Jacky Falcón

Functional development of the zebrafish pineal gland: light-induced expression of period2 is required for onset of the circadian clock.

In zebrafish, the pineal gland is a photoreceptive organ that contains an intrinsic circadian oscillator and exhibits rhythmic arylalkylamine‐N‐acetyltransferase (zfaanat2) mRNA expression. In the present study, we investigated the role of light and of a clock gene, zperiod2 (zper2), in the development of this rhythm. Analysis of zfaanat2 mRNA expression in the pineal gland of 3‐day‐old zebrafish embryos after exposure to different photoperiodic regimes indicated that light is required for proper development of the circadian clock‐controlled rhythmic expression of zfaanat2, and that a 1‐h light pulse is sufficient to initiate this rhythm. Analysis of zper2 mRNA expression in zebrafish embryos exposed to different photoperiodic regimes indicated that zper2 expression is transiently up‐regulated by light but is not regulated by the circadian oscillator. To establish the association between light‐induced zper2 expression and light‐induced clock‐controlled zfaanat2 rhythm, zPer2 knock‐down experiments were performed. The zfaanat2 mRNA rhythm, induced by a 1‐h light pulse, was abolished in zPer2 knock‐down embryos. These experiments indicated that light‐induced zper2 expression is crucial for establishment of the clock‐controlled zfaanat2 rhythm in the zebrafish pineal gland.

Genetic, Temporal and Developmental Differences Between Melatonin Rhythm Generating Systems in the Teleost Fish Pineal Organ and Retina

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N‐acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.

Pineal-retinal relationships: rhythmic biosynthesis and immunocytochemical localization of melatonin in the retina of the pike (Esox lucius)

SummaryThe levels of melatonin and the activities of two enzymes of the melatonin biosynthetic pathway, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were measured throughout the light-dark cycle in the retina of a teleost fish, the pike. HIOMT activity did not display significant variations, whereas NAT activity and melatonin content showed a daily rhythm, high levels occurring during the night. The profiles of the latter two rhythms did not closely match one another and differed from those previously described in the pineal organ of the same species. These results are discussed with respect to a possible paracrine role of retinal melatonin. Melatonin-like immunoreactivity was found in the photoreceptor cell layer and in the Müller cells of the inner nuclear layer. The intensity of the melatonin-like immunoreactivity varied throughout the 24 h light-dark cycle, in good correlation with the variations in the melatonin level as measured by radioimmunoassay.

Regulation of melatonin production by catecholamines and adenosine in a photoreceptive pineal organ. An in vitro study in the pike and the trout

Abstract: The pineal organ of fish contains photoreceptor cells with structural and functional analogies to retinal photoreceptors. In these cells, the light/dark (LD) cycle influences the production of melatonin by controlling the activity of one of its synthetizing enzymes, serotonin N‐acetyltransferase (NAT). The daily rhythm in NAT activity is generated endogenously in the pike but not in the trout pineal. We report here that in addition to the LD information, chemical factors are also involved in the control of melatonin production. Adenosine and two of its analogs stimulated or inhibited NAT activity and melatonin release in cultured pike and trout pineals, depending on the experimental conditions. It is believed that the nucleoside, produced locally, exerts a modulatory role on the neurohormonal output via still enigmatic mechanisms, involving a transmembranous carrier. Nocturnal melatonin production in cultured pike pineals was inhibited by α‐adrenergic agonists and stimulated by a β‐adrenergic agonist. No effect could be induced in trout pineals cultured under similar conditions. Because melatonin production by pineal photoreceptors is apparently regulated by both light and chemical inputs, we propose they might be multieffector cells.

Calcium and melatonin production in dissociated trout pineal photoreceptor cells in culture

Trout pineal cells maintained in primary culture produce melatonin in high amounts during night time and low amounts during daytime. The dark-induced increase in melatonin production was enhanced, in a dose-dependent manner, by elevating extracellular calcium concentration. Low external calcium concentration reduced nocturnal and diurnal melatonin production. Bay K 8644 increased, in a dose-dependent manner, the dark-induced rise in melatonin output, and this effect was antagonized by nifedipine and verapamil. This suggests a role for the dihydropyridine calcium channels in the regulation of the melatonin output. To confirm this, patch-clamp recordings (whole-cell perforated) were run in a 20 mmol/l barium medium at different holding potentials from -80 mV. A voltage-dependent inward current was activated from -30 mV to +40 mV with a maximal amplitude being observed at 0 mV. This current was drastically increased in the presence of Bay K 8644. Nifedipine inhibited the current both in the absence or in the presence of Bay K 8644. Our results are consistent with the idea that extracellular calcium participates in the control of melatonin secretion by photoreceptor cells. It is suggested that activation of the voltage-dependent L-type channel may modulate this secretion.

Transcriptional Regulation of Arylalkylamine-N-Acetyltransferase-2 Gene in the Pineal Gland of the Gilthead Seabream

Pineal serotonin‐N‐acetyltransferase (arylalkylamine‐N‐acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post‐translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real‐time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24‐h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock‐controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light‐entrainable clock‐containing PAC‐2 zebrafish cell line, a stably transfected seabream aanat2 promoter‐luciferase DNA construct exhibited a clock‐controlled circadian rhythm of luciferase activity, characteristic for an E‐box‐driven expression. In NIH‐3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E‐box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal‐specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock‐controlled gene that is regulated by conserved mechanisms.

Pineal photoreceptor cells: photoperiodic control of melatonin production after cell dissociation and culture.

Trout pineal cells were dissociated using a trypsin‐DNase digestion technique. An enriched population of photoreceptor cells was selected from a Percoll gradient centrifugation. The ability of cultured photoreceptor cells (selected or not on a Percoll gradient) to produce melatonin rhythmically was investigated during seven 24 h light/dark cycles. During each cycle, trout pineal photoreceptor cells released low amounts of melatonin during daytime and high amounts during night‐time. Under continuous darkness, melatonin release was continually high. The profile of its rhythm and that of the activity of the hydroxyindole‐O‐methyltransferase—the last enzyme of the melatonin biosynthetic pathway—depended on the substrates and on the culture media used. Some of them appear suitable for short‐ or long‐term culture of photoreceptor cells permitting the study of their neuroendocrine properties.

Immunocytochemical localization and circadian variations of serotonin and N-acetylserotonin in photoreceptor cells. Light and electron microscopic study in the teleost pineal organ.

Using two immunocytochemical procedures (i.e., immunofluorescence and the unlabeled peroxidase-antiperoxidase method), the localization of a serotonin(HT)-like and of a N-acetylserotonin (aHT)-like immunoreactivity in the pineal organ of the pike was studied during winter. It was shown that immunostaining was exclusively restricted to the cells of the receptor line (CRL = typical and modified photoreceptors). The intensity of the reactions varied through the light-dark cycle, HT-like immunoreactivity being high during the photophase and low during the scotophase. In contrast, aHT-like immunoreactivity was highest at the beginning of the scotophase. HT and aHT-like immunoreactivities were detected in all cell types of the pineal epithelium after administration of a monoamine oxidase inhibitor. Up to now, only HT immunoreactivity could be localized at the ultrastructural level. In a number of typical and modified photoreceptors, a HT-positive staining seemed to be confined within the hyaloplasm of the inner segment, particularly with that of the perikaryon and basal pedicle. Our previous and present results strongly suggest that indole compounds, which are involved in the regulation of various neuroendocrine processes in fish, are synthetized within the CRL. Taking into account that the CRL of the pike are also photosensitive, it appears more and more likely that they are photoneuroendocrine cells involved in mediating the effects of the photoperiod on various physiological and behavioral processes.

Melatonin production in organ cultured chicken pineal: modulation by adenosine and its analogs.

The effects of adenosine, of its non-metabolizable analogs, and of compounds related to its metabolism, were investigated in the photosensitive chicken pineal, maintained in static culture or in superfusion. Stimulation or inhibition of melatonin production was obtained, depending on the experimental conditions tested. Endogenous adenosine is involved in the regulation of the melatonin output. The effects of the nucleoside might depend on the balance between its intra- and extracellular pools; (re)uptake mechanisms are most probably involved. It is suggested that cell surface receptors mediate adenosine effects, but intracellular actions (P-site, transmethylation pathways) are not excluded. This investigation is a breakthrough in the field of pineal physiology which offers new perspectives in the study of the control of melatonin production.

Localization of 5-hydroxytryptamine and protein (s) in the secretion granules of the rudimentary photoreceptor cells in the pineal ofLacerta

SummaryIn order to localize 5-hydroxytryptamine (5-HT) stores, the pineal organ ofLacerta was examined by electron microscopy following argentaffin and chromaffin reactions. The results obtained with these ultracytochemical reactions were correlated with those obtained previously using the Falck and Hillarp method and electron microscopic radioautography.The secretion granules (diameter: 60–340 nm) of the secretory rudimentary photoreceptor (SRP) cells are the locus of the argentaffin and chromaffin reactions. The reactivity of the granules disappeared afterp-CPA or reserpine administration and increased after nialamide injection.From the previous and present results it can be concluded that 5-HT in the pineal organ ofLacerta is stored in the secretion granules of SRP cells and coexists with a proteinaceous and/or possibly a glycoproteinaceous component. Whether this secretion is neurohormonal in nature is still not clear.