Daily rhythmic expression patterns of clock1a, bmal1, and per1 genes in retina and hypothalamus of the rainbow trout, Oncorhynchus mykiss.

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.

Changes in plasma melatonin levels and pineal organ melatonin synthesis following acclimation of rainbow trout (Oncorhynchus mykiss) to different water salinities.

Melatonin has been suggested to play a role in fish osmoregulation, and in salmonids has been related to the timing of adaptive mechanisms during smolting. It has been described that acclimation to different environmental salinities alters levels of circulating melatonin in a number of fish species, including rainbow trout. However, nothing is known regarding salinity effects on melatonin synthesis in the pineal organ, which is the main source of rhythmically produced and secreted melatonin in blood. In the present study we have evaluated, in rainbow trout, the effects of acclimation to different salinities on day and night plasma melatonin values and pineal organ melatonin synthesis. Groups of freshwater (FW)-adapted rainbow trout were placed in tanks with four different levels of water salinity (FW, 6, 12, 18 p.p.t.; parts per thousand) and maintained for 6 h or 5 days. Melatonin content in plasma and pineal organs, as well as the pineal content of serotonin (5-HT) and its main oxidative metabolite (5-hydroxyindole-3-acetic acid; 5-HIAA) were measured by high performance liquid chromatography. In addition, day-night changes in pineal organ arylalkylamine N-acetyltransferase (AANAT2) activity and aanat2 gene expression were studied. Plasma osmolalities were found to be higher in rainbow trout exposed to all salinity levels compared with the control FW groups. A salinity-dependent increase in melatonin content was found in both plasma and pineal organs. This effect was observed during the night, and was related to an increase in aanat2 mRNA abundance and AANAT2 enzyme activity, both of which also occurred during the day. Also, the levels of indoles (5-HT, 5-HIAA) in the pineal organ were negatively affected by increasing water salinity, which seems to be related to the higher recruitment of 5-HT as a substrate for the increased melatonin synthesis. A stimulatory effect of salinity on pineal aanat2 mRNA expression was also identified. These results indicate that increased external salinity promotes melatonin synthesis in the pineal organ of rainbow trout by enhancing synthesis of AANAT protein independently of its regulation by light. The possibility that pineal melatonin is a target for hormones involved in the response of fish to osmotic challenge is discussed, as well as the potential role of melatonin in the timing of osmoregulatory processes.

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.

Effects of naphthalene, beta-naphthoflavone and benzo(a)pyrene on the diurnal and nocturnal indoleamine metabolism and melatonin content in the pineal organ of rainbow trout, Oncorhynchus mykiss.

Polycyclic aromatic hydrocarbons (PAHs) have deleterious effects on neuroendocrine systems in teleost fish affecting, among other processes, reproductive function or stress responses. The hormone melatonin, mainly produced in the pineal organ of vertebrates, is involved in the regulation of biological rhythms as well as other important functions, and may also act as an antioxidant molecule. The effects of environmental pollutants on the endocrine and metabolic activity of the pineal organ have been studied only in mammals. We here evaluate the effects of the PAHs naphthalene (NAP) and benzo(a)pyrene (BaP) and the flavonoid beta-naphthoflavone (BNF) on the pineal organ of rainbow trout by quantifying the diurnal and nocturnal pineal content of some indoles and methoxyindoles, including melatonin. NAP mainly induced diurnal increases in the pineal content of melatonin and other methoxyindoles like 5-methoxytryptamine (5-MT), 5-methoxyindole-3-acetic acid (5-MIAA) or 5-methoxytryptophol (5-MTOL). Those increases did not occur at night, when even occasional decreases were observed compared with controls. NAP also induced some diurnal and nocturnal decreases in the levels of indolic compounds like serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA), while pineal content of 5-hydroxytryptophan (5-HTP) was first decreased (few hours after injection) and then increased (few days after injection) during the day. BaP and BNF induced strong increases in diurnal levels of melatonin, whereas other pineal compounds were unaffected. It seems that an increase of the methylation capacity of the pineal organ takes place during the day, and a decrease occurs at night. Those effects could be mediated by changes in the activity of key enzymes involved in pineal melatonin biosynthesis, maybe as a result of the alteration of the cellular phototransduction mechanisms involved in the light-induced inhibition of melatonin synthesis in the pineal photoreceptor cells. These results demonstrate for the first time that environmental pollutants can disrupt the activity of the pineal organ of teleost fish. This disruption could be a threat for the survival of the animals in their natural environment, although the increases observed in melatonin levels could play a relevant role as a toxicity-protection factor.