Mel) rhythms as outputs of the master circadian clock have been studied. The study was performed on bucks kept first under constant conditions of total darkness (DD) and constant Ta, then maintained under DD conditions but exposed to Ta cycles with heat period during subjective day and cold period during subjective night. Finally, the Ta cycles were reversed with highest temperatures during the subjective night and the lowest temperatures during the subjective day. Under constant conditions, the circadian rhythms of Tb and LA were free running with an endogenous period of 25.3 and 25.0 hours, respectively. Ta cycles entrained the rhythms of Tb and LA to a period of exactly 24.0 hours; while when reversed, the Ta cycles led to an inversion of Tb and LA rhythms. Similarly, Ta cycles were also able to entrain Mel rhythm, by adjusting its secretion to the cooling phase before and after Ta cycles inversion. All together, these results show that the Ta cycles entrain the master circadian clock in the goat.

Gene expression profiling during hibernation in the European hamster.

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body's clocks at the beginning of the active period.

Effect of Melatonin Implants during the Non-Breeding Season on the Onset of Ovarian Activity and the Plasma Prolactin in Dromedary Camel.

To examine a possible control of reproductive seasonality by melatonin, continual-release subcutaneous melatonin implants were inserted 4.5 months before the natural breeding season (October-April) into female camels (Melatonin-treated group). The animals were exposed to an artificial long photoperiod (16L:8D) for 41 days prior to implant placement to facilitate receptivity to the short-day signal that is expected with melatonin implants. The treated and control groups (untreated females) were maintained separately under outdoor natural conditions. Ovarian follicular development was monitored in both groups by transrectal ultrasonography and by plasma estradiol-17ß concentrations performed weekly for 8 weeks and then for 14 weeks following implant insertion. Plasma prolactin concentrations were determined at 45 and 15 days before and 0, 14, 28, 56, and 98 days after implant insertion. Plasma melatonin concentration was determined to validate response to the artificial long photoperiod and to verify the pattern of release from the implants. Results showed that the artificial long photoperiod induced a melatonin secretion peak of significantly (P < 0.05) shorter duration (about 2.5 h). Melatonin release from the implants resulted in higher circulating plasma melatonin levels during daytime and nighttime which persisted for more than 12 weeks following implants insertion. Treatment with melatonin implants advanced the onset of follicular growth activity by 3.5 months compared to untreated animals. Plasma estradiol-17ß increased gradually from the second week after the beginning of treatment to reach significantly (P < 0.01) higher concentrations (39.2 ± 6.2 to 46.4 ± 4.5 pg/ml) between the third and the fifth week post insertion of melatonin implants. Treatment with melatonin implants also induced a moderate, but significant (P < 0.05) suppressive effect on plasma prolactin concentration on the 28th day. These results demonstrate that photoperiod appears to be involved in dromedary reproductive seasonality. Melatonin implants may be a useful tool to manipulate seasonality and to improve reproductive performance in this species. Administration of subcutaneous melatonin implants during the transition period to the breeding season following an artificial signal of long photoperiod have the potential to advance the breeding season in camels by about 2.5 months.

The lateral habenula interacts with the hypothalamo-pituitary adrenal axis response upon stressful cognitive demand in rats.

The lateral habenula (LHb) is involved in emotional and cognitive behaviors. Recently, we have shown in rats that blockade of excitatory inputs to the LHb not only induced deficits of memory retrieval in the water maze, but also altered swim strategies (i.e., induced excessive thigmotaxis). The latter observation, although consistent with the occurrence of memory deficits, could also possibly be the consequence of an excessive level of stress, further suggesting a role for the LHb in the stress response in our behavioral paradigm. To test this hypothesis we performed in rats intra-LHb infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 267 ng/side in 0.3 µL), or vehicle, and assessed the responsiveness of the hypothalamo-pituitary adrenal (HPA) axis to environmental stressful or non-stressful situations. We have measured plasma corticosterone (CORT) concentrations at different time points before and following intra-LHb infusion of CNQX - or of the same volume of vehicle - in three conditions: during the probe test of a water maze experiment; in an anxiety test, the elevated plus maze; and in a home cage condition. Whereas there were no differences in the home cage condition and in the elevated plus maze, in the water maze experiment we observed that CNQX-treated rats presented, along with memory deficits, a higher level of blood CORT than vehicle-treated rats. These results suggest that perturbations of the modulation of the HPA axis are consecutive to the alteration of LHb function, whether it is the result of a defective direct control of the LHb over the HPA axis, or the consequence of memory deficits.

Maternal photoperiod programs hypothalamic thyroid status via the fetal pituitary gland.

In wild mammals, offspring development must anticipate forthcoming metabolic demands and opportunities. Within species, different developmental strategies may be used, dependent on when in the year conception takes place. This phenotypic flexibility is initiated before birth and is linked to the pattern of day length (photoperiod) exposure experienced by the mother during pregnancy. This programming depends on transplacental communication via the pineal hormone melatonin. Here, we show that, in the Siberian hamster (Phodopus sungorus), the programming effect of melatonin is mediated by the pars tuberalis (PT) of the fetal pituitary gland, before the fetal circadian system and autonomous melatonin production is established. Maternal melatonin acts on the fetal PT to control expression of thyroid hormone deiodinases in ependymal cells (tanycytes) of the fetal hypothalamus, and hence neuroendocrine output. This mechanism sets the trajectory of reproductive and metabolic development in pups and has a persistent effect on their subsequent sensitivity to the photoperiod. This programming effect depends on tanycyte sensitivity to thyroid stimulating hormone (TSH), which is dramatically and persistently increased by short photoperiod exposure in utero. Our results define the role of the fetal PT in developmental programming of brain function by maternal melatonin and establish TSH signal transduction as a key substrate for the encoding of internal calendar time from birth to puberty.

miR-132/212 Modulates Seasonal Adaptation and Dendritic Morphology of the Central Circadian Clock.

The central circadian pacemaker, the suprachiasmatic nucleus (SCN), encodes day length information by mechanisms that are not well understood. Here, we report that genetic ablation of miR-132/212 alters entrainment to different day lengths and non-24 hr day-night cycles, as well as photoperiodic regulation of Period2 expression in the SCN. SCN neurons from miR-132/212-deficient mice have significantly reduced dendritic spine density, along with altered methyl CpG-binding protein (MeCP2) rhythms. In Syrian hamsters, a model seasonal rodent, day length regulates spine density on SCN neurons in a melatonin-independent manner, as well as expression of miR-132, miR-212, and their direct target, MeCP2. Genetic disruption of Mecp2 fully restores the level of dendritic spines of miR-132/212-deficient SCN neurons. Our results reveal that, by regulating the dendritic structure of SCN neurons through a MeCP2-dependent mechanism, miR-132/212 affects the capacity of the SCN to encode seasonal time.

Like melatonin, agomelatine (S20098) increases the amplitude of oscillations of two clock outputs: melatonin and temperature rhythms.

Depression and biological rhythms disturbances are strongly associated. Agomelatine is an antidepressant with melatoninergic MT1-MT2 agonist and serotoninergic 5-HT2c antagonist properties. Both melatonin and 5-HT are known to modulate circadian rhythmicity controlled by the endogenous clock located in the suprachiasmatic nuclei (SCN). The aim of the present study was to compare the effect of an acute injection of agomelatine (Ago), melatonin (MLT) or an antagonist 5-HT2c (S32006), on the rhythms of two robust clock outputs: the pineal MLT secretion and the body temperature rhythm (Tc). Daily endogenous MLT profiles were measured using transpineal microdialysis over 4 consecutive days in rats maintained on a 12 h light/12 h dark cycle. Simultaneously, Tc was recorded. The drugs were injected subcutaneously at three doses (1, 2.5 or 5 mg/kg) at the onset of darkness. Both Ago and MLT, at the dose of 2.5 mg/kg, increased the amplitude of the peak of MLT secretion and this effect was observed 2 d after injection. Moreover, both drugs induced a dose-dependent advance of the rhythm onset which resulted in lengthening of the MLT peak. S32006 had no effect on the rhythm of MLT. Ago, MLT and S32006 increased the amplitude of the rhythm of Tc. These data suggest a central action of Ago, directly on the SCN, via melatoninergic receptors responsible for both the increased amplitude of MLT rhythm and the phase advance. The increase in the amplitude of the body temperature could involve both MLT agonist and/or 5-HT2c antagonist properties of Ago.

Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius).

In mammals the light-dark (LD) cycle is known to be the major cue to synchronize the circadian clock. In arid and desert areas, the camel (Camelus dromedarius) is exposed to extreme environmental conditions. Since wide oscillations of ambient temperature (Ta) are a major factor in this environment, we wondered whether cyclic Ta fluctuations might contribute to synchronization of circadian rhythms. The rhythm of body temperature (Tb) was selected as output of the circadian clock. After having verified that Tb is synchronized by the LD and free runs in continuous darkness (DD), we submitted the animals to daily cycles of Ta in LL and in DD. In both cases, the Tb rhythm was entrained to the cycle of Ta. On a 12-h phase shift of the Ta cycle, the mean phase shift of the Tb cycle ranged from a few hours in LD (1 h by cosinor, 4 h from curve peaks) to 7-8 h in LL and 12 h in DD. These results may reflect either true synchronization of the central clock by Ta daily cycles or possibly a passive effect of Ta on Tb. To resolve the ambiguity, melatonin rhythmicity was used as another output of the clock. In DD melatonin rhythms were also entrained by the Ta cycle, proving that the daily Ta cycle is able to entrain the circadian clock of the camel similar to photoperiod. By contrast, in the presence of a LD cycle the rhythm of melatonin was modified by the Ta cycle in only 2 (or 3) of 7 camels: in these specific conditions a systematic effect of Ta on the clock could not be evidenced. In conclusion, depending on the experimental conditions (DD vs. LD), the daily Ta cycle can either act as a zeitgeber or not.

Altered circadian rhythm of melatonin concentrations in hypocretin-deficient men.

Hypocretin deficiency causes narcolepsy. It is unknown whether melatonin secretion is affected in this sleep disorder. Therefore, in both narcolepsy patients and matched controls, the authors measured plasma melatonin levels hourly for 24 h before and after 5 days of sodium oxybate (SXB) administration. Although mean melatonin concentrations were similar between patients and controls, in narcoleptics the percentage of 24-h melatonin secreted during the daytime was significantly higher, and melatonin secretion exhibited a weaker coupling to sleep. SXB did not affect melatonin secretion. These findings suggest that hypocretin deficiency might disturb both the circadian control of melatonin release and its temporal association with sleep.

Daily Aa-nat gene expression in the camel (Camelus dromedarius) pineal gland.

Arylalkylamine N-acetyltransferase (AA-NAT) is the rhythm-generating enzyme for the synthesis of pineal melatonin. Molecular investigations have revealed two biological models for the activation of AA-NAT. In rodent species, Aa-nat gene transcription is turned off during the daytime and markedly activated at night. In primates, sheep, and cows, the Aa-nat gene is constitutively transcripted with no visible daily variations. This inter-species difference in Aa-nat gene regulation leads to different daily profiles in melatonin synthesis and release. Thus, the nighttime onset of the rise in circulating melatonin is delayed and slow in rodents, whereas it is fast and sharp in sheep. In the camel (Camelus dromedarius), we have observed that circulating melatonin rises immediately after sunset, suggesting AA-NAT activity is regulated at the post-transcriptional level. In agreement with this hypothesis, we report herein the amount of Aa-nat mRNA in the camel pineal gland is high, during both the day and night with no daily variations, while melatonin concentration in the same pineal tissue is five times higher during the night than daytime.

Turkey retina and pineal gland differentially respond to constant environment.

Dynamics of rhythmic oscillations in the activity of arylalkylamine N-acetyltransferase (AA-NAT, the penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in the retina and pineal gland of turkeys maintained for 7 days in the environment without daily light-dark (LD) changes, namely constant darkness (DD) or continuous light (LL). The two tissues differentially responded to constant environment. In the retina, a circadian AA-NAT activity rhythm disappeared after 5 days of DD, while in the pineal gland it persisted for the whole experiment. No circadian rhythm was observed in the retinas of turkeys exposed to LL, although rhythmic oscillations in both AA-NAT and melatonin content were found in the pineal glands. Both tissues required one or two cycles of the re-installed LD for the full recovery of the high-amplitude AA-NAT rhythm suppressed under constant conditions. It is suggested that the retina of turkey is less able to maintain rhythmicity in constant environment and is more sensitive to changes in the environmental lighting conditions than the pineal gland. Our results indicate that, in contrast to mammals, pineal glands of light-exposed galliformes maintain the limited capacity to rhythmically produce melatonin.

Opposite actions of hypothalamic vasopressin on circadian corticosterone rhythm in nocturnal versus diurnal species.

Relatively little is known about the function of the biological clock and its efferent pathways in diurnal species, despite the fact that its major transmitters and neuronal connections are also conserved in humans. The mammalian biological clock is located in the hypothalamic suprachiasmatic nuclei (SCN). Several lines of evidence suggest that the activity cycle of the SCN itself is similar in nocturnal and diurnal mammals. Previously, we showed that, in the rat, vasopressin (VP) derived from the SCN has a strong inhibitory effect on the release of adrenal corticosterone and is an important component in the generation of a daily rhythm in plasma corticosterone concentrations. In the present study we investigated the role of VP in the control of the daily corticosterone rhythm in a diurnal rodent, i.e. Arvicanthis ansorgei. Contrary to our previous (rat) results, VP administered to the hypothalamic paraventricular nucleus in A. ansorgei had a stimulatory effect on the release of corticosterone. Moreover, both the morning and evening rise in corticosterone were blocked by the administration of a VP receptor antagonist. These results show that with regard to the circadian control of the corticosterone rhythm in diurnal and nocturnal rodents, temporal information is carried along the same pathway from the SCN to its target areas, but the response of the target area may be quite different. We propose that the reversed response to VP is due to a change in the phenotype of the target neurons that are contacted by the SCN efferents, i.e. glutamatergic instead of gamma-aminobutyric acid (GABA)ergic.

Trans-pineal microdialysis in the Djungarian hamster (Phodopus sungorus): a tool to study seasonal changes of circadian clock activities.

The Djungarian hamster is a highly seasonal small mammal. The rhythmic secretion of melatonin by the pineal gland is under control of the circadian clock, conveying the photoperiodic message to the organism. Trans-pineal microdialysis permits the in vivo study of this well-defined and precise clock output by measuring melatonin release directly in the pineal gland. The aim of this study was to adapt this method to the Djungarian hamster in order to monitor clock properties during photoperiodic changes. Male adult Djungarian hamsters were kept in a long photoperiod (LD 16:8) and melatonin release was measured hourly during the dark period for several weeks. Melatonin showed a regular secretion between ZT 17 and ZT 23.5 whereas the amplitude became stable only after the third day of perfusion. To test how quickly changes in melatonin profile can be measured, 15-min light pulses were given at different time points throughout the scotophase. Light-pulses immediately interrupted melatonin secretion at any time point during the scotophase and the temporal resolution for measurement could be reduced to 30 min. In accordance with studies in the rat, long-term effects of light on the clock could only be observed when a light pulse was administered in the second half of the night. For the first time we established a method to measure precisely a direct and reliable clock-output in a highly seasonal species which allows us now to study the circadian and seasonal properties of the clock in detail.

A functional subdivision of the circadian clock is revealed by differential effects of melatonin administration.

The biological clock of the suprachiasmatic nuclei drives numerous physiological and behavioural circadian rhythms. In this study, we addressed the question as to whether different components of the clock may control separately various circadian functions. Using the rat transpineal microdialysis tool, we analysed the effect of clock perturbation by exogenous melatonin injection on two hormonal clock outputs: pineal melatonin and adrenal corticosterone secretions. As already reported, a single melatonin injection at the light/dark transition induces a marked increase in the endogenous pineal melatonin peak for the two following days. In the same animals, by contrast, the amplitude of the corticosterone rhythm was not altered following melatonin injection. These data show that the melatonin injection does not display an overall effect on the circadian clock, but rather influences a subpopulation of melatonin-sensitive neurons involved, among other functions, in the circadian control of the pineal pathway.

Environmental control and adrenergic regulation of pineal activity in the diurnal tropical rodent, Arvicanthis ansorgei.

Like nocturnal rodents, the diurnal tropical rodent Arvicanthis ansorgei shows a daily rhythm in pineal melatonin content. Seasonal and photoperiodic variations in the biosynthetic activity of the pineal gland: arylalkylamine-N-acetyltransferase (AA-NAT), hydroxyindole-O-methyltransferase (HIOMT) activities and melatonin content were measured in male and female A. ansorgei captured near Samaya, Mali, and kept either under artificial laboratory photoperiods [light-dark (LD) cycles: LD 14:10, LD 12:12 or LD 10:14 or caught in the field in Mali and killed at four different times of the year (January, April, June and November). Under artificial photoperiod, the duration of the nocturnal peak of AA-NAT activity and melatonin content increased with the duration of the dark period while the amplitude did not significantly change. In the field, annual variations in the amplitude of the nocturnal melatonin peak were observed with a maximum in April (highest temperature, low humidity and no grass availability, only seeds) and a minimum in November (high humidity, maximum green grass availability). The variations in the amplitude of the melatonin peak were not correlated with changes in AA-NAT HIOMT activities, suggesting that seasonal variations in the amplitude of the melatonin peak are not driven by these enzymes. Daytime injections of the beta-adrenergic agonist, isoproterenol, stimulated melatonin synthesis in January, April and June, but not in November. The annual differences in the amplitude of the melatonin peak as well as the seasonal differences in the response to an adrenergic stimulation suggest that environmental factors other than photoperiod, such as temperature, humidity and consequent food availability, could be important in the regulation of the annual variations in the pineal biosynthetic activity in this species.

Pineal melatonin synthesis and release are not altered throughout the estrous cycle in female rats.

Melatonin times reproduction with seasons in many photoperiodic mammalian species. Whether sexual hormones reflect on melatonin synthesis is still debated. The aim of this work was to study, using a large panel of technical approaches, whether the daily profile of pineal melatonin synthesis and release varies with the estrous cycle in the female rat. The mRNA levels and enzyme activities of the melatonin synthesizing enzymes, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase were similar at the four stages of the rat estrous cycle. The endogenous release of melatonin, followed by transpineal microdialysis during six consecutive days in cycling female rats, displayed no significant variation during this interval. Taken together, the present results demonstrate that there is no regular fluctuation in the pineal metabolism leading to melatonin synthesis and release throughout the estrous cycle in female rats.

In the rat, exogenous melatonin increases the amplitude of pineal melatonin secretion by a direct action on the circadian clock.

The effect of exogenous melatonin on pineal melatonin synthesis was studied in the rat in vivo. Daily melatonin profiles were measured by transpineal microdialysis over 4 consecutive days in rats maintained on a 12-h light : 12-h dark schedule (LD 12 : 12). Curve-fitting was used to determine the amplitude of the peak of melatonin production, and the times of its onset (IT50) and offset (DT50). A subcutaneous injection of melatonin (1 mg/kg) at the onset of darkness (ZT12) induced an advance of IT50 on the second day after the treatment, in 50% of the animals kept in LD. When the animals were switched to constant darkness, the treatment caused no detectable advance of IT50, while 70% of individuals showed a significant delay in DT50 2 days after the injection. Locally infusing the drug by reverse microdialysis into the suprachiasmatic nuclei (SCN) failed to enhance the shift in melatonin onset. Following subcutaneous melatonin injection, a significant increase ( approximately 100%) in melatonin peak amplitude was observed. This increase persisted over 2 days and occurred only when the melatonin was applied at ZT12, but not at ZT6, 17 or 22. The effect was also observed when the drug was infused directly into the SCN, but not into the pineal. Thus, the SCN are the target site for the effect of exogenous melatonin on the amplitude of the endogenous melatonin rhythm, with a similar window of sensitivity as its phase-shifting effect on the pacemaker.