Time-Lag in Feeding Schedule Acts as a Stressor That Alters Circadian Oscillators in Goldfish

The circadian system controls temporal homeostasis in all vertebrates. The light-dark (LD) cycle is the most important zeitgeber (“time giver”) of circadian system, but feeding time also acts as a potent synchronizer in the functional organization of the teleost circadian system. In mammals is well known that food intake during the rest phase promotes circadian desynchrony which has been associated with metabolic diseases. However, the impact of a misalignment of LD and feeding cycles in the entrainment of fish circadian oscillators is largely unknown. The objective of this work was to investigate how a time-lag feeding alters temporal homeostasis and if this could be considered a stressor. To this aim, goldfish maintained under a 12 h light-12 h darkness were fed at mid-photophase (SF6) or mid-scotophase (SF18). Daily rhythms of locomotor activity, clock genes expression in hypothalamus, liver, and head kidney, and circulating cortisol were studied. Results showed that SF6 fish showed daily rhythms of bmal1a and clock1a in all studied tissues, being in antiphase with rhythms of per1 genes, as expected for proper functioning clocks. The 12 h shift in scheduled feeding induced a short phase advance (4–5-h) of the clock genes daily rhythms in the hypothalamus, while in the liver the shift for clock genes expression rhythms was the same that the feeding time shift (∼12 h). In head kidney, acrophases of per genes underwent a 12-h shift in SF18 animals, but only 6 h shift for clock1a. Plasma cortisol levels showed a significant daily rhythm in animals fed at SF6, but not in SF18 fish fed, which displayed higher cortisol values throughout the 24-h. Altogether, results indicate that hypothalamus, liver, and head kidney oscillate in phase in SF6 fish, but these clocks are desynchronized in SF18 fish, which could explain cortisol alterations. These data reinforce the hypothesis that the misalignment of external cues (daily photocycle and feeding time) alters fish temporal homeostasis and it might be considered a stressor for the animals.

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title: Circadian Rhythmicity and Light Sensitivity of the Zebrafish Brain
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title: Feeding entrainment of locomotor activity rhythms, digestive enzymes and neuroendocrine factors in goldfish
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title: Physiological significance of a peripheral tissue circadian clock
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title: Feeding during the rest phase promotes circadian conflict in nuclei that control energy homeostasis and sleep–wake cycle in rats
abstract: Food intake during the rest phase promotes circadian desynchrony, which has been associated with metabolic diseases. However, the link between circadian rhythm and metabolic alterations is not well understood. To investigate this issue, we explored the circadian rhythm of c‐Fos immunoreactivity (IR) in rats fed during the day, during the night or with free access to food for 3 weeks. The analysis was focused on the hypothalamic nuclei, which are interconnected and involved in the control of energy homeostasis and/or arousal: lateral hypothalamus (LH), perifornical area, arcuate, ventrolateral pre‐optic (VLPO) and tuberomammillary nuclei. The results show that food intake during the rest phase flattened the circadian c‐Fos expression in the LH and perifornical area, and induced a phase shift in the VLPO area. In addition, c‐Fos expression was analyzed in the orexin and melanin‐concentrating hormone (MCH) neurons of the LH, which are involved in the control of food intake and arousal, and in α‐melanin‐stimulating hormone and neuropeptide Y (NPY) cells in the arcuate nucleus, all of which are involved in feeding–fasting cycles, energy homeostasis and sending projections to the LH. The results indicate that feeding during the rest phase decreased orexin neuron activation in the light in comparison with the other groups. Feeding during this phase also flattened the activity rhythm of MCH and α‐melanin‐stimulating hormone neurons and increased NPY IR when the light was turned on. This evidence indicates that mealtime differentially affected the hypothalamic nuclei under investigation leading to a circadian conflict that might account for metabolic impairment.

title: Influence of Light on Feeding Anticipatory Activity in Goldfish
abstract: Most animals when presented with food on a periodic basis develop feeding anticipatory activity (FAA). The objective of the present study was to investigate the synchronization of activity rhythms to light and feeding in single goldfish given a single daily meal and exposed to different light regimes. In the 1st experiment, the group of fish fed at the end of the day showed a longer FAA (228 ± 27 min) than the group fed 1 h after lights-on (97 ± 31 min). In the 2nd experiment, goldfish under conflicting zeitgebers, LD (T = 26) and feeding (T = 24) cycles, initially synchronized to the light cycle although feeding time gradually assumed greater importance as the experiment progressed. In the 3rd experiment, after altering the feeding cycles by advancing or delaying feeding time by 4 h in LD 0.25:23.75, most goldfish resynchronized to the feeding stimulus and developed FAA. In conclusion, the authors' results revealed that FAAin goldfish is driven by an endogenous timing system that is entrainable by periodic feeding and influenced by the light regime.

title: A Blind Circadian Clock in Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception
abstract: Evolution during millions of years in perpetual darkness leads to mutations in non-visual opsin genes (Melanopsin and TMT opsin) and an aberrant, blind circadian clock in cavefish.

title: Feeding Time Synchronizes Clock Gene Rhythmic Expression in Brain and Liver of Goldfish (Carassius auratus)
abstract: Little is known about the feeding time dependence of clock gene expression in fish. The aim of the present study was to investigate whether a scheduled feeding time can entrain the rhythmic expression of several clock genes (period and cryptocrome) in the brain and liver of a teleost, the goldfish. Fish maintained under continuous light (LL) conditions were divided into 3 groups. Two groups were fed daily at 1000 h and 2200 h, respectively, and the third group was subjected to a random schedule regime. After 30 days, the fishes under 24-h food deprivation were sacrificed through a 24-h cycle, and clock gene expression in the optic tectum, hypothalamus, and liver was quantified by real-time PCR. The findings pointed to differences between the central and peripheral tissues studied. In the absence of a light-dark cycle (constant light), a scheduled feeding regime was necessary and sufficient to maintain both the rhythmic expression of several clock genes in the optic tectum and hypothalamus, as well as daily rhythms in locomotor activity. In contrast, neither locomotor activity nor clock gene expression in brain tissues was synchronized in randomly fed fish. However, in the liver, most of the clock genes studied presented significant daily rhythms in phase (related to the time of the last meal) in all 3 experimental groups, suggesting that the daily rhythm of clock genes in this organ only depends on the last meal time. The data suggest that, as in mammals, the smooth running of the food entrainable oscillator (FEO) in fish involves the rhythmic expression of several clock genes (Per1 and Cry3) in the central and peripheral structures. The results also indicate that the food anticipatory activity (FAA) in goldfish is not only the result of rhythmic clock gene expression in the liver because rhythmic clock gene expression was observed in randomly fed fishes, while FAA was not observed.

title: SYNCHRONIZATION TO LIGHT AND RESTRICTED-FEEDING SCHEDULES OF BEHAVIORAL AND HUMORAL DAILY RHYTHMS IN GILTHEAD SEA BREAM (SPARUS AURATA)
abstract: Food is not continuously available in the wild, and so most animals show a wide variety of circadian rhythms that can be entrained to feeding time. The aim of this research was to evaluate the effect of time-restricted feeding on the daily rhythms of gilthead sea bream, with food being provided during the day or night under a 12:12 h light-dark (LD) cycle or constant light (LL) conditions. Self-feeding and locomotor activity, as well as daily rhythms of cortisol, glucose, and melatonin, were evaluated. Fish synchronized their feeding behavior to the feeding phase, so that in LD they displayed 78% nocturnal feeding activity under night-feeding and 81% diurnal feeding activity under day-feeding, while under LL-feeding they displayed 72% of their daily activity during the 12 h feeding phase. In contrast, locomotor activity was mostly diurnal (66–71%), regardless of the feeding schedule, and it became arrhythmic under LL. Cortisol showed daily rhythms that peaked at different times, depending on the light and feeding schedule: one peak several hours before feeding under day-feeding and night-feeding, and two peaks under LL-feeding. Glucose displayed low-amplitude variations, with no daily rhythms being detected. Melatonin, however, showed a nocturnal rhythm, regardless of the feeding schedule, while the rhythm became attenuated under LL. Taken together, these results highlight the role of feeding on endocrine and metabolic rhythms, suggesting that feeding behavior should be considered when studying these variables.

title: Light-dark cycle and feeding time differentially entrains the gut molecular clock of the goldfish (Carassius auratus).
abstract: The aim of the present study was to investigate how photocycle and feeding-time cues regulate the daily expression of Per1a, Per2a, Per3, and Cry3 in the goldfish hindgut. For this purpose, we studied the daily rhythmicity of these genes in fish maintained under different lighting conditions and under different feeding regimes (scheduled or not). We also studied whether the timing of just one meal is able to reset the hindgut molecular clock. In a first experiment, randomly fed fish were divided into four groups and kept under different light conditions for 30 d: 12 h light and 12 h dark (12L:12D), an inverted photoperiod (12D:12L), constant darkness (24D), and constant light (24L). In a second study, fish maintained under 24L were divided into four groups fed at different time points for 35 d: (1) fish scheduled-fed once a day (at 10:00 h); (2) fish fed with a 12-h shifted schedule (at 22:00 h), (3) fish fed at 10:00 h throughout the experiment, except the last day when fed at 22:00 h; and (4) a randomly fed group of fish. Fish were sacrificed every 6 h throughout a 24-h cycle. In both experiments, gPer1a, gPer2a, gPer3, and gCry3 transcripts were quantified using Real Time-qPCR in the hindgut. Results show the clock genes gPer1a, gPer2a, and gCry3 are synchronized by both zeitgebers, the photocycle and feeding regime, in goldfish hindgut. Moreover, such clock genes anticipate light-on and food delivery, when these cues appear in a cyclic manner. In the absence of both zeitgebers, gCry3 and gPer2a rhythmicity disappeared. In contrast, the gPer1 rhythm was maintained under 24L and random feeding conditions, but not always, suggesting that food when randomly supplied is able to reset the clock depending on other factors, such as the energetic and metabolic conditions of the fish. The expression of gPer2a was not activated during the light phase of the cycle, suggesting the hindgut of goldfish is a non-direct photosensitive organ. In contrast to the other three genes, gPer3 expression in the goldfish hindgut seemed to be dependent on the timing of the last food delivery, even in the presence of a photocycle. This gene was the only one that maintained daily rhythms under both constant lighting conditions (24D and 24L), although with lower amplitude than when a photocycle was present. This indicates that, although the acrophase (peak time) of the gPer3 expression rhythm seems to be driven by feeding time, there is an interaction of both zeitgebers, food and light, to regulate its expression. In conclusion, present data indicate: (1) the hindgut of goldfish can be synchronized in vivo by both the photocycle and feeding time; (2) food is a potent signal that entrains this peripheral oscillator; and (3) both environmental cues seems to target different elements of the molecular clock.

title: Synchronization of daily rhythms of locomotor activity and plasma glucose, cortisol and thyroid hormones to feeding in Gilthead seabream (Sparus aurata) under a light–dark cycle
abstract: Food availability is far from constant but tends to be cyclic, and fish therefore show a variety of circadian rhythms which can be entrained to feeding time. The aim of this study was to investigate the synchronization to mealtimes of behavioral (locomotor activity), metabolic (glucose) and endocrine (cortisol and thyroid hormones) daily rhythms in gilthead seabream. To this end, fish were reared under a 12:12 LD cycle and fed 1% of their body weight once a day either at mid-light (ML) or at mid-dark (MD) of the LD cycle. Fish synchronized their locomotor activity to the phase in which food was delivered, ML and MD fish displaying 86+/-3% and 81+/-1% of their total daily activity during daytime and nighttime, respectively. Daily variations of blood glucose were strongly synchronized to feeding time in both experimental groups, peaking 8h after the meal. A postprandial cortisol peak was observed in both groups. In fish fed at MD the cortisol values were high during the 8h following feeding, whereas in fish fed at ML cortisol levels returned to basal values within 4h. In addition, MD fish showed a higher average daily cortisol value (24.75+/-2.65 ng/ml) than ML fish (10.30+/-2.18 ng/ml). Feeding time affected the magnitude of daily variations in the thyroid hormones. When the time of feeding was delayed by 12h, a shift of the same magnitude could be observed in the glucose daily rhythm and a dramatic change in the cortisol levels of the ML-fed group compared with results mentioned above. In summary, gilthead seabream synchronized their locomotor activity to the phase when food was provided and showed different degrees of synchronization of their behavioral, metabolic and endocrine rhythms to feeding time. Since these parameters are used to evaluate stress responses and welfare in seabream, their daily rhythm and synchronization to light and feeding time should be taken into account.

title: Rhythms in the endocrine system of fish: a review
abstract: The environment which living organisms inhabit is not constant and many factors, such as light, temperature, and food availability, display cyclic and predictable variations. To adapt to these cyclic changes, animals present biological rhythms in many of their physiological variables, timing their functions to occur when the possibility of success is greatest. Among these variables, many endocrine factors have been described as displaying rhythms in vertebrates. The aim of the present review is to provide a thorough review of the existing knowledge on the rhythms of the endocrine system of fish by examining the hormones that show rhythmicity, how environmental factors control these rhythms and the variation in the responses of the endocrine system depending on the time of the day. We mainly focused on the hypothalamic–pituitary axis, which can be considered as the master axis of the endocrine system of vertebrates and regulates a great variety of functions, including reproduction, growth, metabolism, energy homeostasis, stress response, and osmoregulation. In addition, the rhythms of other hormones, such as melatonin and the factors, produced in the gastrointestinal system of fish are reviewed.

title: The role of clock genes and rhythmicity in the liver
abstract: The liver is the important organ to maintain energy homeostasis of an organism. To achieve this, many biochemical reactions run in this organ in a rhythmic fashion. An elegant way to coordinate the temporal expression of genes for metabolic enzymes relies in the link to the circadian timing system. In this fashion not only a maximum of synchronization is achieved, but also anticipation of daily recurring events is possible. Here we will focus on the input and output pathways of the hepatic circadian oscillator and discuss the recently found flexibility of its circadian transcriptional networks.

title: Glucocorticoid hormones are both a major circadian signal and major stress signal: How this shared signal contributes to a dynamic relationship between the circadian and stress systems
abstract: Glucocorticoid hormones are a powerful mammalian systemic hormonal signal that exerts regulatory effects on almost every cell and system of the body. Glucocorticoids act in a circadian and stress-directed manner to aid in adaptation to an ever-changing environment. Circadian glucocorticoid secretion provides for a daily waxing and waning influence on target cell function. In addition, the daily circadian peak of glucocorticoid secretion serves as a timing signal that helps entrain intrinsic molecular clock phase in tissue cells distributed throughout the body. Stress-induced glucocorticoid secretion also modulates the state of these same cells in response to both physiological and psychological stressors. We review the strong functional interrelationships between glucocorticoids and the circadian system, and discuss how these interactions optimize the appropriate cellular and systems response to stress throughout the day. We also discuss clinical implications of this dual aspect of glucocorticoid signaling, especially for conditions of circadian and HPA axis dysregulation.

title: Melatonin reduces locomotor activity and circulating cortisol in goldfish
abstract: The present study focused on the effects of a subchronic melatonin treatment on locomotor activity and cortisol plasma levels in goldfish. We compared two different administration routes: peripheral (10 microg/g body weight) versus central (1 microg/microl) injections of melatonin for 7 or 4 days, respectively. Daily locomotor activity, including both diurnal and nocturnal activities, food anticipatory activity and circulating cortisol at 11:00 (under 24 h of food deprivation and 17 h postinjection) were significantly reduced after repeated intraperitoneal injections with melatonin for 7 days, but not after intracerebroventricular treatment. Taking in mind the anoretic effect of melatonin in this species, we investigated if such feeding reduction is directly responsible for the reduction in motor activity induced by melatonin treatment. Food restriction (50%) for 10 days did not significantly modify either daily locomotor activity or plasma cortisol levels in goldfish, indicating that the peripheral action of melatonin diminishing locomotor activity in goldfish is not a direct consequence of its anoretic action. In summary, our results indicate that, as previously described in other vertebrate species, melatonin can regulate locomotor activity and cortisol levels in goldfish, suggesting a sedative effect of this hormone in this teleost.

title: A nonlinear regression program for small computers.
abstract: A BASIC computer program for performing weighted nonlinear regression is described and a listing of the program is given. The program, which is small and simple to use, has been designed to be run by users with little knowledge of mathematics or computers. Robust methods of analysis are described which may be applied to data in which experimental errors are not normally distributed, and the program incorporates one such method. It is shown that the program is useful for the analysis of data conforming to the Michaelis-Menten equation, a single exponential, and to binding equations, and other applications are discussed.

title: Serotonin N-acetyltransferase (NAT) activity and melatonin levels in the frog retina are not correlated during the seasonal cycle.
abstract: The seasonal pattern of daily serotonin N-acetyltransferase (NAT) and melatonin rhythms in the lateral eye of Rana perezi has been characterized to assess any seasonal relationship between enzymatic activity and melatonin production and to evaluate the photothermal responses of such rhythms. In winter there was no daily rhythm either in NAT activity or in melatonin production, although the highest values of NAT activity and lowest melatonin concentrations were measured at this time. Ocular melatonin showed a high-amplitude diurnal rhythm during the summer, whereas a low-amplitude rhythm was observed in spring and autumn. Diurnal melatonin levels were temperature-independent; however, nocturnal melatonin production significantly increased with temperature. Ocular NAT was always higher at lower than at higher temperatures (two- to threefold), both at night and during the day. As suggested for pineal melatonin production in other poikilotherm species, photoperiod controls the duration of the melatonin nocturnal rise, whereas temperature regulates the amplitude of the daily melatonin rhythm in the frog retina. The lack of correlation between daily NAT and melatonin rhythms in the frog retina may result from the high thermal sensitivity of the ocular NAT and a temperature-dependent inhibition substrate of NAT activity.

title: Restricted feeding entrains liver clock without participation of the suprachiasmatic nucleus
abstract: There are two main stimuli that entrain the circadian rhythm, the light‐dark cycle (LD) and restricted feeding (RF). Light‐induced entrainment requires induction of the Per1 and Per2 genes in the suprachiasmatic nucleus (SCN), the locus of a main oscillator. In this experiment, we determined whether RF resets the expression of circadian clock genes in the mouse liver with or without participation of the SCN.

title: The “Other” Circadian System: Food as a Zeitgeber
abstract: It is not surprising that limiting food access to a particular time of day has profound effects on the behavior and physiology of animals. It has been clear for some time that pre-meal behavioral activation, a rise in core temperature, elevated serum corticosterone, and an increase in duodenal disaccharidases are under circadian control and that the observed circadian properties are not abolished by lesions of the suprachiasmatic nucleus (SCN), but the search for the locus of a separate food-entrainable oscillator (FEO) has not been successful. The cloning of circadian clock genes and the discovery that these genes are expressed in many central nervous system structures outside the SCN and in peripheral tissues have led to new strategies for investigating potential loci of an FEO. Recent findings concerning the entrainment of clock gene expression in the central nervous system and in peripheral tissues by periodic food access are presented, and the implications of these findings for a better understanding of a circadian system that entrains to meals, rather than to light, are discussed.

title: Circadian Clock Genes of Goldfish, Carassius auratus: cDNA Cloning and Rhythmic Expression of Period and Cryptochrome Transcripts in Retina, Liver, and Gut
abstract: Clock genes are known to be the molecular core of biological clocks of vertebrates. They are expressed not only in those tissues considered central pacemakers, but also in peripheral tissues. In the present study, partial cDNAs for 6 of the principal clock genes (Period 1-3 and Cryptochrome 1-3) were cloned from a teleost fish, the goldfish (Carassius auratus ). These genes showed high homology (approximately 90%) with the respective cDNAs of zebrafish (Danio rerio), the only other teleost from which clock genes have been cloned. The daily expression pattern of each gene in retina, gut, and liver of goldfish was investigated using quantitative RT-PCR and cosinor analysis. All clock genes analyzed in the retina showed circadian rhythmicity; however, only Per 2-3 and Cry 2-3 were rhythmic in goldfish liver and gut. The amplitude and phase of the expression in liver and gut were different from those found in goldfish retina. Such differences suggest that other cues, such as feeding time, may contribute to the entrainment of oscillators in goldfish liver and gut. Our results support the use of goldfish as a teleost model to investigate the location and functioning of the circadian oscillators.

title: Anatomical distribution and daily profile of gper1b gene expression in brain and peripheral structures of goldfish (Carassius auratus)
abstract: The functional organization of the circadian system and the location of the main circadian oscillators vary through phylogeny. Present study investigates by in situ hybridization the anatomical location of the clock gene gPer1b in forebrain and midbrain, pituitary, and in two peripheral locations, the anterior intestine and liver, in a teleost fish, the goldfish (Carassius auratus). Moreover, the daily expression profiles of this gene were also studied by quantitative Real Time-PCR. Goldfish were maintained under a 12L–12D photoperiod and fed daily at 2 h after lights were switched on. A wide distribution of gPer1b mRNA in goldfish brain and pituitary was found in telencephalon, some hypothalamic nuclei (including the homologous to mammalian SCN), habenular nucleus, optic tectum, cerebellum and torus longitudinalis. Moreover, gPer1b expression was observed, for the first time in teleosts, in the pituitary, liver and anterior intestine. Day/night differences in gper1b mRNA abundance were found by in situ hybridization, with higher signal at nighttime that correlates with the results obtained by RT-PCR, where a rhythmic gPer1b expression was found in all tissues with acrophases at the end of the night. Amplitudes of gper1b rhythms vary among tissues, being higher in liver and intestine than in the brain, maybe because different cues entrain clocks in these locations. These results support the existence of functional clocks in many central and peripheral locations in goldfish coordinated, ticking at the same time.

title: FEEDING ENTRAINMENT OF DAILY RHYTHMS OF LOCOMOTOR ACTIVITY AND CLOCK GENE EXPRESSION IN ZEBRAFISH BRAIN
abstract: Light and feeding cycles strongly synchronize daily rhythms in animals, which may, as a consequence, develop food anticipatory activity (FAA). However, the light/food entraining mechanisms of the central circadian oscillator remain unknown. In this study, we investigate the existence of FAA in seven groups of zebrafish subjected to a light/dark (LD) cycle or constant light (LL) and different feeding regimes (random, fasting, and feeding in the middle of the light phase or dark phase). The aim was to ascertain whether the daily rhythm of behavior and clock gene (per1 and cry1) expression in the zebrafish brain was entrained by the light and feeding regime. The results revealed that FAA developed in zebrafish fed daily at a fixed time, under LD and under LL. Zebrafish displayed locomotor activity mostly during the daytime, although the percentage of activity during the light phase varied depending on feeding time (ranging from 93.2% to 63.1% in the mid-light and mid-dark fed groups, respectively). However, the different feeding regimes failed to modify the daily rhythm of per1 and cry1 expression in the zebrafish brain under LD (approximate acrophases [peak times] at ZT22 and ZT4, respectively; lights-on = ZT0). Under LL, per1 and cry1 expression did not show significant daily rhythmicity, regardless of the feeding regime. These findings indicate that, although schedule-fed zebrafish developed FAA as regards locomotor activity, feeding had little effect on clock gene expression in whole brain homogenates, suggesting the feeding-entrainable oscillator may be located elsewhere or at specific brain sites.

title: MOLECULAR CLONING, TISSUE DISTRIBUTION, AND DAILY RHYTHMS OF EXPRESSION OF PER1 GENE IN EUROPEAN SEA BASS (DICENTRARCHUS LABRAX)
abstract: Circadian rhythms are controlled by interlocked autoregulatory feedback loops consisting of interactions of a group of circadian clock genes and their proteins. The Period family is a group of genes that are essential components of the molecular clock. In the present study, we cloned a period gene (per1) of the European sea bass, a marine teleost of chronobiological interest. The cloned sequence encoded a protein consisting of 1436 amino acids that homology and phylogenic analyses showed to be related with fish PER1 proteins possessing very high identity with Oryzias latipes (Medaka) per1. Polymerase chain reaction screening of per1 expression showed that this gene is expressed in all the tissues analyzed (brain, heart, liver, gill, muscle, digestive tract, adipose tissue, spleen, and retina). In addition, a daily expression rhythm, with an acrophase (peak time) ∼ZT0 (lights-on), was found in the two tissue types investigated: neural (brain) and peripheral (liver and heart). In conclusion, identification and characterization of the gene encoding sea bass per1 provide valuable information for understanding the circadian mechanism at the molecular level in this species, although further research is needed to clarify the exact role that per1 plays in the circadian oscillator and the dual behavior of European sea bass.

title: Daily Rhythmic Expression Patterns of Clock1a, Bmal1, and Per1 Genes in Retina and Hypothalamus of the Rainbow Trout, Oncorhynchus Mykiss
abstract: Living organisms show daily rhythms in physiology, behavior, and gene expression, which are due to the presence of endogenous clocks that synchronize biological processes to the 24-h light/dark cycle. In metazoans, generation of circadian rhythmicity is a consequence of specialized tissues known as “master clocks,” having different locations among species. A few studies have described clock-gene expression in fish neural tissues, but none of them assessed clock-gene expression in different discrete regions. The present study was designed to explore the presence/absence of circadian clock-gene expression in the rainbow trout (Oncorhynchus mykiss) retina and hypothalamus. Juvenile fish were acclimated to a 12:12 light (L)-dark (D) cycle. Then, retina and hypothalamus were collected from animals kept under LD conditions or constant darkness (DD) for 24 h. Real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) assays were performed to quantify expression of the core circadian genes Clock1a, Bmal1, and Per1 as representative members of the circadian oscillator. All clock genes analyzed in the retina and hypothalamus showed circadian fluctuations. However, gene expression peaked in the rainbow trout hypothalamus with a 3-h (Clock1a and Bmal1) or 6-h (Per1) delay relative to that observed in the retina, the latter showing highest expression levels at zeitgeber times 9 (ZT9) for Clock1a and Bmal1, and at ZT21 for Per1. When exposed to DD, the rhythmic gene expression pattern was maintained for all genes in the rainbow trout retina, but only for Clock1a and Per1 in the hypothalamus. Bmal1 failed to cycle under DD, suggesting that hypothalamic clock function might depend on either several clock-gene isoforms or regulation from external inputs. Overall, these data indicate that representative molecular members of the core circadian clock are present in both the retina and hypothalamus of rainbow trout.

title: Cloning, tissue expression pattern and daily rhythms of Period1, Period2, and Clock transcripts in the flatfish Senegalese sole, Solea senegalensis
abstract: An extensive network of endogenous oscillators governs vertebrate circadian rhythmicity. At the molecular level, they are composed of a set of clock genes that participate in transcriptional–translational feedback loops to control their own expression and that of downstream output genes. These clocks are synchronized with the environment, although entrainment by external periodic cues remains little explored in fish. In this work, partial cDNA sequences of clock genes representing both positive (Clock) and negative (Period1, Period2) elements of the molecular feedback loops were obtained from the nocturnal flatfish Senegalese sole, a relevant species for aquaculture and chronobiology. All of the above genes exhibited high identities with their respective teleost clock genes, and Per–Arnt–Sim or basic helix–loop–helix binding domains were recognized in their primary structure. They showed a widespread distribution through the animal body and some of them displayed daily mRNA rhythms in central (retina, optic tectum, diencephalon, and cerebellum) and peripheral (liver) tissues. These rhythms were most robust in retina and liver, exhibiting marked Period1 and Clock daily oscillations in transcript levels as revealed by ANOVA and cosinor analysis. Interestingly, expression profiles were inverted in retina and optic tectum compared to liver. Such differences suggest the existence of tissue-dependent zeitgebers for clock gene expression in this species (i.e., light for retina and optic tectum and feeding time for liver). This study provides novel insight into the location of the molecular clocks (central vs. peripheral) and their different phasing and synchronization pathways, which contributes to better understand the teleost circadian systems and its plasticity.

title: Comparison of light, food, and temperature as environmental synchronizers of the circadian rhythm of activity in mice
abstract: Although entrainment (and masking) of circadian rhythms by light has been extensively studied, much less attention has been given to other environmental cycles that can modulate circadian rhythms in mammals. In this study in mice, the entraining strength of different environmental cycles was compared. Running-wheel activity was monitored before, after, and while the animals were under one of four environmental cycles: a full light–dark cycle with 12 h of light and 12 h of darkness each day, a cycle of 1 h of light per day, a cycle of food availability consisting of 80 % of the baseline free-feeding amount presented once a day, and an ambient temperature cycle consisting of 23 h at 24 °C and 1 h at 12 °C each day. Four measures of zeitgeber strength were used: percentage of animals that entrained, rhythm robustness in the entrained state, stability of activity onsets, and stability of acrophases. The results indicate that, at least in mice, a full light–dark cycle is the most powerful modulator of the circadian rhythm of locomotor activity, as a consequence of both entrainment and masking. When entrainment alone is considered, temperature seems to be as strong a modulator as light, while food restriction is a weaker modulator and affects primarily a food-anticipatory component of the activity rhythm.

title: Light and feeding entrainment of the molecular circadian clock in a marine teleost (Sparus aurata)
abstract: Daily light and feeding cycles act as powerful synchronizers of circadian rhythmicity. Ultimately, these external cues entrain the expression of clock genes, which generate daily rhythmic behavioral and physiological responses in vertebrates. In the present study, we investigated clock genes in a marine teleost (gilthead sea bream). Partial cDNA sequences of key elements from both positive (Bmal1, Clock) and negative (Per2, Cry1) regulatory loops were cloned before studying how feeding time affects the daily rhythms of locomotor activity and clock gene expression in the central (brain) and peripheral (liver) oscillators. To this end, all fish were kept under a light-dark (LD) cycle and were divided into three experimental groups, depending on the time of their daily meal: mid-light (ML), mid-darkness (MD), or at random (RD) times. Finally, the existence of circadian control on gene expression was investigated in the absence of external cues (DD + RD). The behavioral results showed that seabream fed at ML or RD displayed a diurnal activity pattern (>91% of activity during the day), whereas fish fed at MD were nocturnal (89% of activity during the night). Moreover, seabream subjected to regular feeding cycles (ML and MD groups) showed food-anticipatory activity (FAA). Regardless of the mealtime, the daily rhythm of clock gene expression in the brain peaked close to the light-dark transition in the case of Bmal1 and Clock, and at the beginning of the light phase in the case of Per2 and Cry1, showing the existence of phase delay between the positive and negative elements of the molecular clock. In the liver, however, the acrophases of the daily rhythms differed depending on the feeding regime: the maximum expression of Bmal1 and Clock in the ML and RD groups was in antiphase to the expression pattern observed in the fish fed at MD. Under constant conditions (DD + RD), Per2 and Cry1 showed circadian rhythmicity in the brain, whereas Bmal1, Clock, and Per2 did in the liver. Our results indicate that the seabream clock gene expression is endogenously controlled and in liver it is strongly entrained by food signals, rather than by the LD cycle, and that scheduled feeding can shift the phase of the daily rhythm of clock gene expression in a peripheral organ (liver) without changing the phase of these rhythms in a central oscillator (brain), suggesting uncoupling of the light-entrainable oscillator (LEO) from the food-entrainable oscillator (FEO). These findings provide the basis and new tools for improving our knowledge of the circadian system and entraining pathways of this fish species, which is of great interest for the Mediterranean aquaculture.

title: Daily and seasonal expression of clock genes in the pituitary of the European sea bass (Dicentrarchus labrax).
abstract: The expression of select clock genes (clock, bmal, per1, per2, cry1, cry2) was investigated throughout the day and across the four seasons for two consecutive years in the pituitary of adult sea bass (Dicentrarchus labrax). A rhythmic pattern of daily expression was consistently observed in summer and autumn, while arrhythmicity was observed for some clock genes during spring and winter, concomitant with low water temperatures. The expression of clock and bmal showed highest values at the end of the day and during the night, while that of per and cry was mostly antiphasic, with high values during the day. Melatonin affects clock-gene expression in the pituitary of mammals. We therefore sought to test the effect of melatonin on clock-gene expression in the pituitary of sea bass both in vivo and in vitro. Melatonin modestly affected the expression of some clock genes (in particular cry genes) when added to the fish diet or the culture medium of pituitary glands. Our data show that clock genes display rhythmic daily expression in the pituitary of adult sea bass, which are profoundly modified according to the season. We suggest that the effect of photoperiod on clock gene expression may be mediated, at least in part, by melatonin, and that temperature may have a key role adjusting seasonal variations.

title: The light-induced transcriptome of the zebrafish pineal gland reveals complex regulation of the circadian clockwork by light
abstract: Light constitutes a primary signal whereby endogenous circadian clocks are synchronized (‘entrained’) with the day/night cycle. The molecular mechanisms underlying this vital process are known to require gene activation, yet are incompletely understood. Here, the light-induced transcriptome in the zebrafish central clock organ, the pineal gland, was characterized by messenger RNA (mRNA) sequencing (mRNA-seq) and microarray analyses, resulting in the identification of multiple light-induced mRNAs. Interestingly, a considerable portion of the molecular clock (14 genes) is light-induced in the pineal gland. Four of these genes, encoding the transcription factors dec1, reverbb1, e4bp4-5 and e4bp4-6, differentially affected clock- and light-regulated promoter activation, suggesting that light-input is conveyed to the core clock machinery via diverse mechanisms. Moreover, we show that dec1, as well as the core clock gene per2, is essential for light-entrainment of rhythmic locomotor activity in zebrafish larvae. Additionally, we used microRNA (miRNA) sequencing (miR-seq) and identified pineal-enhanced and light-induced miRNAs. One such miRNA, miR-183, is shown to downregulate e4bp4-6 mRNA through a 3′UTR target site, and importantly, to regulate the rhythmic mRNA levels of aanat2, the key enzyme in melatonin synthesis. Together, this genome-wide approach and functional characterization of light-induced factors indicate a multi-level regulation of the circadian clockwork by light.

title: Mutations in blind cavefish target the light-regulated circadian clock gene, period 2
abstract: Light represents the principal signal driving circadian clock entrainment. However, how light influences the evolution of the clock remains poorly understood. The cavefish Phreatichthys andruzzii represents a fascinating model to explore how evolution under extreme aphotic conditions shapes the circadian clock, since in this species the clock is unresponsive to light. We have previously demonstrated that loss-of-function mutations targeting non-visual opsins contribute in part to this blind clock phenotype. Here, we have compared orthologs of two core clock genes that play a key role in photic entrainment, cry1a and per2, in both zebrafish and P. andruzzii. We encountered aberrantly spliced variants for the P. andruzzii per2 transcript. The most abundant transcript encodes a truncated protein lacking the C-terminal Cry binding domain and incorporating an intronic, transposon-derived coding sequence. We demonstrate that the transposon insertion leads to a predominantly cytoplasmic localization of the cavefish Per2 protein in contrast to the zebrafish ortholog which is distributed in both the nucleus and cytoplasm. Thus, it seems that during evolution in complete darkness, the photic entrainment pathway of the circadian clock has been subject to mutation at multiple levels, extending from opsin photoreceptors to nuclear effectors.

title: Entrainment of the circadian clock in the liver by feeding.
abstract: Circadian rhythms of behavior are driven by oscillators in the brain that are coupled to the environmental light cycle. Circadian rhythms of gene expression occur widely in peripheral organs. It is unclear how these multiple rhythms are coupled together to form a coherent system. To study such coupling, we investigated the effects of cycles of food availability (which exert powerful entraining effects on behavior) on the rhythms of gene expression in the liver, lung, and suprachiasmatic nucleus (SCN). We used a transgenic rat model whose tissues express luciferase in vitro. Although rhythmicity in the SCN remained phase-locked to the light-dark cycle, restricted feeding rapidly entrained the liver, shifting its rhythm by 10 hours within 2 days. Our results demonstrate that feeding cycles can entrain the liver independently of the SCN and the light cycle, and they suggest the need to reexamine the mammalian circadian hierarchy. They also raise the possibility that peripheral circadian oscillators like those in the liver may be coupled to the SCN primarily through rhythmic behavior, such as feeding.

title: Regulation of circadian gene expression in liver by systemic signals and hepatocyte oscillators.
abstract: The mammalian circadian timing system has a hierarchical structure, in that a master pacemaker located in the suprachiasmatic nuclei (SCN) coordinates slave oscillators present in virtually all body cells. In both the SCN and peripheral organs, the rhythm-generating oscillators are self-sustained and cell-autonomous, and it is likely that the molecular makeup of master and slave oscillators is nearly identical. However, due to variations in period length, the phase coherence between peripheral oscillators in intact animals must be established by daily signals emanating directly or indirectly from the SCN master clock. The synchronization of individual cellular clocks in peripheral organs is probably accomplished by immediate-early genes that interpret the cyclic systemic signals and convey this phase information to core clock components. This model predicts that circadian gene expression in peripheral organs can be influenced either by systemic signals emanating from the SCN master clock, local oscillators, or both. We developed a transgenic mouse strain in which hepatocyte clocks are only operative when the tetracycline analog doxycycline is added to the food or drinking water. The genome-wide mapping of genes whose cyclic expression in liver does not depend on functional hepatocyte oscillators unveiled putative signaling pathways that may participate in the phase entrainment of peripheral clocks.

title: Circadian rhythms of gene expression of lipid metabolism in Gilthead Sea bream liver: Synchronisation to light and feeding time
abstract: This research aimed at investigating circadian rhythm expression of key genes involved in lipid metabolism in the liver of a teleost fish (Sparus aurata), and their synchronisation to different light-dark (L-D) and feeding cycles. To this end, 90 gilthead sea bream were kept in 12:12 h (light:dark, LD, lights on at ZT0) and fed a single daily meal at mid-light (ML = ZT6), mid-darkness (MD = ZT18) and randomly (RD) at a 1.5% body weight ration. A total of 18 tanks were used, six tanks per feeding treatment with five fishes per tank; locomotor activity was recorded in each tank. After 25 days of synchronisation to these feeding regimes, fishes were fasted for one day and liver samples were taken every 4 hours during a 24 h cycle (ZT2, 6, 10, 14, 18 and 22) and stored at −80 °C until analysis. To determine whether the rhythm expression presented an endogenous control, another experiment was performed using 30 fish kept in complete darkness and fed randomly (DD/RD). Samples were taken following the same procedure as above. The results revealed that all genes investigated exhibited well defined daily rhythms. The lipolysis-related and fatty acid turnover genes (hormone-sensitive lipase (hsl) and peroxisome proliferator-activated receptor-α (pparα)) exhibited a nocturnal achrophase (Ø = ZT18:03–19:21); lipoprotein lipase (lpl) also showed the same nocturnal achrophase (Ø = ZT20:04–21:36). In contrast, lipogenesis-related gene, fatty acid synthase (fas), and of fatty acid turnover, cyclooxygenase (cox-2), showed a diurnal rhythm (Ø = ZT2:27–8:09); while pparγ was nocturnal (Ø = ZT16:16–18:05). Curiously, feeding time had little influence on the phase of these daily rhythms, since all feeding groups displayed similar achrophases. Furthermore, under constant conditions pparα and hsl showed circadian rhythmicity. These findings suggest that lipid utilisation in the liver is rhythmic and strongly synchronised to the LD cycle, regardless of feeding time, which should be taken into consideration when investigating fish nutrition and the design of feeding protocols.

title: Daily rhythms of lipid metabolic gene expression in zebra fish liver: Response to light/dark and feeding cycles
abstract: Despite numerous studies about fish nutrition and lipid metabolism, very little is known about the daily rhythm expression of lipogenesis and lipolysis genes. This research aimed to investigate the existence of daily rhythm expressions of the genes involved in lipid metabolism and their synchronization to different light/dark (LD) and feeding cycles in zebra fish liver. For this purpose, three groups of zebra fish were submitted to a 12:12 h LD cycle. A single daily meal was provided to each group at various times: in the middle of the light phase (ML); in the middle of the dark phase (MD); at random times. After 20 days of acclimation to these experimental conditions, liver samples were collected every 4 h in one 24-h cycle. The results revealed that most genes displayed a significant daily rhythm with an acrophase of expression in the dark phase. The acrophase of lipolytic genes (lipoprotein lipase – lpl, peroxisome proliferator-activated receptor – pparα and hydroxyacil CoA dehydrogenase – hadh) was displayed between ZT 02:17 h and ZT 18:31 h. That of lipogenic genes (leptin-a – lepa, peroxisome proliferator-activated receptor – pparγ, liver X receptor – lxr, insulin-like growth factor – igf1, sterol regulatory element-binding protein – srebp and fatty acid synthase – fas) was displayed between ZT 15:25 h and 20:06 h (dark phase). Feeding time barely influenced daily expression rhythms, except for lxr in the MD group, whose acrophase shifted by about 14 h compared with the ML group (ZT 04:31 h versus ZT 18:29 h, respectively). These results evidence a strong synchronization to the LD cycle, but not to feeding time, and most genes showed a nocturnal acrophase. These findings highlight the importance of considering light and feeding time to optimize lipid metabolism and feeding protocols in fish farming.

title: Adrenal peripheral clock controls the autonomous circadian rhythm of glucocorticoid by causing rhythmic steroid production
abstract: Glucocorticoid (GC) is an adrenal steroid with diverse physiological effects. It undergoes a robust daily oscillation, which has been thought to be driven by the master circadian clock in the suprachiasmatic nucleus of the hypothalamus via the hypothalamus–pituitary–adrenal axis. However, we show that the adrenal gland has its own clock and that the peripheral clockwork is tightly linked to steroidogenesis by the steroidogenic acute regulatory protein. Examination of mice with adrenal-specific knockdown of the canonical clock protein BMAL1 reveals that the adrenal clock machinery is required for circadian GC production. Furthermore, behavioral rhythmicity is drastically affected in these animals, together with altered expression of Period1, but not Period2, in several peripheral organs. We conclude that the adrenal peripheral clock plays an essential role in harmonizing the mammalian circadian timing system by generating a robust circadian GC rhythm.

title: Mammalian Molecular Clocks
abstract: As a consequence of the Earth's rotation, almost all organisms experience day and night cycles within a 24-hr period. To adapt and synchronize biological rhythms to external daily cycles, organisms have evolved an internal time-keeping system. In mammals, the master circadian pacemaker residing in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus generates circadian rhythmicity and orchestrates numerous subsidiary local clocks in other regions of the brain and peripheral tissues. Regardless of their locations, these circadian clocks are cell-autonomous and self-sustainable, implicating rhythmic oscillations in a variety of biochemical and metabolic processes. A group of core clock genes provides interlocking molecular feedback loops that drive the circadian rhythm even at the single-cell level. In addition to the core transcription/translation feedback loops, post-translational modifications also contribute to the fine regulation of molecular circadian clocks. In this article, we briefly review the molecular mechanisms and post-translational modifications of mammalian circadian clock regulation. We also discuss the organization of and communication between central and peripheral circadian oscillators of the mammalian circadian clock.

title: Peripheral Circadian Oscillators in Mammals: Time and Food
abstract: Peripheral cells from mammalian tissues, while perfectly capable of circadian rhythm generation, are not light sensitive and thus have to be entrained by nonphotic cues. Feeding time is the dominant zeitgeber for peripheral mammalian clocks: Daytime feeding of nocturnal laboratory rodents completely inverts the phase of circadian gene expression in many tissues, including liver, heart, kidney, and pancreas, but it has no effect on the SCN pacemaker. It is thus plausible that in intact animals, the SCN synchronizes peripheral clocks primarily through temporal feeding patterns that are imposed through behavioral restactivity cycles. In addition, body temperature rhythms, which are themselves dependent on both feeding patterns and rest-activity cycles, can sustain circadian, clock gene activity in vivo and in vitro. The SCN may also influence the phase of rhythmic gene expression in peripheral tissues through direct chemical pathways. In fact, many chemical signals induce circadian gene expression in tissue culture cells. Some of these have been shown to elicit phase shifts when injected into intact animals and are thus candidates for physiologically relevant timing cues. While the response of the SCN to light is strictly gated to respond only during the night, peripheral oscillators can be chemically phase shifted throughout the day. For example, injection of dexamethasone, a glucocorticoid receptor agonist, resets the phase of circadian liver gene expression during the entire 24-h day. Given the bewildering array of agents capable of influencing peripheral clocks, the identification of physiologically relevant agents used by the SCN to synchronize peripheral clocks will clearly be an arduous undertaking. Nevertheless, we feel that experimental systems by which this enticing problem can be tackled are now at hand.

title: Shifting the feeding of mice to the rest phase creates metabolic alterations, which, on their own, shift the peripheral circadian clocks by 12 hours
abstract: peripheral circadian clocks (CCs) and metabolism are intimately linked, as pathologies occur on perturbation of their coupling. In mice, shifting the feeding time from the phase of activity (night) to the phase of rest (day) is known to act as a time cue for peripheral CCs, leading to a 12-hour shift of the time at which their CC components are expressed. However, the molecular mechanisms that underlie this shift are largely unknown. Here, we reveal both the origin and the identity of the metabolic signals that are generated on shifting eating to the rest phase and how these signals directly alter the expression of CC components to generate the shift that ultimately leads to metabolic syndrome-like pathology. The molecular mechanisms underlying the events through which alterations in diurnal activities impinge on peripheral circadian clocks (PCCs), and reciprocally how the PCCs affect metabolism, thereby generating pathologies, are still poorly understood. Here, we deciphered how switching the diurnal feeding from the active to the rest phase, i.e., restricted feeding (RF), immediately creates a hypoinsulinemia during the active phase, which initiates a metabolic reprogramming by increasing FFA and glucagon levels. In turn, peroxisome proliferator-activated receptor alpha (PPARα) activation by free fatty acid (FFA), and cAMP response element-binding protein (CREB) activation by glucagon, lead to further metabolic alterations during the circadian active phase, as well as to aberrant activation of expression of the PCC components nuclear receptor subfamily 1, group D, member 1 (Nr1d1/RevErbα), Period (Per1 and Per2). Moreover, hypoinsulinemia leads to an increase in glycogen synthase kinase 3β (GSK3β) activity that, through phosphorylation, stabilizes and increases the level of the RevErbα protein during the active phase. This increase then leads to an untimely repression of expression of the genes containing a RORE DNA binding sequence (DBS), including the Bmal1 gene, thereby initiating in RF mice a 12-h PCC shift to which the CREB-mediated activation of Per1, Per2 by glucagon modestly contributes. We also show that the reported corticosterone extraproduction during the RF active phase reflects an adrenal aberrant activation of CREB signaling, which selectively delays the activation of the PPARα–RevErbα axis in muscle and heart and accounts for the retarded shift of their PCCs.

title: The circadian rhythm of glucocorticoids is regulated by a gating mechanism residing in the adrenal cortical clock.
abstract: In mammals, the master clock of the suprachiasmatic nuclei (SCN) and subordinate clocks found throughout the body coordinate circadian rhythms of behavior and physiology. We characterize the clock of the adrenal, an important endocrine gland that synchronizes physiological and metabolic rhythms. Clock gene expression was detected in the outer adrenal cortex prefiguring a role of the clock in regulating gluco- and mineral corticoid biogenesis. In Per2/Cry1 double mutant mice, which lack a circadian clock, hypothalamus/pituitary/adrenal axis regulation was defective. Organ culture and tissue transplantation suggest that the adrenal pacemaker gates glucocorticoid production in response to adrenocorticotropin (ACTH). In vivo the adrenal circadian clock can be entrained by light. Transcriptome profiling identified rhythmically expressed genes located at diverse nodes of steroid biogenesis that may mediate gating of the ACTH response by the adrenal clock.

title: Daily Variation in Concentration of Cortisol in Plasma in Intact and Hypophysectomized Gulf Killifish
abstract: A daily variation in concentration of cortisol in plasma is synchronized by a 12-hour daily photoperiod in intact as well as in hypophysectomized fish. The daily rhythm in concentration of the adrenal steroid does not depend on a daily rhythm in the concentration of pituitary adrenocorticotropic hormone.

title: Daily variation in concentration of plasma corticosteroid in hypophysectomized rats.
abstract: A daily variation in the concentration of plasma corticosteroid hormone is present in hypophysectomized rats implanted with pellets of adrenocorticotropic hormone (ACTH) and thyroxine. Although ACTH is necessary to maintain a functional adrenal, and thyroxine is required to permit the expression of the plasma corticosteroid rhythm, a normal daily variation in plasma corticosteroid concentration does not appear to depend on the periodic release of pituitary hormones.

title: Diurnal Rhythmicity of the Canonical Clock Genes Per1, Per2 and Bmal1 in the Rat Adrenal Gland is Unaltered after Hypophysectomy
abstract: Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus (SCN), and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. Rhythmic expression of clock genes in the adrenal glands has previously been reported. Since the central clock in the SCN communicates with the adrenal glands via circadian release of adrenocorticotrophic hormone, we quantified the mRNAs for the canonical clock genes, Per1, Per2 and Bmal1 in the adrenal glands by real‐time reverse transcription‐polymerase chain reaction during a 24‐h‐cycle in normal and hypophysectomised rats. The mRNAs for all the three clock genes disclosed rhythmic oscillations with a period of 24 h and the phase did not differ between the hypophysectomised and intact rats. The expression pattern of Per1 and Bmal1 was in antiphase in both groups of animals. In situ hybridisation histochemistry using antisense RNA probes demonstrated that, at times of peak expression, mRNAs for all the three clock genes were expressed in the adrenal cortex with a particularly strong labelling in the zona reticularis. In accordance with the mRNA localisation, immunostaining for PER1 protein was visualised in cells of the adrenal cortex, being most intense in the inner zone. The immunostaining also demonstrated a translocation of PER1 protein from the cytoplasm to the nucleus during the daily cycle, supporting the existence of a core oscillator in the individual adrenal gland cells. Our findings support the existence of a circadian core oscillator in cells of the rat adrenal cortex and indicate that the activity of the oscillator is independent of SCN signalling via the pituitary gland. The adrenal cortical clock could be involved in rhythmic transcriptional activation of genes associated with hormonal biosynthesis, involved in gating of the response of the adrenal cortex to external cues or involved in apoptosis of adrenal cortical cells.