Circadian functioning of Locus Cœruleus of the nocturnal rat and diurnal rodent Arvicanthis.

The noradrenergic Locus Cœruleus is one of the major arousal structures involved in inducing wakefulness. While brain noradrenaline (NA) amounts display 24-h variations, the origin of NA rhythm is currently unknown. In this study, we tested the hypothesis that NA rhythm could result from its rhythmic synthesis. Therefore, we investigated the 24-h expression profile of NA rate-limiting enzyme, tyrosine hydroxylase (th), in the Locus Cœruleus (LC) of the nocturnal rat and the diurnal rodent Arvicanthis, under 12 h:12 h light/dark (LD) and constant darkness (DD) conditions. In both species, th mRNA levels vary significantly over 24-h. In nocturnal rats, th mRNA profiles show a unimodal rhythm, with peak values in late day in LD, and in the middle of the subjective day in DD. In contrast, th mRNA rhythm in Arvicanthis is characterized by a bimodal profile, with higher levels at the beginning of the day and of the night in LD, and in the middle of the subjective day and night in DD. The rhythmic pattern of th expression may be dependent on a LC clock machinery. Therefore, we investigated the expression of three clock genes, namely bmal1, per1, and per2, and found that their mRNAs display significant variations between day and nighttime points in both species, but in opposite directions. These data show that NA rhythm may be related to circadian expression of th gene in both species, but differs between nocturnal and diurnal rodents. Furthermore, the phase opposition of clock gene expression in the rat compared to Arvicanthis suggests that the clock machinery might be one of the mechanisms involved in th rhythmic expression.

Bimodal serotonin synthesis in the diurnal rodent, Arvicanthis ansorgei.

In mammals, behavioral activity is regulated both by the circadian system, orchestrated by the suprachiasmatic nucleus (SCN), and by arousal structures, including the serotonergic system. While the SCN is active at the same astronomical time in diurnal and nocturnal species, little data are available concerning the serotonergic (5HT) system in diurnal mammals. In this study, we investigated the functioning of the 5HT system, which is involved both in regulating the sleep/wake cycle and in synchronizing the SCN, in a diurnal rodent, Arvicanthis ansorgei. Using in situ hybridization, we characterized the anatomical extension of the raphe nuclei and we investigated 24 h mRNA levels of the serotonin rate-limiting enzyme, tryptophan hydroxylase 2 (tph2). Under both 12 h:12 h light/dark (LD) and constant darkness (DD) conditions, tph2 mRNA expression varies significantly over 24 h, displaying a bimodal profile with higher values around the (projected) light transitions. Furthermore, we considered several SCN outputs, namely melatonin, corticosterone, and locomotor activity. In both LD and DD, melatonin profiles display peak levels during the biological night. Corticosterone plasma levels show a bimodal rhythmic profile in both conditions, with higher levels preceding the two peaks of Arvicanthis locomotor activity, occurring at dawn and dusk. These data demonstrate that serotonin synthesis in Arvicanthis is rhythmic and reflects its bimodal behavioral phenotype, but differs from what has been previously described in nocturnal species.

Pinealectomy and gonadectomy modulate amplitude, but not photoperiodic modulation of Clock gene expression in the Syrian hamster suprachiasmatic nuclei.

The duration of daytime light phase (photoperiod) controls reproduction in seasonal mammals. Syrian hamsters are sexually active when exposed to long photoperiod, while gonadal atrophy is observed after exposure to short photoperiod. The photorefractory period, or photorefractoriness, is a particular state of spontaneous recrudescence of sexual activity that occurs after a long-term exposure to short photoperiod. Expression of core clock genes in the master circadian clock contained in the suprachiasmatic nuclei depends on photoperiodic conditions. Interestingly, the expression of the Clock gene is also modified in photorefractory Syrian hamsters. Since melatonin and testosterone levels in seasonal species are dependent on photoperiod, photoperiodic variations of Clock mRNA levels in the suprachiasmatic clock could be a consequence of these hormonal changes. To test this hypothesis, we analysed the effects of pinealectomy on Clock mRNA changes due to long to short photoperiod transition and of gonadectomy on Clock mRNA levels in photorefractory period. Our data show that the suprachiasmatic integration of the short photoperiod (assessed by a rhythmic expression profile of Clock) is independent of the presence of melatonin. Furthermore, constitutively low expression of Clock observed during the photorefractory period does not require the presence of either melatonin or testosterone. However, we show that both hormones provide positive feedback on average levels of Clock expression. Thus, our data support the hypothesis that daily variations of Clock levels in the suprachiasmatic nuclei are influenced by photoperiodic changes and the time spent in short photoperiod, independently of seasonal modifications of melatonin or testosterone levels.

The dromedary camel displays annual variation in hypothalamic kisspeptin and Arg-Phe-amide-related peptide-3 according to sex, season, and breeding activity.

The dromedary camel (Camelus dromedarius) is a desert mammal whose cycles in reproductive activity ensure that the offspring's birth and weaning coincide with periods of abundant food resources and favorable climate conditions. In this study, we assessed whether kisspeptin (Kp) and arginine-phenylalanine (RF)-amide related peptide-3 (RFRP-3), two hypothalamic peptides known to regulate the mammalian hypothalamo-pituitary gonadal axis, may be involved in the seasonal control of camel's reproduction. Using specific antibodies and riboprobes, we found that Kp neurons are present in the preoptic area (POA), suprachiasmatic (SCN), and arcuate (ARC) nuclei, and that RFRP-3 neurons are present in the paraventricular (PVN), dorsomedial (DMH), and ventromedial (VMH) hypothalamic nuclei. Kp fibers are found in various hypothalamic areas, notably the POA, SCN, PVN, DMH, VMH, supraoptic nucleus, and the ventral and dorsal premammillary nucleus. RFRP-3 fibers are found in the POA, SCN, PVN, DMH, VMH, and ARC. POA and ARC Kp neurons and DMH RFRP-3 neurons display sexual dimorphism with more neurons in female than in male. Both neuronal populations display opposed seasonal variations with more Kp neurons and less RFRP-3 neurons during the breeding (December-January) than the nonbreeding (July-August) season. This study is the first describing Kp and RFRP-3 in the camel's brain with, during the winter period lower RFRP-3 expression and higher Kp expression possibly responsible for the HPG axis activation. Altogether, our data indicate the involvement of both Kp and RFRP-3 in the seasonal control of the dromedary camel's breeding activity.

An Ultradian Feeding Schedule in Rats Affects Metabolic Gene Expression in Liver, Brown Adipose Tissue and Skeletal Muscle with Only Mild Effects on Circadian Clocks.

Restricted feeding is well known to affect expression profiles of both clock and metabolic genes. However, it is unknown whether these changes in metabolic gene expression result from changes in the molecular clock or in feeding behavior. Here we eliminated the daily rhythm in feeding behavior by providing 6 meals evenly distributed over the light/dark-cycle. Animals on this 6-meals-a-day feeding schedule retained the normal day/night difference in physiological parameters including body temperature and locomotor activity. The daily rhythm in respiratory exchange ratio (RER), however, was significantly phase-shifted through increased utilization of carbohydrates during the light phase and increased lipid oxidation during the dark phase. This 6-meals-a-day feeding schedule did not have a major impact on the clock gene expression rhythms in the master clock, but did have mild effects on peripheral clocks. In contrast, genes involved in glucose and lipid metabolism showed differential expression. In conclusion, eliminating the daily rhythm in feeding behavior in rats does not affect the master clock and only mildly affects peripheral clocks, but disturbs metabolic rhythms in liver, skeletal muscle and brown adipose tissue in a tissue-dependent manner. Thereby, a clear daily rhythm in feeding behavior strongly regulates timing of peripheral metabolism, separately from circadian clocks.

Short-term propofol anaesthesia down-regulates clock genes expression in the master clock.

BACKGROUND: Propofol anesthesia triggers phase-advances of circadian rhythms controlled by the suprachiasmatic nuclei (SCN), the master clock. Besides, inhalational anesthesia has been associated with a subsequent reduction of Per2 mRNA levels in the whole brain of rodents. The acute effects of propofol anesthesia per se on the SCN molecular clockwork remain unclear. Here we aim to study the expression of Per1 and Per2 clock genes in the SCN of rats exposed to constant darkness after a single dose of propofol. METHODS: Thirty 2-months old rats were randomly divided into 2 groups receiving a single dose of either 120 mg/kg propofol 1% (n=15), or intralipid® 10% (n=15) in late day (projected circadian time (CT) 10, i.e., 10h after the expected time of lights on). Thereafter, rat brains were sampled in darkness 1h, 2h or 3h after the treatment (projected CT11, CT12 or CT13). Expression of Per1 and Per2 mRNA was analyzed by in situ hybridization in SCN coronal sections. RESULTS: Per1 expression was affected by time and treatment. Per1 expression in the SCN after propofol treatment decreased at CT11 and CT12 when compared to the vehicle group. For Per2 expression, we observed only a treatment effect. Observed in dark conditions without hypothermia or/and concomitant surgery, such down-regulation of clock genes Per is only correlated to propofol treatment. This may explain "jet-lag-like" symptoms described by patients after anesthesia. CONCLUSION: We show here for the first time that short-term propofol anesthesia leads to a transient down-regulation of Per1 and Per2 expression in the SCN.

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.

Sex differences in the photoperiodic regulation of RF-Amide related peptide (RFRP) and its receptor GPR147 in the syrian hamster.

RF-(Arg-Phe) related peptides (RFRP-1 and -3) are considered to play a role in the seasonal regulation of reproduction; however, the effect of the peptides depends on species and gender. This study aimed at comparing the RFRP system in male and female Syrian hamsters over long and short photoperiods to investigate the neuroanatomical basis of these differential effects. The neuroanatomical distribution of RFRP neurons and fibers, revealed using an antiserum recognizing RFRP-1 and -3, as well as GPR147 mRNA, are similar in male and female Syrian hamsters. RFRP neurons are mainly found in the medial hypothalamus, whereas RFRP projections and GPR147 mRNA are observed in the preoptic area, anteroventral-periventricular nucleus, suprachiasmatic nucleus, paraventricular nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamus, habenular nucleus, and arcuate nucleus. The number of RFRP neurons is higher in females than in males, and in both sexes, the number of RFRP neurons is reduced in short photoperiods. GPR147 mRNA levels are higher in females than in males and are downregulated in short photoperiods, particularly in females. Interestingly, the number of RFRP-positive fibers in the anteroventral-periventricular nucleus is higher only in females adjusted to a short photoperiod. Our results suggest that the RFRP system, which is strongly regulated by photoperiod in both male and female Syrian hamsters, is particularly important in females, with a distinct role in the anteroventral-periventricular nucleus, possibly in the regulation of the preovulatory luteinizing hormone surge via kisspeptin neurons.

Evidence for a Putative Circadian Kiss-Clock in the Hypothalamic AVPV in Female Mice.

The kisspeptin (Kp) neurons in the anteroventral periventricular nucleus (AVPV) are essential for the preovulatory LH surge, which is gated by circulating estradiol (E2) and the time of day. We investigated whether AVPV Kp neurons in intact female mice may be the site in which both E2 and daily signals are integrated and whether these neurons may host a circadian oscillator involved in the timed LH surge. In the afternoon of proestrous day, Kp immunoreactivity displayed a marked and transient decrease 2 hours before the LH surge. In contrast, Kp content was stable throughout the day of diestrus, when LH levels are constantly low. AVPV Kp neurons expressed the clock protein period 1 (PER1) with a daily rhythm that is phase delayed compared with the PER1 rhythm measured in the main clock of the suprachiasmatic nuclei (SCN). PER1 rhythm in the AVPV, but not in the SCN, exhibited a significant phase delay of 2.8 hours in diestrus as compared with proestrus. Isolated Kp-expressing AVPV explants from PER2::LUCIFERASE mice displayed sustained circadian oscillations of bioluminescence with a circadian period (23.2 h) significantly shorter than that of SCN explants (24.5 h). Furthermore, in AVPV explants incubated with E2 (10 nM to 1 µM), the circadian period was lengthened by 1 hour, whereas the SCN clock remained unaltered. In conclusion, these findings indicate that AVPV Kp neurons display an E2-dependent daily rhythm, which may possibly be driven by an intrinsic circadian clock acting in combination with the SCN timing signal.

Kisspeptins and RFRP-3 Act in Concert to Synchronize Rodent Reproduction with Seasons.

Seasonal mammals use the photoperiodic variation in the nocturnal production of the pineal hormone melatonin to synchronize their reproductive activity with seasons. In rodents, the (SD) short day profile of melatonin secretion has long been proven to inhibit reproductive activity. Lately, we demonstrated that melatonin regulates the expression of the hypothalamic peptides kisspeptins (Kp) and RFamide-related peptide-3 (RFRP-3), recently discovered as potent regulators of gonadotropin-releasing hormone (GnRH) neuron activity. In the male Syrian hamster, Kp expression in the arcuate nucleus is down-regulated by melatonin independently of the inhibitory feedback of testosterone. A central or peripheral administration of Kp induces an increase in pituitary gonadotropins and gonadal hormone secretion, but most importantly a chronic infusion of the peptide reactivates the photo-inhibited reproductive axis of Syrian hamsters kept in SD conditions. RFRP-3 expression in the dorsomedial hypothalamus is also strongly inhibited by melatonin in a SD photoperiod. Although RFRP-3 is usually considered as an inhibitory component of the gonadotropic axis, a central acute administration of RFRP-3 in the male Syrian hamster induces a marked increase in gonadotropin secretion and testosterone production. Furthermore, a chronic central infusion of RFRP-3 in SD-adapted hamsters reactivates the reproductive axis, in the same manner as Kp. Both Kp and RFRP-3 neurons project onto GnRH neurons and both neuropeptides regulate GnRH neuron activity. In addition, central RFRP-3 infusion was associated with a significant increase in arcuate Kp expression. However, the actual sites of action of both peptides in the Syrian hamster brain are still unknown. Altogether our findings indicate that Kp and RFRP neurons are pivotal relays for the seasonal regulation of reproduction, and also suggest that RFRP neurons might be the primary target of the melatoninergic message.

Regulating amyloid precursor protein synthesis through an internal ribosomal entry site.

Expression of amyloid precursor protein (APP) is critical to the etiology of Alzheimer's disease (AD). Consequently, regulating APP expression is one approach to block disease progression. To this end, APP can be targeted at the levels of transcription, translation, and protein stability. Little is currently known about the translation of APP mRNA. Here, we report that endogenous APP mRNA is translated in neural cell lines via an internal ribosome entry site (IRES) located in the 5'-untranslated leader. The functional unit of the APP IRES is located within the 5' 50 nucleotides of the 5'-leader. In addition, we found that the APP IRES is positively regulated by two conditions correlated with AD, increased intracellular iron concentration and ischemia. Interestingly, the enhancement of APP IRES activity is dependent upon de novo transcription. Taken together, our data suggest that internal initiation of translation of the APP mRNA is an important mode for synthesis of APP, a mechanism which is regulated by conditions that also contribute to AD.

Melatonin induces Cry1 expression in the pars tuberalis of the rat.

In mammals, interacting transcriptional/post-translational feedback loops involving 'clock genes' and their protein products control circadian organisation. These genes are not only expressed in the master circadian clock of the suprachiasmatic nuclei (SCN) but also in many peripheral tissues where they exhibit similar but not identical dynamic to that seen in the SCN. Among these peripheral tissues, the pars tuberalis (PT) of the pituitary expresses clock genes. We show here that the PT of the rat, like that of other rodents, rhythmically expresses Per1. We also report rhythmic expression of another clock gene, Cry1. The peak of Cry1 mRNA expression occurs during the night concomitantly with rising blood plasma melatonin concentrations. Using an acute injection paradigm, we demonstrate that Cry1 expression is directly induced by melatonin in the PT. Melatonin injection at the end of the subjective day also affects Per1 expression, leading to diminished mRNA levels. These data support the existence of a time-measurement model in the PT based on direct opposite actions of melatonin on Per1 and Cry1 expression.

MT1 melatonin receptor mRNA tissular localization by PCR amplification.

OBJECTIVES: The pineal gland transduces photoperiodic informations to the neuroendocrine axis through the nocturnally melatonin secretion. This hormonal message plays a major role in the biological rhythm regulation. By autoradiography, more than 130 melatonin putative targets have been reported in the central nervous system (CNS) and in peripheral tissues. However, cross-species consensus concern only a few of them like the suprachiasmatic nuclei (SCN), the master circadian clock, and the pars tuberalis of the pituitary. Recently, MT1 melatonin receptor cDNA have been cloned in several mammals providing us with new tools to investigate its tissular location at the gene level. In the present study, we report a screening for MT1 mRNA by RT-PCR amplification of numerous tissue mRNA. METHOD: mRNA were extracted from a large variety of rat tissues. To semi-quantify the melatonin receptor mRNA expression level, each cDNA was amplified concomitantly with both beta-actin and MT1 specific primers. RESULTS: In central and peripheral tissues previously reported to bind melatonin, strong PCR signals were logically observed. More surprisingly, a vast majority of studied tissues express MT1 mRNA and then might be responsive to melatonin. CONCLUSION: Numerous biological functions express diurnal rhythmicity and internal-synchronization. As, most of them apparently do not receive any out-coming neuronal message from the SCN, endocrine communication was proposed to support biological rhythm synchronization. Our present data strengthen the idea that the nocturnally restricted melatonin secretion could be one internal zeitgeber that putatively distributes the endogenous circadian rhythmicity to all tissues expressing melatonin receptors.

Light exposure during daytime modulates expression of Per1 and Per2 clock genes in the suprachiasmatic nuclei of mice.

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the master circadian clock in mammals. Nocturnal light pulses that reset the circadian clock also lead to rapid increases in levels of Per1 and Per2 mRNA in the SCN, suggesting that these genes are involved in the synchronization to light. During the day, when light has no phase-shifting effects in nocturnal rodents, the consequences of light exposure for Per expression have been less thoroughly studied. Therefore, the effects of light exposure during the day were assessed on Per1 and Per2 mRNA in the SCN of mice. Expression of Per1 and Per2 was generally increased by 30-min light pulses during the subjective day, with more pronounced effects in the morning. One exception was noted for a transient decrease in Per2 expression after a short light pulse applied at midday. Prolonged light exposure (up to 3 hr) starting at midday markedly increased Per2 expression but not that of Per1. Moreover, the amplitude of the daily variations of both Per and the duration of Per1 peak was increased in mice exposed to a light-dark cycle compared with those transferred to constant darkness. Finally, the amplitude of the daily variations of both Per and the basal level of Per1 were increased in mice under a light-dark cycle compared with animals synchronized to a skeleton photoperiod (i.e., with daily dawn and dusk 1-hr exposures to light). Taken together, the results indicate that prolonged light exposure during daytime positively modulates daily levels of Per1 and Per2 mRNA in the SCN of mice.

Per and neuropeptide expression in the rat suprachiasmatic nuclei: compartmentalization and differential cellular induction by light.

Per1 and Per2, two clock genes rhythmically expressed in the suprachiasmatic nucleus (SCN), are implicated in the molecular mechanism of the circadian pacemaker and play a major role in its entrainment by light. To date, it is not known if every cell of the SCN, a heterogeneous structure in respect of neuropeptide content, expresses clock genes equally. The aim of this study was to identify, by single and double non-radioactive and/or radioactive hybridizations, the cell types (AVP, VIP and GRP) expressing Per1 or Per2 in the SCN of rats, (1) when Per are highly expressed during the daytime, and (2) after induction of Per expression by a light pulse at night. Our results indicate that, during the daytime, Per1 and Per2 genes are both mainly expressed in the AVP cells of the dorso-median part of the SCN, whereas only a few VIP cells in the ventral part of the SCN exhibit Per gene expression. In contrast, following a light pulse at night, there is differential induction of the two Per genes. Per1 expression essentially occurs in the ventro-lateral GRP cells, while Per2 expression is not restricted to the retinorecipient part of the SCN as it also occurs in AVP cells. Altogether, our results suggest that Per1 and Per2 are mainly expressed in AVP cells during the daytime and suggest that GRP cells play an important role in resetting of the clock by light.

MT1 melatonin receptor mRNA expression exhibits a circadian variation in the rat suprachiasmatic nuclei.

The aim of the present study was to investigate the daily regulation of both MT1 and MT2 melatonin receptor subtype mRNA expression in the rat SCN in order to clarify their role in the daily variation of SCN melatonin receptors. Existing MT1 and MT2 partial clones were extended by PCR to 982 and 522 bp, respectively. However, while the MT1 clone allowed us to set up a highly sensitive in situ hybridization (ISH) method, we could not detect MT2 expression within the SCN. Therefore, our results suggest that only MT1 mRNA can be correlated with 2-iodo-melatonin binding sites in the rat SCN. Investigation of MT1 mRNA expression throughout the 24 h light/dark cycle or in constant darkness clearly showed that in the two conditions, mRNA expression showed a robust rhythm with two peaks, one after the day/night and one after the night/day transitions in LD, and at the beginning of the subjective night and day in DD, respectively. Furthermore, these variations were not linked to the daily changes in melatonin receptor density. Thus, the transcriptional regulation of MT1 receptors does not appear to play a role in the daily regulation of melatonin binding sites availability.