Seasonal variations of clock gene expression in the suprachiasmatic nuclei and pars tuberalis of the European hamster (Cricetus cricetus)

In mammals, day length (photoperiod) is read and encoded in the main circadian clock, the suprachiasmatic nuclei (SCN). In turn, the SCN control the seasonal rhythmicity of various physiological processes, in particular the secretion pattern of the pineal hormone melatonin. This hormone then operates as an essential mediator for the control of seasonal physiological functions on some tissues, especially the pars tuberalis (PT). In the European hamster, both hormonal (melatonin) and behavioral (locomotor activity) rhythms are strongly affected by season, making this species an interesting model to investigate the impact of the seasonal variations of the environment. The direct (on SCN) and indirect (via melatonin on PT) effect of natural short and long photoperiod was investigated on the daily expression of clock genes, these being expressed in both tissues. In the SCN, photoperiod altered the expression of all clock genes studied. In short photoperiod, whereas Clock mRNA levels were reduced, Bmal1 expression became arrhythmic, probably resulting in the observed dramatic reduction in the rhythm of Avp expression. In the PT, Per1 and Rev-erbalpha expressions were anchored to dawn in both photoperiods. The daily profiles of Cry1 mRNA were not concordant with the daily variations in plasma melatonin although we confirmed that Cry1 expression is regulated by an acute melatonin injection in the hamster PT. The putative role of such seasonal-dependent changes in clock gene expression on the control of seasonal functions is discussed.     

Circulating levels of glucocorticoid hormones in WHHL and NZW rabbits: circadian cycle and response to repeated social encounter

Social environment influences the progression of atherosclerosis in an important experimental model of disease, the Watanabe Heritable Hyperlipidemic rabbit (WHHL). Although the hypothalamic-pituitary-adrenocortical (HPA) system is likely to play an important role in the behavioral modulation of disease, relatively little is known about the glucocorticoid responses in these animals, or in other strains of rabbits. The purpose of the present study was to: (1) evaluate the rabbit glucocorticoid circadian rhythm, (2) compare plasma cortisol and corticosterone responses to social stress, and (3) examine strain differences (i.e., WHHL vs. New Zealand White (NZW)) in rabbit glucocorticoid responses to assess whether WHHLs have an aberrant HPA system. It was found that male rabbits secrete both corticosterone and cortisol in a circadian rhythm that peaks in the afternoon and reaches a nadir at 0600 h, i.e., approximately 12 h out-of-phase with the human glucocorticoid rhythm. Both glucocorticoids responded similarly to social stress induced by repeated daily 4 h pairings with another male rabbit; after 10 days of pairings, glucorticoid values were significantly correlated with the amount of defensive agonistic behavior exhibited. Finally, there were no significant strain differences in glucocorticoid circadian rhythms, baselines, or responses to social stress. These data suggest that glucocorticoid responses (i.e., circadian rhythms, responses to social stress) in the WHHL are similar to glucocorticoid responses in standard laboratory white rabbits.     

Absence of pineal-independent mediation of seasonal differences in suprachiasmatic nucleus AVP and VIP mRNA expression in Siberian hamsters

Assessment of seasonal variations in expression of brain neuropeptide mRNA is complicated by concurrent circadian variations. Because entrainment of suprachiasmatic nucleus (SCN) based rhythms differs in long versus short day lengths, valid seasonal comparisons must be made at equivalent circadian phases. We used a novel experimental design which permitted sampling at identical circadian phases of animals exhibiting opposite seasonal reproductive responses to the same intermediate day length. This allowed us to test whether seasonal changes in arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) mRNA expression in the SCN occur in the absence of the pineal gland. Juvenile Siberian hamsters were gestated and maintained postnatally in either a long photoperiod (16 h light/day) or short photoperiod (10 h light/day). At the time of weaning (18 days of age), the hamsters were pinealectomized and either transferred to a new photoperiod (10-, 16- or 14-h light/day) or left in the original photoperiod. Hamsters from 10L had substantially smaller and lighter testes than those from 16L. If photoperiodic modulation of AVP and VIP mRNA expression occurs in the absence of the pineal, then transfer of pinealectomized hamsters from a longer (16L) or shorter (10L) photoperiod to an intermediate photoperiod (14L) should result in a differential response with respect to SCN AVP and VIP mRNA expression but not testis size. When sampled at an identical circadian phase (3 h after lights on) in 14L there was no difference in the expression of AVP or VIP mRNA in the SCN between animals previously housed in long versus short day lengths. In contrast to a previous study that did not carefully control for circadian phase, the present findings suggest that seasonal photoperiodic control of SCN neuropeptide mRNA expression depends upon the pineal gland. In addition, the present findings demonstrate a significant, negative correlation between AVP mRNA expression in the SCN and the length of the daily active phase (alpha).     

Complex regional influence of photoperiod on the nycthemeral functioning of the dorsal and median raphé serotoninergic system in the Syrian hamster

The Syrian hamster ( Mesocricetus auratus ) is a widely used species for the study of biological clock synchronization and photoperiodism. The serotoninergic system arising from the median (MnR) and the dorsal raphé (DR) is a major actor in circadian clock synchronization. This serotoninergic system is also associated with functions and behaviours influenced by seasonal changes. The aim of the present study was to assess the influence of photoperiod on the daily functioning of the MnR and DR serotoninergic system. The morphology of both raphé nuclei was analysed in hamsters kept under long and short photoperiod by immunocytochemical detection of two markers of the serotoninergic system, serotonin and tryptophan hydroxylase (TPH, the rate-limiting enzyme of serotonin synthesis). The morphological analysis revealed a fairly complex morphological organization of the DR and MnR along their caudo-rostral extent. This morphological organisation was similar in the two photoperiods. However, quantification of several markers of serotonin (5-HT) synthesis in the DR and MnR revealed a time-dependent functioning of serotoninergic cells that was locally influenced by photoperiod. In particular, the significant daily variations of tph2 -mRNA and TPH levels in the rostral MnR, and of the 5-HT/5-HIAA (5 - hydroxyindoleacetic acid) ratio within the suprachiasmatic nuclei, were abolished under short photoperiod. The results are discussed with regard to the known physiological role of the serotoninergic system on the biological clock.     

Methamphetamine-induced,suprachiasmatic nucleus-independent circadian rhythms of activity and mPer gene expression in the striatum of the mouse

While the suprachiasmatic nucleus (SCN) coordinates the majority of daily rhythms, some circadian patterns of expression are controlled from outside of the SCN. These include responses to daily methamphetamine (MAP) injection, or daily restricted feeding. The mechanisms underlying these SCN-independent circadian rhythms are unknown. A circadian oscillation in the expression of mPer1 and/or mPer2, mouse period, in the SCN is considered necessary to generate an SCN-dependent circadian rhythm. Therefore, in this experiment, we examined the association between mPer gene expression and the MAP-induced, SCN-independent circadian rhythm. Acute injection of MAP caused an elevation of mPer1, mBmal1, and mNpas2 gene expression in the striatum and mPer1 in the liver. Daily MAP injection at a fixed time for 6 days shifted the rhythmic mPer1 and mPer2 expression in the striatum from a nocturnal to a diurnal rhythm, but failed to affect that in the SCN. Although lesion of the SCN 'flattened'mPer gene oscillation in the striatum and liver, daily MAP injection caused both behavioural and mPer gene expression rhythms. Daily MAP injection at variable injection intervals (12-36 h) for 6 days, however, failed to produce mPer gene rhythm in the striatum. Daily repeated MAP signals may strengthen the oscillatory force of SCN-independent circadian behavioural and molecular rhythms. The present results suggest that daily oscillation of mPer genes outside the SCN is closely associated with the regulation of SCN-independent rhythms. Thus, the present experiment highlights strongly the important role of clock gene expression, in the brain, that underlies the circadian behavioural rhythm.     

Circadian organization and neural mediation of hamster reproductive rhythms.

(1) Circadian rhythms mediate various cycles of the hamster's reproductive physiology and behavior. (2) Destruction of the suprachiasmatic nuclei interfered with the integration and/or generation of coherent circadian rhythms of wheel-running behavior and also abolished photoperiodic regulation of the testes, which remain large and functional after the lesion regardless of photoperiod or presence of the eyes. (3) The retinohypothalamic tract is the principal visual projection for the entrainment of circadian activity rhythms to the light: dark cycle and also appears necessary and sufficient for mediating the effects of photoperiod on the reproductive physiology of the male hamster. (4) The hamster estrous cycle free-ran under constant dim illumination with a period significantly different from 4 days; regression analyses of concurrently recorded free-running heat onsets and wheel-running behavior showed that the period of the estrous cycle was a multiple (× 4) of the circadian activity period. This implies a circadian organization of the estrous cycle and mediation of both activity and estrous behaviors by a common circadian system. Implications of these findings for contemporary views of sexual differentiation of gonadotrophin release are considered. (5) Estradiol influenced the phase angle of entrainment of wheel-running behavior by ovariectomized hamsters. The period of the free-running activity rhythm was significantly shortened in blind ovariectomized hamsters implanted with Silastic capsules containing estradiol benzoate. The functional significance of these hormone-mediated changes in rhythm periodicity is discussed. (6) Exposure of male hamsters to short daily photoperiods reduced the frequency of copulation. Castrated males maintained on short daily photoperiods were less responsive than those on long days to the activational effects of testosterone on copulation. The significance of this photoperiodically induced refractoriness to hormones, which in this species appears limited to the male, is discussed.     

Glucocorticoid Regulation of Clock Gene Expression in the Mammalian Limbic Forebrain

The cyclical expression of the clock protein PERIOD2 (PER2) in select regions of the limbic forebrain is contingent upon the rhythmic secretion of the adrenal glucocorticoid, corticosterone. Daily rhythmic PER2 expression in the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and the central nucleus of the amygdala (CEA) is abolished with the removal of the adrenal glands but restored with rhythmic hormone replacement via the drinking water at a time corresponding to the endogenous peak of circulating glucocorticoids. Here, we investigated the effects of serial or acute systemic injections of corticosterone on the expression of PER2 in the BNSTov and CEA of both adrenalectomized (ADX) and intact rats. We sought to determine whether there is a temporal window of corticosterone sensitivity by delivering the hormone at a time corresponding to trough levels of circulating glucocorticoids, at lights on. We found that daily morning injections of corticosterone induced PER2 expression in the BNSTov and CEA of ADX rats, with levels peaking 1 h after injection. In intact rats, daily morning injections significantly upregulated the expression of PER2 in the BNSTov and CEA 1 h after injection and dampened the evening peak, while a single injection abolished the rhythm of PER2 expression in the CEA but had no effect on PER2 in the BNSTov. Our findings suggest that despite the potential masking effect of signals from the light-entrained master clock, daytime chronic and acute corticosterone administration can alter the rhythmic expression of PER2 in the BNSTov and CEA, and that the response is region-specific and dependent on the duration of treatment.     

Exogenous Corticosterone Induces the Expression of the Clock Protein,PERIOD2, in the Oval Nucleus of the Bed Nucleus of the Stria Terminalis and the Central Nucleus of the Amygdala of Adrenalectomized and Intact Rats

The cyclical expression of the clock protein PERIOD2 (PER2) in select regions of the limbic forebrain is contingent upon the rhythmic secretion of the adrenal glucocorticoid, corticosterone. Daily rhythmic PER2 expression in the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and the central nucleus of the amygdala (CEA) is abolished with the removal of the adrenal glands but restored with rhythmic hormone replacement via the drinking water at a time corresponding to the endogenous peak of circulating glucocorticoids. Here, we investigated the effects of serial or acute systemic injections of corticosterone on the expression of PER2 in the BNSTov and CEA of both adrenalectomized (ADX) and intact rats. We sought to determine whether there is a temporal window of corticosterone sensitivity by delivering the hormone at a time corresponding to trough levels of circulating glucocorticoids, at lights on. We found that daily morning injections of corticosterone induced PER2 expression in the BNSTov and CEA of ADX rats, with levels peaking 1 h after injection. In intact rats, daily morning injections significantly upregulated the expression of PER2 in the BNSTov and CEA 1 h after injection and dampened the evening peak, while a single injection abolished the rhythm of PER2 expression in the CEA but had no effect on PER2 in the BNSTov. Our findings suggest that despite the potential masking effect of signals from the light-entrained master clock, daytime chronic and acute corticosterone administration can alter the rhythmic expression of PER2 in the BNSTov and CEA, and that the response is region-specific and dependent on the duration of treatment.     

Variable Restricted Feeding Disrupts the Daily Oscillations of Period2 Expression in the Limbic Forebrain and Dorsal Striatum in Rats

Predictable restricted feeding schedules limit food availability to a single meal at the same time each day, lead to the induction and entrainment of circadian rhythms in food-anticipatory activity, and shift daily rhythms of clock gene expression in areas of the brain that are important in the regulation of motivational and emotional state. In contrast, when food is delivered under a variable restricted feeding (VRF) schedule, at a new and unpredictable mealtime each day, circadian rhythms in food-anticipatory activity fail to develop. Here, we study the effects of VRF on the daily rhythm of plasma corticosterone and of clock gene expression in the limbic forebrain and dorsal striatum, of rats provided a 2-h access to a complete meal replacement (Ensure Plus) at an unpredictable time each day. VRF schedules varied the mealtimes within the 12 h of light (daytime VRF), the 12 h of dark (nighttime VRF), or across the 24 h light–dark cycle (anytime VRF). Our results show that contrary to the synchronizing effects of predictable restricted feeding, VRF blunts the daily corticosterone rhythm and disrupts daily rhythms of PER2 expression in a region-specific and mealtime-dependent manner.     

5-hydroxyoxindole, an indole metabolite, is present at high concentrations in brain

5-Hydroxyoxindole has been identified as a urinary metabolite of indole, which is produced from tryptophane via the tryptophanase activity of gut bacteria. We have demonstrated recently that 5-hydroxyoxindole is an endogenous compound in blood and tissues of mammals, including humans. To date, 5-hydroxyoxindole's role is unknown. The aim of this study was to compare 5-hydroxyoxindole levels in plasma and cerebrospinal fluid (CSF) during day-night and seasonal changes, as a common approach to pilot physiological characterization of any compound. Simultaneous blood and CSF sampling was performed in the ewe, because its size allows collection in quantities suitable for 5-hydroxyoxindole assay (HPLC-ED) in awake animals, without obvious physiological or behavioral disturbance. 5-Hydroxyoxindole concentration was quite stable in plasma (2-6 nM range), whereas, in CSF, it displayed marked day-night and photoperiodic variations (4-116 nM range). 5-Hydroxyoxindole levels in CSF were twofold higher at night than during the day and at least one order of magnitude higher during the long compared with the short photoperiod. These day/night and photoperiodic variations persisted after pinealectomy, indicating that 5-hydroxyoxindole rhythms in CSF are independent of melatonin formation. In conclusion, high levels of 5-hydroxyoxindole in the CSF during long photoperiod and its daily modulation suggest physiological involvement of 5-hydroxyoxindole in rhythmic adjustments in the brain, independently of the pineal gland.     

The suprachiasmatic nuclei as a seasonal clock

In mammals, the suprachiasmatic nucleus (SCN) contains a central clock that synchronizes daily (i.e., 24-h) rhythms in physiology and behavior. SCN neurons are cell-autonomous oscillators that act synchronously to produce a coherent circadian rhythm. In addition, the SCN helps regulate seasonal rhythmicity. Photic information is perceived by the SCN and transmitted to the pineal gland, where it regulates melatonin production. Within the SCN, adaptations to changing photoperiod are reflected in changes in neurotransmitters and clock gene expression, resulting in waveform changes in rhythmic electrical activity, a major output of the SCN. Efferent pathways regulate the seasonal timing of breeding and hibernation. In humans, seasonal physiology and behavioral rhythms are also present, and the human SCN has seasonally rhythmic neurotransmitter levels and morphology. In summary, the SCN perceives and encodes changes in day length and drives seasonal changes in downstream pathways and structures in order to adapt to the changing seasons.     

Experimental jetlag disrupts circadian clock genes but improves performance in racehorses after light-dependent rapid resetting of neuroendocrine systems and the rest-activity cycle

Abrupt alterations in the 24-h light : dark cycle, such as those resulting from transmeridian air travel, disrupt circadian biological rhythms in humans with detrimental consequences on cognitive and physical performance. In the present study, a jetlag-simulated phase shift in photoperiod temporally impaired circadian peaks of peripheral clock gene expression in racehorses but acutely enhanced athletic performance without causing stress. Indices of aerobic and anaerobic capacities were significantly increased by a phase-advance, enabling prolonged physical activity before fatigue occurred. This was accompanied by rapid re-entrainment of the molecular clockwork and the circadian pattern of melatonin, with no disturbance of the adrenal cortical axis, but a timely rise in prolactin, which is a hormone known to target organs critical for physical performance. Subsequent studies showed that, unlike the circadian pattern of melatonin, and in contrast to other species, the daily rhythm of locomotor activity was completely eliminated under constant darkness, but it was restored immediately upon the reintroduction of a light : dark cycle. Resetting of the rhythm of locomotion was remarkably fast, revealing a rapid mechanism of adaptation and a species dependency on light exposure for the expression of daily diurnal activity. These results show that horses are exquisitely sensitive to sudden changes in photoperiod and that, unlike humans, can benefit from them; this appears to arise from powerful effects of light underlying a fast and advantageous process of adjustment to the phase shift.     

Suprachiasmatic nuclei influence hibernation rhythms of golden-mantled ground squirrels

Hibernation and body mass rhythms were studied in 13 golden-mantled ground squirrels maintained in an LD 12:12 photoperiod at 5 °C. Complete or partial ablation of the suprachiasmatic nuclei (SCN) disrupted normal hibernation rhythms. Over the course of 2 years, several animals progressed through 4 hibernation cycles, one squirrel manifested two abnormally long hibernation seasons, and another failed to hibernate. Squirrels with intact SCN exhibited normal circannual hibernation rhythms at intervals of 11.5 ± 0.3 months. Hibernation coincided with the weight loss phase of the body mass cycle in control squirrels, but these two rhythms were dissociated in animals with lesions of the SCN. The annual plasma testosterone rhythm was normal or slightly phase-delayed in squirrels with SCN lesions maintained at 23 °C. The SCN may be part of a neural circuit that mediates circannual organization of hibernation rhythms.     

Photoperiodic modulation of the hepatic clock by the suprachiasmatic nucleus and feeding regime in mice

Changes in photoperiod modulate the central circadian clock in the suprachiasmatic nucleus (SCN) as well as the peripheral clocks. Consequently, the SCN-driven output rhythms in activity and feeding are also modulated by the photoperiod. The aim of the present study was to elucidate whether photoperiodic modulation of the hepatic clock is mediated by changes in feeding or by another SCN-driven pathway. Five days after the change from short photoperiod (SP) to long photoperiod (LP), the profiles of Per2 and Rev-erbα expression in the rostral, middle and caudal regions of the SCN were desynchronized and those in the liver were modulated as in mice fully entrained to LP. The SCN profiles were not affected in mice left under SP and subjected to the 6-h night-time feeding regime for 5 days. In the liver, the profiles were shifted to the same phase, but their waveforms were not modulated compared with those under LP. In mice subjected to the change from SP to LP and fed twice daily during the daytime, the profiles in the SCN were not affected, whereas the waveforms and phases of those in the liver were affected. The data demonstrate that the adjustment of gene expression profiles in the rostral, middle and caudal SCN to the change from SP to LP proceeds within 5 days and is not affected by changes in the feeding regime. The results also suggest that the photoperiod-modulated SCN affects waveforms of gene expression profiles in the liver by food-independent signals.     

Characterization of Glucocorticoid Binding Capacity in Human Mononuclear Lymphocytes: Increase by Metyrapone is Prevented by Dexamethasone Pretreatment

Autoregulation of receptor systems by their own ligands is a well established biological phenomenon. While down-regulation of the glucocorticoid binding capacity by glucocorticoids has been shown in animals and humans, data on up-regulation processes in humans are lacking. To further explore glucocorticoid receptor plasticity in relation to endogenous ligands, glucocorticoid binding parameters were assessed in 15 healthy controls before and after oral administration of 1.5 g metyrapone with and without dexamethasone pretreatment. Administration of metyrapone resulted in blockade of the feedback of the hypothalamic-pituitary-adrenal system as shown by the rise in adrenocorticotropin levels, while pretreatment with 1 mg dexamethasone completely suppressed adrenocorticotropin concentrations. Glucocorticoid binding sites per lymphocyte exhibited an increase of 63% following metyrapone administration, which was prevented by dexamethasone pretreatment. Comparison of morning and afternoon glucocorticoid binding sites per cell in 11 healthy volunteers further revealed a diurnal rhythm of glucocorticoid receptor sites. These data suggest that human lymphocyte glucocorticoid receptors are under autoregulatory control.     

Aging and photoperiod regulate glutamic acid decarboxylase(67) messenger RNA expression.

Aging and short photoperiod exposure both induce similar long-term changes in circadian rhythms, including alterations in the timing and the amplitude of rhythms. Furthermore, these chronic conditions affect the function of the circadian pacemaker in the suprachiasmatic nuclei (SCN) by altering rhythmic expression of neuropeptide messenger RNAs (mRNAs). Because GABA modulates SCN neuronal activity, and GABAergic neurons innervate peptidergic neurons in the SCN, the present study investigated whether photoperiod or aging affect the expression of mRNA for GAD(67), the enzyme responsible for regulating the tonic levels of GABA. As a control for regional specificity, the reticular thalamic nucleus (RTN) was also examined. In situ hybridization for GAD(67) mRNA was performed on brain sections derived from Siberian hamsters exposed to a long day or a short day photoperiod for 15 days, and on brain sections from young (3-4 months old) and old (12-17 months old) Syrian hamsters exposed to a long photoperiod. The results showed that photoperiod and aging have different effects on GAD(67) mRNA expression. Exposure to short day photoperiod significantly increased GAD(67) mRNA expression in both the SCN and RTN of Siberian hamsters, while aging significantly decreased GAD(67) mRNA expression in the RTN of Syrian hamsters but had no effect on GAD(67) mRNA expression in the SCN. These findings suggest that modulation of GAD(67) mRNA expression in the SCN is associated with photoperiodic regulation of neuropeptide mRNA expression, but is not a common mechanism for chronic regulation of circadian rhythms. Also, GAD(67) mRNA expression in the RTN is differentially affected by photoperiod and aging.