Unraveling the periprandial changes in brain serotonergic activity and its correlation with food intake-related neuropeptides in rainbow trout Oncorhynchus mykiss.

This study explored changes in brain serotonin content and activity together with hypothalamic neuropeptide mRNA abundance around feeding time in rainbow trout, as well as the effect of one-day fasting. Groups of trout fed at two (ZT2) and six (ZT6) hours after lights on were sampled from 90 minutes before to 240 minutes after feeding, while additional groups of non-fed trout were also included in the study. Changes in brain amine and metabolite contents were measured in hindbrain, diencephalon and telencephalon, while in the diencephalon the mRNA abundance of tryptophan hydroxylase (tph1, tph2), serotonin receptors (5htr1a, 5htr1b and 5htr2c) and several neuropeptides (npy, agrp1, cartpt, pomca1, crfb) involved in the control of food intake were also assessed. The results showed changes in the hypothalamic neuropeptides that were consistent with the expected role for each in the regulation of food intake in rainbow trout. Serotonergic activity increased rapidly at the time of food intake in the diencephalon and hindbrain and remained high for much of the postprandial period. This increase in serotonin abundance was concomitant with elevated levels of pomca1 mRNA in the diencephalon, suggesting that serotonin might act on brain neuropeptides to promote a satiety profile. Furthermore, serotonin synthesis and neuronal activity appear to increase already before the time of feeding, suggesting additional functions for this amine before and during food intake. Exploration of serotonin receptors in the diencephalon revealed only small changes for gene expression of 5htr1b and 5htr2c receptors during the postprandial phase. Therefore, the results suggest that serotonin may play a relevant role in the regulation of feeding behavior in rainbow trout during periprandial time, but a better understanding of its interaction with brain centers involved in receiving and processing food-related signals is still needed.

Differential circadian and light-driven rhythmicity of clock gene expression and behaviour in the turbot, Scophthalmus maximus.

In fish, the circadian clock represents a key regulator of many aspects of biology and is controlled by combinations of abiotic and biotic factors. These environmental factors are frequently manipulated in fish farms as part of strategies designed to maximize productivity. The flatfish turbot, Scophthalmus maximus, represents one of the most important species within the aquaculture sector in Asia and Europe. Despite the strategic importance of this species, the function and regulation of the turbot circadian system remains poorly understood. Here, we have characterized the core circadian clock genes, clock1, per1, per2 and cry1 in turbot and have studied their daily expression in various tissues under a range of lighting conditions and feeding regimes. We have also explored the influence of light and feeding time on locomotor activity. Rhythmic expression of the four core clock genes was observed in all tissues studied under light dark (LD) cycle conditions. Rhythmicity of clock gene expression persisted upon transfer to artificial free running, constant conditions confirming their endogenous circadian clock control. Furthermore, turbot showed daily cycles of locomotor activity and food anticipatory activity (FAA) under LD and scheduled-feeding, with the activity phase as well as FAA coinciding with and being dependent upon exposure to light. Thus, while FAA was absent under constant dark (DD) conditions, it was still detected in constant light (LL). In contrast, general locomotor activity was arrhythmic in both constant darkness and constant light, pointing to a major contribution of light, in concert with the circadian clock, in timing locomotor activity in this species. Our data represents an important contribution to our understanding of the circadian timing system in the turbot and thereby the optimization of rearing protocols and the improvement of the well-being of turbot within fish farming environments.

Response of rainbow trout's (Oncorhynchus mykiss) hypothalamus to glucose and oleate assessed through transcription factors BSX, ChREBP, CREB, and FoxO1.

We aimed to obtain information regarding mechanisms that link glucose- and fatty acid-sensing systems to expression of neuropeptides that regulate food intake in the fish brain. We assessed the relative expression and protein levels of the transcription factors BSX, ChREBP, FoxO1, and CREB in the hypothalamus of rainbow trout (Oncorhynchus mykiss) treated for 6 h with either glucose or oleate in vivo (intra-cerebroventricular treatment with 1 µl 100 g- 1 body weight of 40 µg glucose or 1 µmol oleate) or in vitro (incubation with 4-8 mM glucose or 100-500 µM oleate). BSX levels decreased after oleate treatment for mRNA (10% in vitro and 47% in vivo) and protein (25%), while minor changes occurred after glucose treatment. CREB values generally decreased after glucose or oleate treatment for mRNA (50% in vivo) as well as the phosphorylation status of protein (80%). Foxo1 mRNA levels increased in vivo with glucose (129%) and decreased in vivo with oleate (60%), and protein phosphorylation status increased with glucose (in vivo) and oleate. mRNA values of chrebpα decreased in response to glucose and oleate, while protein levels decreased with oleate and increased with glucose. The results support the association of several transcription factors with metabolic control of food intake in fish.

Evolution shapes the responsiveness of the D-box enhancer element to light and reactive oxygen species in vertebrates.

The circadian clock is a highly conserved cell-autonomous mechanism that directs daily rhythms in most aspects of biology. Daily entrainment by environmental signals, notably light, is essential for its function. However, our understanding of the mechanisms and the evolution of photic entrainment remains incomplete. Fish represent attractive models for exploring how light regulates the circadian clock due to the direct light sensitivity of their peripheral clocks. Central to this property is the light induced expression of clock genes that is mediated by D-box enhancer elements. Here, using zebrafish cells, we reveal that the light responsive D-box enhancer serves as a nuclear target for reactive oxygen species (ROS). We demonstrate that exposure to short wavelengths of visible light triggers increases in ROS levels via NADPH oxidase activity. Elevated ROS activates the JNK and p38 MAP kinases and in turn, induces clock gene expression via the D-box. In blind cavefish and mammals, where peripheral clocks are no longer entrained by direct illumination, ROS levels are still increased upon light exposure. However, in these species ROS no longer induces D-box driven clock gene transcription. Thus, during evolution, alterations in ROS-responsive signal transduction pathways underlie fundamental changes in peripheral clock photoentrainment.

Evidence for the presence in rainbow trout brain of amino acid-sensing systems involved in the control of food intake.

To assess the hypothesis of central amino acid-sensing systems involved in the control of food intake in fish, we carried out two experiments in rainbow trout. In the first one, we injected intracerebroventricularly two different branched-chain amino acids (BCAAs), leucine and valine, and assessed food intake up to 48 h later. Leucine decreased and valine increased food intake. In a second experiment, 6 h after similar intracerebroventricular treatment we determined changes in parameters related to putative amino acid-sensing systems. Different areas of rainbow trout brain present amino acid-sensing systems responding to leucine (hypothalamus and telencephalon) and valine (telencephalon), while other areas (midbrain and hindbrain) do not respond to these treatments. The decreased food intake observed in fish treated intracerebroventricularly with leucine could relate to changes in mRNA abundance of hypothalamic neuropeptides [proopiomelanocortin (POMC), cocaine- and amphetamine-related transcript (CART), neuropeptide Y (NPY), and agouti-related peptide (AgRP)]. These in turn could relate to amino acid-sensing systems present in the same area, related to BCAA and glutamine metabolism, as well as mechanistic target of rapamycin (mTOR), taste receptors, and general control nonderepressible 2 (GCN2) kinase signaling. The treatment with valine did not affect amino acid-sensing parameters in the hypothalamus. These responses are comparable to those characterized in mammals. However, clear differences arise when comparing rainbow trout and mammals, in particular with respect to the clear orexigenic effect of valine, which could relate to the finding that valine partially stimulated two amino acid-sensing systems in the telencephalon. Another novel result is the clear effect of leucine on telencephalon, in which amino acid-sensing systems, but not neuropeptides, were activated as in the hypothalamus.

Pigment patterns in adult fish result from superimposition of two largely independent pigmentation mechanisms.

Dorso-ventral pigment pattern differences are the most widespread pigmentary adaptations in vertebrates. In mammals, this pattern is controlled by regulating melanin chemistry in melanocytes using a protein, agouti-signalling peptide (ASIP). In fish, studies of pigment patterning have focused on stripe formation, identifying a core striping mechanism dependent upon interactions between different pigment cell types. In contrast, mechanisms driving the dorso-ventral countershading pattern have been overlooked. Here, we demonstrate that, in fact, zebrafish utilize two distinct adult pigment patterning mechanisms - an ancient dorso-ventral patterning mechanism, and a more recent striping mechanism based on cell-cell interactions; remarkably, the dorso-ventral patterning mechanism also utilizes ASIP. These two mechanisms function largely independently, with resultant patterns superimposed to give the full pattern.

Critical role of the matricellular protein SPARC in mediating erythroid progenitor cell development in zebrafish.

Sparc (osteonectin) is a multifunctional matricellular glycoprotein expressed by many differentiated cells. Members of this family mediate cell-matrix interactions rather than acting as structural components of the extracellular matrix (ECM); therefore, they can influence many remodelling events, including haematopoiesis. We have investigated the role of sparc in embryonic haematopoiesis using a morpholino antisense oligonucleotide-based knockdown approach. Knockdown of sparc function resulted in specific erythroid progenitor cell differentiation defects that were highlighted by changes in gene expression and morphology, which could be rescued by injection of sparc mRNA. Furthermore, a comparison of blood phenotypes of sparc and fgfs knockdowns with similar defects and the sparc rescue of the fgf21 blood phenotype places sparc downstream of fgf21 in the genetic network regulating haematopoiesis in zebrafish. These results establish a role for an ECM protein (Sparc) as an important regulator of embryonic haematopoiesis during early development in zebrafish.

Diurnal rhythms in hypothalamic/pituitary AVT synthesis and secretion in rainbow trout: evidence for a circadian regulation.

Arginine vasotocin (AVT) and isotocin (IT) are two neurohypophysial peptide hormones for which a role in adaptation to environmental changes has been suggested in fish. In teleosts, there are only a few available studies about circadian changes of AVT and IT levels, and a role of those peptides in the circadian system has been mainly suggested on the basis of the role of the homologous hormone AVP in mammals. Herein, we evaluated the diurnal rhythms in plasma AVT, pituitary AVT and IT content and the hypothalamic pro-vasotocin (pro-VT) expression in rainbow trout kept under a natural photoperiod, as well as their persistence in constant darkness as a tool for defining circadian dependence. Trout kept under a natural light cycle showed clear diurnal rhythms in both circulating and pituitary AVT levels with peak values around the last hours of the light phase. Hypothalamic pro-VT mRNA was also rhythmically expressed with similar peak characteristics. These rhythms persisted in fish kept under constant darkness for nearly two consecutive days, although peaks were progressively attenuated and phase-advanced. An IT rhythm was also found in pituitary of the trout maintained under a natural photoperiod, but not in those kept under continuous darkness. These results suggest that rhythms of hypothalamic AVT synthesis might be regulated by endogenous circadian mechanisms, and these rhythms contribute to maintain a similar fluctuation in pituitary AVT secretion into the blood. A potential role for AVT in the circadian and seasonal time-keeping system of teleost fish, either as a component of the neural machinery that participates in the adaptation to cyclic environmental changes, or as a circadian/seasonal output signal, is also discussed.

A simple and sensitive method for determination of melatonin in plasma, bile and intestinal tissues by high performance liquid chromatography with fluorescence detection.

This paper describes the development of a simple and sensitive method for routine quantification of melatonin in low sample amounts by using standard equipment of HPLC with fluorescence detection. A double chloroform extraction with an intermediate cleaning step with 0.1N NaOH allowed to concentrate melatonin and to avoid interferences in extracts of the different tissues assayed. The analytical procedure was found to be precise and linear for a wide range of melatonin concentrations. The retention time of melatonin was about 9 min and the recoveries were in the range of 89-94%. The lower limit of quantification estimated on extracted samples was /ml. This method was validated in daytime and nighttime samples of plasma, bile and intestinal tissues of trout.

Food deprivation and refeeding effects on pineal indoles metabolism and melatonin synthesis in the rainbow trout Oncorhynchus mykiss.

The effects of food deprivation and refeeding on daily rhythms of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and melatonin contents, as well as on arylalkylamine N-acetyltransferase (AANAT) activity were evaluated in the pineal organ of rainbow trout. In addition, changes in circulating melatonin and cortisol levels were tested at one single point at day and night. Immature rainbow trout were distributed in 3 experimental groups: fish fed, fish fasted (7 days), and fish fasted for 7 days and refed for 5 days. All fish were sampled from each treatment group at different times of the day-night cycle. Pineal melatonin levels and AANAT activity showed daily variations in either fed, fasted and refed trout, displaying highest values at night. Fasted trout showed reduced melatonin content throughout the 24-h cycle, which was associated with decreased AANAT activity. Rhythms of pineal 5-HT and 5-HIAA levels were evident in all groups and were negatively correlated to melatonin in fed fish groups, but not in fasted fish. A higher content of 5-HT and 5-HIAA was observed in fasted fish during the night with no apparent changes during daytime for 5-HT and increased 5-HIAA levels. Furthermore, decreased circulating levels of melatonin were observed at midday, but not at night, in food deprived trout. Refeeding for 5 days generally counteracted the effects of food deprivation. Cortisol levels in plasma were reduced after food deprivation and remained low in refed fish. The results show that food deprivation impairs daily rhythms of melatonin content in trout pineal organ by affecting the activity of melatonin synthesizing enzymes rather than by a deficiency in substrate availability.

Evidence for arylalkylamine N-acetyltransferase (AANAT2) expression in rainbow trout peripheral tissues with emphasis in the gastrointestinal tract.

In fish, melatonin is reported to be produced mainly in the pineal organ, but there is also evidence for the presence of melatonin in a number of extrapineal sites where it could act as an intracellular mediator or paracrine signal. The present study use the reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of the enzyme arylalkylamine-N-acetyltransferase (AANAT2), which catalyzes the limiting step for melatonin synthesis, in different peripheral tissues of rainbow trout, with emphasis in the gastrointestinal tract (GIT). The results show AANAT2 gene expression in almost all peripheral tissues tested, including gills, kidney, muscle, skin, liver, Brockmann bodies, gall bladder, spleen and GIT, but not in adipose tissue. Furthermore, in trout GIT we observed that AANAT2 is expressed only in the muscular layer of all segments tested (esophagus, stomach, pyloric ceca, foregut, midgut and hindgut), but not in the mucosal layer. No significant differences were obtained among the different GIT segments evaluated. These results support an almost ubiquitous synthesis of melatonin in peripheral organs of rainbow trout, which can be related with a local role of the hormone as autocrine or paracrine factor. In addition, our data support the existence of a local synthesis of melatonin in trout GIT, which is discussed and could be involved in some sort of endocrine regulation of feeding and digestive activity, acting as an antioxidant or contributing to maintain melatonin levels in plasma.

Daily changes in parameters of energy metabolism in brain of rainbow trout: dependence on feeding.

We assessed the daily patterns of parameters involved in energy metabolism in plasma and brain of rainbow trout. Where daily rhythms were found, we analyzed the potential influence of feeding. Immature rainbow trout were randomly distributed in 3 groups: fish fed for 7 days, fish fasted for 7 days, and fish fasted for 7 days and refed for 4 days. On sampling day, fish of fed and refed groups were fed at 11.00 h, and all fish were sampled from each treatment group using the following time schedule: 14.00, 18.00, 21.00, 00.00, 04.00, 07.00, 10.00 and 14.00 h. The results obtained from metabolic parameters assessed in plasma and brain can be grouped into three different categories, such as (i) those displaying no 24 h changes in fed fish such as plasma lactate, protein or acetoacetate levels, as well as brain amino acid and protein levels, and lowKm(glucose) hexokinase, and aspartate aminotransferase activities, (ii) those displaying 24 h changes that were apparently dependent on feeding since they disappeared in fasted fish such as the case of plasma cortisol, glucose and triglyceride levels, as well as brain glycogen, glucose, and lactate levels, and pyruvate kinase and hexokinase IV activities, and (iii) those parameters displaying 24 h changes apparently not dependent on feeding such as plasma amino acids, brain acetoacetate levels as well as several enzyme activities measured in brain such as glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, glutamate dehydrogenase, and lactate dehydrogenase-oxidase. In general, 24 h changes dependent on feeding indicate an increased use of glucose in brain several hours post-feeding whereas those changes not dependent on feeding were characterized by reduced levels/activity at the night period suggesting a metabolic depression in brain during darkness.

Indoleamines and 5-methoxyindoles in trout pineal organ in vivo: daily changes and influence of photoperiod.

This study describes the diel rhythms in several indoleamines, melatonin, and related 5-methoxyindoles in the pineal organ of rainbow trout in vivo. In addition, the effect of different photoperiod conditions was evaluated. Melatonin levels displayed clear daily rhythms in the pineal organ of rainbow trout kept experimentally under long (LD 16:08), neutral (LD 12:12), and short (LD 08:16) photoperiods. Duration of melatonin signal was dependent on the night length of prevailing photoperiod, while peak amplitude was higher when lengthening the photoperiod. Significant daily rhythms in 5-HT content, the precursor of melatonin synthesis, were found in neutral and short photoperiod with increases of the amine content just after the light-dark interphase and decreases in the middle of the night, which were more important under short photoperiod. In contrast, no significant 24-h cyclic variation was found in pineal 5-HT content under long photoperiod. Daily profiles in the content of the main 5-HT oxidative metabolite, the 5-hydroxyindoleacetic acid (5-HIAA), outlined those of the amine precursor. The chronograms of both aminergic compounds contrast with those of 5-hydroxytryptophan content, which displayed a net tendency to increase at night. This study also provides evidence for the existence of daily cyclic changes in the content of 5-methoxytryptamine (5-MT), 5-methoxyindoleacetic acid (5-MIAA), and 5-methoxytryptophol (5-MTOL) in trout pineal organ, which were also dependent on photoperiod. The 24-h profiles in 5-MT content correlated well with those of 5-HT, showing a peak at the first hour of darkness in all photoperiodic conditions, and a decay at midnight only in both neutral and long photoperiods. Similarly, the content of 5-MTOL also displayed high values during the day-night transition in trout kept under neutral and long photoperiods, followed by a slow decay all along the night. Finally, levels of 5-MIAA increased in all photoperiods when lights were turned off, being this nocturnal increase maximal in fish kept under LD 16:08. These results suggest that light-dark cycle modulates daily rhythms in pineal indoles and non-melatonin 5-methoxyindoles by acting mainly through the melatonin synthesis activity, which limits the availability of 5-HT for the oxidative and direct methylation pathways. In addition, it seems that a nocturnally increased synthesis of 5-HT might be a requirement for the optimal formation of melatonin and other 5-methoxyindoles in the pineal organ when trout remain under short photoperiods.