Fatty Acid Sensing in the Gastrointestinal Tract of Rainbow Trout: Different to Mammalian Model?

It is well established in mammals that the gastrointestinal tract (GIT) senses the luminal presence of nutrients and responds to such information by releasing signaling molecules that ultimately regulate feeding. However, gut nutrient sensing mechanisms are poorly known in fish. This research characterized fatty acid (FA) sensing mechanisms in the GIT of a fish species with great interest in aquaculture: the rainbow trout (Oncorhynchus mykiss). Main results showed that: (i) the trout GIT has mRNAs encoding numerous key FA transporters characterized in mammals (FA transporter CD36 -FAT/CD36-, FA transport protein 4 -FATP4-, and monocarboxylate transporter isoform-1 -MCT-1-) and receptors (several free FA receptor -Ffar- isoforms, and G protein-coupled receptors 84 and 119 -Gpr84 and Gpr119-), and (ii) intragastrically-administered FAs differing in their length and degree of unsaturation (i.e., medium-chain (octanoate), long-chain (oleate), long-chain polyunsaturated (α-linolenate), and short-chain (butyrate) FAs) exert a differential modulation of the gastrointestinal abundance of mRNAs encoding the identified transporters and receptors and intracellular signaling elements, as well as gastrointestinal appetite-regulatory hormone mRNAs and proteins. Together, results from this study offer the first set of evidence supporting the existence of FA sensing mechanisms n the fish GIT. Additionally, we detected several differences in FA sensing mechanisms of rainbow trout vs. mammals, which may suggest evolutionary divergence between fish and mammals.

Economic Improvement of Artisanal Fishing by Studying the Survival of Discarded Plectorhinchus mediterraneus.

Europe calls for the end to fisheries discards, which means bringing all caught fish (subject to minimum sizes or quotas) to land. This decision is beneficial to the ecosystem, since it forces the selectivity of the fishing gears to improve. However, artisanal fishermen find themselves in a vulnerable situation where their subsistence depends on catches with small profit margins. An exemption to this landing obligation exists, as it is also ruled that those animals whose survival is scientifically guaranteed may be returned to the sea. Here we study the survival of Plectorhinchus mediterraneus captured by hookline and gillnet, as well as their physiological recovery. Survival exceeds 93% in both cases. The physiological assessment of primary (cortisol) and secondary (energy mobilization, acid-base and hydromineral balance, and immune system) stress responses indicates that surviving animals are able to recover after fishing. Thus, we propose the optimal size of capture of this species to achieve greater economic benefit. For this, we rely on the prices according to size in recent years, as well as on the growth curves of the species. In this way, by releasing fish of less than 1 kg, the current benefits could be multiplied between 2.3 and 9.6 times. This pilot study lays the groundwork for regulating artisanal fisheries through scientific data related to survival of discards along with information on the sale prices.

Nuclear Receptors (PPARs, REV-ERBs, RORs) and Clock Gene Rhythms in Goldfish (Carassius auratus) Are Differently Regulated in Hypothalamus and Liver.

The circadian system is formed by a network of oscillators located in central and peripheral tissues that are tightly linked to generate rhythms in vertebrates to adapt the organism to the cyclic environmental changes. The nuclear receptors PPARs, REV-ERBs and RORs are transcription factors controlled by the circadian system that regulate, among others, a large number of genes that control metabolic processes for which they have been proposed as key genes that link metabolism and temporal homeostasis. To date it is unclear whether these nuclear receptors show circadian expression and which zeitgebers are important for their synchronization in fish. Therefore, the objective of this study was to investigate whether the two main zeitgebers (light-dark cycle and feeding time) could affect the synchronization of central (hypothalamus) and peripheral (liver) core clocks and nuclear receptors in goldfish. To this aim, three experimental groups were established: fish under a 12 h light-12 h darkness and fed at Zeitgeber Time 2; fish with the same photoperiod but randomly fed; and fish under constant darkness and fed at Circadian Time 2. After one month, clock genes and nuclear receptors expression in hypothalamus and liver and circulating glucose were studied. Clock genes displayed daily rhythms in both tissues of goldfish if the light-dark cycle was present, with shifted-acrophases of negative and positive elements, as expected for proper functioning clocks. In darkness-maintained fish hypothalamic clock genes were fully arrhythmic while the hepatic ones were still rhythmic. Among studied nuclear receptors, in the hypothalamus only nr1d1 was rhythmic and only when the light-dark cycle was present. In the liver all nuclear receptors were rhythmic when both zeitgebers were present, but only nr1d1 when one of them was removed. Plasma glucose levels showed significant rhythms in fish maintained under random fed regimen or constant darkness, with the highest levels at 1-h postprandially in all groups. Altogether these results support that hypothalamus is mainly a light-entrained-oscillator, while the liver is a food-entrained-oscillator. Moreover, nuclear receptors are revealed as clear outputs of the circadian system acting as key elements in the timekeeping of temporal homeostasis, particularly in the liver.

First evidence of nocturnin in fish: two isoforms in goldfish differentially regulated by feeding.

Nocturnin (NOC) is a unique deadenylase with robust rhythmic expression involved in the regulation of metabolic processes in mammals. Currently, the possible presence of NOC in fish is unknown. This report aimed to identify NOC in a fish model, the goldfish ( Carassius auratus), and to study the possible regulation of its expression by feeding. Two partial-length cDNAs of 293 and 223 bp, named nocturnin-a ( noc-a) and nocturnin-b ( noc-b), were identified and found to be highly conserved among vertebrates. Both mRNAs show a similar widespread distribution in central and peripheral tissues, with higher levels detected for noc-a compared with noc-b. The periprandial expression profile revealed that noc-a mRNAs rise sharply after a meal in hypothalamus, intestinal bulb, and liver, whereas almost no changes were observed for noc-b. Food deprivation was found to exert opposite effects on the expression of both NOCs (generally inhibitory for noc-a, and stimulatory for noc-b) in the three mentioned tissues. A single meal after a 48-h food deprivation period reversed (totally or partially) the fasting-induced decreases in noc-a transcripts in all studied tissues and the increases in noc-b expression in the intestinal bulb. Together, this study offers the first report of NOC in fish and shows a high dependence of its expression on feeding and nutritional status. The differential responses to feeding of the two NOCs raise the possibility that they might be underlying different physiological mechanisms (e.g., food intake, lipid mobilization, energy homeostasis) in fish.

Ghrelin induces clock gene expression in the liver of goldfish in vitro via protein kinase C and protein kinase A pathways.

The liver is the most important link between the circadian system and metabolism. As a food-entrainable oscillator, the hepatic clock needs to be entrained by food-related signals. The objective of the present study was to investigate the possible role of ghrelin (an orexigenic peptide mainly synthesized in the gastrointestinal tract) as an endogenous synchronizer of the liver oscillator in teleosts. To achieve this aim, we first examined the presence of ghrelin receptors in the liver of goldfish. Then, the ghrelin regulation of clock gene expression in the goldfish liver was studied. Finally, the possible involvement of the phospholipase C/protein kinase C (PLC/PKC) and adenylate cyclase/protein kinase A (AC/PKA) intracellular signalling pathways was investigated. Ghrelin receptor transcripts, ghs-r1a, are present in the majority of goldfish hepatic cells. Ghrelin induced the mRNA expression of the positive (gbmal1a, gclock1a) and negative (gper genes) elements of the main loop of the molecular clock machinery, as well as grev-erbα (auxiliary loop) in cultured liver. These effects were blocked, at least in part, by a ghrelin antagonist. Incubation of liver with a PLC inhibitor (U73122), a PKC activator (phorbol 12-myristate 13-acetate) and a PKC inhibitor (chelerythrine chloride) demonstrated that the PLC/PKC pathway mediates such ghrelin actions. Experiments with an AC activator (forskolin) and a PKA inhibitor (H89) showed that grev-erbα regulation could be due to activation of PKA. Taken together, the present results show for the first time in vertebrates a direct action of ghrelin on hepatic clock genes and support a role for this hormone as a temporal messenger in the entrainment of liver circadian functions.

Interplay between the endocrine and circadian systems in fishes.

The circadian system is responsible for the temporal organisation of physiological functions which, in part, involves daily cycles of hormonal activity. In this review, we analyse the interplay between the circadian and endocrine systems in fishes. We first describe the current model of fish circadian system organisation and the basis of the molecular clockwork that enables different tissues to act as internal pacemakers. This system consists of a net of central and peripherally located oscillators and can be synchronised by the light-darkness and feeding-fasting cycles. We then focus on two central neuroendocrine transducers (melatonin and orexin) and three peripheral hormones (leptin, ghrelin and cortisol), which are involved in the synchronisation of the circadian system in mammals and/or energy status signalling. We review the role of each of these as overt rhythms (i.e. outputs of the circadian system) and, for the first time, as key internal temporal messengers that act as inputs for other endogenous oscillators. Based on acute changes in clock gene expression, we describe the currently accepted model of endogenous oscillator entrainment by the light-darkness cycle and propose a new model for non-photic (endocrine) entrainment, highlighting the importance of the bidirectional cross-talking between the endocrine and circadian systems in fishes. The flexibility of the fish circadian system combined with the absence of a master clock makes these vertebrates a very attractive model for studying communication among oscillators to drive functionally coordinated outputs.

Performing a hepatic timing signal: glucocorticoids induce gper1a and gper1b expression and repress gclock1a and gbmal1a in the liver of goldfish.

Glucocorticoids have been recently proposed as input signals of circadian system, although the underlying molecular mechanism remains unclear. This work investigates the role of glucocorticoids as modulators of clock genes expression in the liver of goldfish. In fish maintained under a 12L:12D photoperiod, an intraperitoneal injection at Zeitgeber Time 2 of a glucocorticoid analog, dexamethasone (1 µg/g body weight) induced per1 genes while decreased gbmal1a and gclock1a expression in the liver at 8 h post-injection. A 4-h in vitro exposure of goldfish liver to cortisol (0.1-10 µM) also induced gper1 genes in a concentration-dependent manner. Similarly, the exposure of the goldfish cultured liver to dexamethasone produced a concentration-dependent induction of gper1 genes. Moreover, this glucocorticoid analog led to a decrease in gbmal1a and gclock1a transcripts, while the other clock genes analyzed were unaffected. The induction of gper1a and gper1b by dexamethasone in vitro was observed at short times (2 h), whereas the reductions of gbmal1a and gclock1a transcripts needed longer exposure times (8 h) to the glucocorticoid to be significant. Additionally, a 2-h exposure to dexamethasone in the liver culture was enough to extend the induction of per genes for more than 12 h. Present results indicate that gper1 genes are targets for glucocorticoids in the regulation of goldfish hepatic oscillator, as previously reported in mammals, suggesting a conserved role of glucocorticoids in the functional organization of the peripheral circadian system in vertebrates. The repression of clock1a and bmal1a is not so well established, and suggests that other clock genes could be glucocorticoid targets in the goldfish liver.

In Situ Localization and Rhythmic Expression of Ghrelin and ghs-r1 Ghrelin Receptor in the Brain and Gastrointestinal Tract of Goldfish (Carassius auratus).

Ghrelin is a gut-brain peptide hormone, which binds to the growth hormone secretagogue receptor (GHS-R) to regulate a wide variety of biological processes in fish. Despite these prominent physiological roles, no studies have reported the anatomical distribution of preproghrelin transcripts using in situ hybridization in a non-mammalian vertebrate, and its mapping within the different encephalic areas remains unknown. Similarly, no information is available on the possible 24-h variations in the expression of preproghrelin and its receptor in any vertebrate species. The first aim of this study was to investigate the anatomical distribution of ghrelin and GHS-R1a ghrelin receptor subtype in brain and gastrointestinal tract of goldfish (Carassius auratus) using immunohistochemistry and in situ hybridization. Our second aim was to characterize possible daily variations of preproghrelin and ghs-r1 mRNA expression in central and peripheral tissues using real-time reverse transcription-quantitative PCR. Results show ghrelin expression and immunoreactivity in the gastrointestinal tract, with the most abundant signal observed in the mucosal epithelium. These are in agreement with previous findings on mucosal cells as the primary synthesizing site of ghrelin in goldfish. Ghrelin receptor was observed mainly in the hypothalamus with low expression in telencephalon, pineal and cerebellum, and in the same gastrointestinal areas as ghrelin. Daily rhythms in mRNA expression were found for preproghrelin and ghs-r1 in hypothalamus and pituitary with the acrophase occurring at nighttime. Preproghrelin, but not ghs-r1a, displayed a similar daily expression rhythm in the gastrointestinal tract with an amplitude 3-fold higher than the rest of tissues. Together, these results described for the first time in fish the mapping of preproghrelin and ghrelin receptor ghs-r1a in brain and gastrointestinal tract of goldfish, and provide the first evidence for a daily regulation of both genes expression in such locations, suggesting a possible connection between the ghrelinergic and circadian systems in teleosts.

Anatomical distribution and daily profile of gper1b gene expression in brain and peripheral structures of goldfish (Carassius auratus).

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.

The liver of goldfish as a component of the circadian system: Integrating a network of signals.

The circadian system drives daily physiological and behavioral rhythms that allow animals to anticipate cyclic environmental changes. The discovery of the known as "clock genes", which are very well conserved through vertebrate phylogeny, highlighted the molecular mechanism of circadian oscillators functioning, based on transcription and translation cycles (∼ 24 h) of such clock genes. Studies in goldfish have shown that the circadian system in this species is formed by a net of oscillators distributed at central and peripheral locations, as the retina, brain, gut and liver, among others. In this work we review the existing information about the hepatic oscillator in goldfish due to its relevance in metabolism, and its key role as target of a variety of humoral signals. Different input signals modify the molecular clockwork in the liver of goldfish. Among them, there are environmental cues (photocycle and feeding regime) and different encephalic and peripheral endogenous signals (orexin, ghrelin and glucocorticoids). Per clock genes seem to be a common target for different signals. Thus, this genes family might be important for shifting the hepatic oscillator. The physiological relevance of the crosstalking between metabolic and feeding-related hormones and the hepatic clock sets the stage for the hypothesis that these hormones could act as "internal zeitgebers" communicating oscillators in the goldfish circadian system.

Light-dark cycle and feeding time differentially entrains the gut molecular clock of the goldfish (Carassius auratus).

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.

Dopamine D4 receptors regulate intracellular calcium concentration in cultured chicken cone photoreceptor cells: relationship to dopamine receptor-mediated inhibition of cAMP formation.

Dopamine is a retinal neuromodulator secreted from amacrine and interplexiform cells. Activation of dopamine D4 receptors on photoreceptor cells reduces a light-sensitive pool of cAMP. The aim of the present study was to evaluate the role of dopamine receptors and cAMP in the regulation of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in photoreceptor cells of chick retina. Retinal cells from 6 day-old chicken embryos were isolated and cultured for 5-7 days prior to experiments. Cone photoreceptors were the predominant cell type in these cultures. Dopamine and agonists of dopamine D4 receptors suppressed K(+)-stimulated uptake of (45)Ca(2+) and [Ca(2+)](i), measured with the Ca(2+)-sensitive fluorescent dye fura-2AM. The effects of the agonists were blocked by dopamine D2/D4 receptor antagonists or by pertussis toxin. 8Br-cAMP, a cell-permeable analog of cAMP, had no effect on inhibition of K(+)-stimulated (45)Ca(2+) influx or [Ca(2+)](i) by dopamine D2/D4 receptor agonists. Quinpirole inhibited the increase in cAMP level elicited by K(+), which requires Ca(2+) influx through voltage-gated Ca(2+) channels, but not that induced by the calcium ionophore A23187. Moreover, dopamine had no effect on either forskolin-stimulated or Ca(2+)/calmodulin-stimulated adenylyl cyclase activity in cell membranes prepared from the cultured cells. These data indicate that the decrease of cAMP elicited by dopamine D4 receptor stimulation may be secondary to decreased [Ca(2+)](i).

Retinal, pineal and diencephalic expression of frog arylalkylamine N-acetyltransferase-1.

The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in Teleost fish (Aanat1 in the retina and Aanat2 in the pineal organ) but only Aanat1 is found in tetrapods. This study reports the cloning of Aanat1 from R. perezi. Transcripts were mainly expressed in the retina, diencephalon, intestine and testis. In the retina and pineal organ, Aanat1 expression was in the photoreceptor cells. Expression was also seen in ependymal cells of the 3rd ventricle and discrete cells of the suprachiasmatic area. The expression of Aanat1 in both the retina and pineal organ, and the absence of Aanat2 suggests that green frog resembles more to birds and mammals than to Teleost fish, as far as Aanat is concerned. The significance of Aanat1 in extra-pineal and extra-retinal tissues remains to be elucidated; in the diencephalon, it might be associated to the so-called deep brain photoreceptor cells.

Effects of temperature on 2-[125I]-iodomelatonin binding to melatonin receptors in the neural retina of the frog Rana perezi.

The present study analyzes the effect of temperature-dependent modifications on the binding of the analog 2-[125I]-melatonin to melatonin receptors in isolated neural retina membranes from the greenfrog Rana perezi. Association and dissociation rate constants (K+1, K-1) were exponentially increased by the assay temperature. At 15 degrees C, association and dissociation required several hours; meanwhile, at 35 degrees C, rate constants were 100- and 34-fold faster, respectively. However, the Kd constant calculated as K-1/K+1 was unmodified by the assay temperature. When frogs were acclimated at either 5 or 22 degrees C for 1 month, K+1, and K-1 constants determined at 15 and 25 degrees C were identical in both cold- and warm-acclimated groups. Thus, the binding kinetics of melatonin receptors in frog retinas did not shown any thermal compensation. Results from saturation curves and pharmacological profiles of melatonin binding sites support a lack of effect of assay temperature on the affinity of melatonin receptors in the frog retina. The inhibition of [125I]Mel binding by GTPgammaS showed clearly that the coupling of melatonin receptors to G proteins is temperature-dependent. Higher concentrations of the GTP analog were needed to inhibit specific binding when temperature decreased. The temperature effect on binding kinetics and on the G protein coupling to melatonin receptors suggests that the melatonin signal could be transduced distinctly depending on the temperature. Thus, temperature plays a major role, not only on melatonin synthesis, but also in the transduction of melatonin signal in ectotherms.

2-[125I]-melatonin binding sites in the central nervous system and neural retina of the frog Rana perezi: regulation by light and temperature.

The objective of the present study is to test daily and seasonal changes in 2-[125I]-Melatonin ([125I]-Mel) binding in different brain areas and the retina of the frog Rana perezi as well as the possible effect of light and temperature on melatonin receptors. During the day-night cycle, binding of [125I]-Mel showed a clear rhythm in the optic tectum, diencephalon, telencephalon, and neural retina, the binding being higher in the light phase than in the dark phase. By contrast, melatonin receptors did not show any significant summer-winter differences in any of the four tissues studied. In the neural retina, but not in the brain, exposure of frogs to 24 h darkness for one week leads to significantly less [125I]-Mel binding than 24 h light exposure. This darkness-induced reduction of [125I]-Mel binding is not due to a desensitisation of binding sites by high melatonin levels. Thermal acclimation to either 5 or 22 degrees C for one month did not change the affinity (Kd) and density (Bmax) of [125I]-Mel binding sites either in the brain or the retina. All these results indicate that there is a daily rhythm in melatonin receptors in the frog brain and retina, and that the light/dark cycle can drive this rhythm in [125I]-Mel binding in the retina. Temperature apparently did not modify [125I]-Mel binding in frogs.

Characterization of melatonin binding sites in the brain and retina of the frog Rana perezi.

The aim of this study was to characterize 2-[125I]iodomelatonin binding sites in the neural retina and central nervous system (telencephalon, diencephalon, and optic tectum) of the anuran amphibian Rana perezi. Saturation and kinetic studies and pharmacological characterization revealed the existence of a unique melatonin-binding site that belongs to the Mel 1 receptor subtype. The affinity of this site is similar in all tissues studied (Kd, 10.5-12.8 pM), but the density varied from diencephalon and optic tectum, which exhibit the highest density, to telencephalon with the lowest. Neural retina showed an intermediate receptor density. This melatonin-binding site fulfills the requirements of a real hormone receptor; the binding is saturable, reversible, and inhibited by different melatonin agonists and antagonists. The affinity order of ligands is: 2-phenyl-melatonin = 2-I-melatonin > 6-Cl-melatonin = melatoninz >> luzindole. Additionally, specific binding is decreased by non-hydrolysable GTP analogue, sodium, and by pretreatment of membranes with pertussis toxin. All these results suggest the existence of a widely distributed and pharmacologically homogeneous melatonin receptor of the subfamily Mel 1 in the nervous system of Rana perezi coupled to a Gi/o protein.

Diurnal regulation of arylalkylamine N-acetyltransferase activity in chicken retinal cells in vitro: analysis of culture conditions.

PURPOSE: Arylalkylamine N-acetyltransferase (AANAT) is a key regulatory enzyme in the synthesis of melatonin, which displays daily fluctuations in chicken retinal photoreceptors in vivo. The purpose of the present study was to determine if cultures of embryonic neural retina cells express diurnal rhythms of AANAT activity. METHODS: Cell cultures were prepared from chick embryonic day 6 neural retina and incubated for 4 to 8 days in vitro (DIV). Cells were incubated under a daily light-dark (LD) cycle and were harvested day and night. Culture conditions were modified to test the effects of cell density, serum concentration, incubation temperature, S-(4-nitrobenzyl)-6-thioinosine (NBTI), and taurine on AANAT activity. AANAT activity was assayed in cell homogenates by measuring the catalytic formation of N-acetyltryptamine from tryptamine and acetyl coenzyme A. RESULTS: Cells cultured in medium containing 10% fetal bovine serum (FBS) failed to show any diurnal fluctuation in AANAT activity on DIV 5 and 6. However, if the culture medium was replaced on DIV 4 with one containing 1% FBS, and 5 microM NBTI or 5 mM taurine, the cells expressed significant diurnal rhythms of enzyme activity. NBTI was more potent and effective than taurine. Culture conditions were optimized with respect to cell density, serum concentration, incubation temperature, and NBTI concentration. Under optimized conditions, overall cell survival and the density of photoreceptor cells were increased relative to that with the other culture conditions tested. CONCLUSIONS: The results indicate that diurnal rhythms of AANAT activity are expressed in embryonic retinal cells incubated under particular culture conditions. The results show that the mechanisms regulating melatonin synthesis in chicken retinal cells are established during early embryonic life. This culture preparation will be useful in elucidating the photic control mechanisms involved in regulation of melatonin biosynthesis in photoreceptor cells.