Development of a circadian melatonin rhythm in embryonic zebrafish.

We investigated the time course of circadian system development in zebrafish and the role of environmental light cycles in this process, using a rhythm in melatonin content of embryos and larvae as a marker of circadian function. When zebrafish were raised in a cycle of 14 h light and 10 h dark at 28.5 degrees C, nocturnal increases in melatonin content were detectable beginning on the second night post-fertilization (PF). When embryos were transferred to constant darkness (DD) at the end of the second light period, a circadian rhythm of melatonin content persisted for at least three cycles. However, when embryos were transferred from light to DD at 14 h PF, no rhythm was detectable in the population. Phase-locked circadian melatonin rhythms were measurable after embryos were exposed to a transition from constant light (LL) to darkness at 26 or 32 h PF, but not at 20 h. These data indicate that a circadian oscillator that regulates melatonin synthesis becomes functional and responsive by light between 20 and 26 h PF. At this stage, pineal photoreceptors have begun to differentiate, but retinal photoreceptors have not, suggesting that the first circadian melatonin rhythms are of pineal origin. The absence of melatonin rhythms in populations of embryos exposed to DD beginning at earlier stages indicates that there is no timed developmental event that sets the circadian clock in the absence of environmental input. Exposure to DD starting at 14 or 20 h PF did not retard overall development as determined by gross morphological staging criteria, and did not prevent later synchronization of melatonin rhythms by light-dark (LD) cycles.

A role for cyclic AMP in entrainment of the circadian oscillator in Xenopus retinal photoreceptors by dopamine but not by light.

The circadian oscillator in Xenopus retinal photoreceptor layers can be reset in similar ways by light and agonists of D2-like dopamine receptors. Treatments that increase cyclic AMP levels act on this oscillator in an opposite fashion, mimicking darkness in the induction of phase shifts. Light and dopamine have each been reported to inhibit adenylate cyclase in photoreceptors. Together, these data suggest that the transduction pathways for entrainment by dopamine and/or light include suppression of cyclic AMP or a cyclic AMP-sensitive step. In these studies, we examined this hypothesis by measuring the effects of treatment with a cyclic AMP analogue on the phase shifts induced in photoreceptor melatonin rhythms by light or a D2 receptor agonist (quinpirole). When photoreceptor layers were treated simultaneously with 8-(4-chlorophenylthio)cyclic AMP (8-CPT-cAMP) and quinpirole at any of three different phases of the circadian cycle, the resulting phase shifts of the melatonin rhythm were always the same as those caused by 8-CPT-cAMP alone. This indicates that there is a cyclic AMP-sensitive step in the dopamine entrainment pathway. In contrast, light pulses did reset the oscillator in the presence of elevated cyclic AMP. This suggests a separate cyclic AMP-insensitive transduction pathway for entrainment by light. Quinpirole reduced basal levels of cyclic AMP in photoreceptors, but light did not. These data suggest that cyclic AMP plays a role in the entrainment pathway activated by dopamine but not in the entrainment pathway activated by light.

Modulation of rhythmic melatonin synthesis in Xenopus retinal photoreceptors by cyclic AMP.

Cyclic AMP regulates melatonin synthesis in vertebrate photoreceptor cells. In the present study, we investigated whether the circadian rhythm of melatonin synthesis in Xenopus retinal photoreceptor layers is driven by rhythmic changes in cyclic AMP. When the photoreceptor layers were continuously treated with 8-(4-chlorophenylthio)-cyclic AMP (8-CPT-cAMP) at a saturating concentration (1 mM), melatonin release was increased at all times of the day, but robust melatonin rhythms were maintained for 2 days in constant darkness (DD). We also measured cyclic AMP efflux and melatonin release simultaneously from photoreceptor layers that were continuously treated with forskolin and/or 3-isobutyl-1-methylxanthine (IBMX) in light-dark (LD) and DD. Circadian rhythmicity was observed in melatonin release, but not in cyclic AMP efflux, suggesting that changes of melatonin levels are not always caused by the changes of the cyclic AMP levels. In addition, the simultaneous treatment of forskolin and IBMX appeared to saturate sensitivity of melatonin synthesis to cyclic AMP, but this treatment did not abolish melatonin rhythms. These results suggest that circadian rhythms of melatonin can be driven without rhythmic changes of cyclic AMP, and that cyclic AMP regulates melatonin in parallel with the output pathways from the circadian oscillator.

Circadian rhythmicity in the locomotor activity of larval zebrafish.

The zebrafish (Danio rerio) may be useful for mutational analyses of vertebrate circadian clock mechanisms if efficient assays of circadian rhythmicity are available. Using an automated video image analysis system, we found robust circadian rhythms in the locomotor activity of larval (10- to 15-day-old) zebrafish maintained in constant conditions. Activity was rhythmic in > 95% of the animals tested. The timing of peak activity in constant conditions was determined by the prior light:dark cycle, with highest activity during the subjective day. The mean freerunning period of the activity rhythms was 25.6 h, and the within-experiment standard deviation of freerunning period ranged from 0.5 to 1.0 h. Therefore, it should be possible to detect mutations that lengthen or shorten the freerunning circadian period of zebrafish activity rhythms by 1-2 h.

Spectral sensitivity of melatonin synthesis suppression in Xenopus eyecups.

Melatonin synthesis in retinal photoreceptors is stimulated at night by a circadian oscillator and suppressed acutely by light. To identify photoreceptor mechanisms involved in the acute suppression of melatonin synthesis, an action spectrum was measured for dark-adapted Xenopus laevis eyecups at night. Intensity-response curves at six wavelengths from 400 to 650 nm were parallel, suggesting that a single photopigment predominates in melatonin suppression. Half-saturating intensities at 400, 440, 480, and 533 nm were not significantly different from one another, at 1-2 x 10(8) quanta cm(-2) s(-1). Significantly higher intensities of 580- and 650-nm light were required for melatonin suppression. These results indicate a predominant role for the principal green-absorbing rods in acute regulation of retinal melatonin synthesis in response to light, and argue against an important role for the red-absorbing cones. Higher than expected sensitivity at short wavelengths suggests that photoreceptors sensitive to blue and/or violet light may also contribute to melatonin suppression.

Cyclic AMP resets the circadian clock in cultured Xenopus retinal photoreceptor layers.

The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180 degrees out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.

Transcripts encoding two melatonin synthesis enzymes in the teleost pineal organ: circadian regulation in pike and zebrafish, but not in trout.

In this report the photosensitive teleost pineal organ was studied in three teleosts, in which melatonin production is known to exhibit a daily rhythm with higher levels at night; in pike and zebrafish this increase is driven by a pineal clock, whereas in trout it occurs exclusively in response to darkness. Here we investigated the regulation of messenger RNA (mRNA) encoding serotonin N-acetyltransferase (AA-NAT), the penultimate enzyme in melatonin synthesis, which is thought to be primarily responsible for changes in melatonin production. AA-NAT mRNA was found in the pineal organ of all three species and in the zebrafish retina. A rhythm in AA-NAT mRNA occurs in vivo in the pike pineal organ in a light/dark (L/D) lighting environment, in constant lighting (L/L), or in constant darkness (D/D) and in vitro in the zebrafish pineal organ in L/D and L/L, indicating that these transcripts are regulated by a circadian clock. In contrast, trout pineal AA-NAT mRNA levels are stable in vivo and in vitro in L/D, L/L, and D/D. Analysis of mRNA encoding the first enzyme in melatonin synthesis, tryptophan hydroxylase, reveals that the in vivo abundance of this transcript changes on a circadian basis in pike, but not in trout. A parsimonious hypothesis to explain the absence of circadian rhythms in both AA-NAT and tryptophan hydroxylase mRNAs in the trout pineal is that one circadian system regulates the expression of both genes and that this system has been disrupted by a single mutation in this species.

Circadian rhythms of locomotor activity in zebrafish.

As part of an effort to characterize the circadian system of the zebrafish, we examined the circadian regulation of locomotor activity in adult males and females. Gross locomotor activity was measured using infrared movement detectors. The effects of light, dark, and temperature on the amplitude, phase, and free-running periods of locomotor rhythms were determined. When zebrafish were maintained in a 12-h light:12 h dark cycle at 25 degrees C, 86% of the fish were most active during the light phase of the cycle. The phases of free-running rhythms measured after transfer of fish from light cycles to constant conditions indicate that this diurnal activity profile reflects entrained circadian rhythmicity. When animals were maintained in constant conditions, the proportion that expressed significant circadian rhythmicity depended on ambient temperature. At 21 degrees C, 73% of the animals were rhythmic in constant darkness, and 65% were rhythmic in constant light. Fewer (28-59%) were rhythmic at 18 degrees, 25 degrees, and 28.5 degrees C. The free-running period of rhythmic animals was not affected by temperature within this range. The average period was shorter in constant light (LL; 12 lx) than in constant darkness (DD) in all but one experiment, and the difference was statistically significant for animals held at 21 degrees C. These data indicate that zebrafish locomotor activity is regulated by a circadian clock that is temperature compensated. Because rhythmicity is most robust at 21 degrees C, this would be the optimal temperature for future studies of the physiological basis of zebrafish behavioral rhythms.

Circadian oscillators in vertebrate retinal photoreceptor cells.

Recent progress in research on retinal circadian rhythmicity is reviewed. Important advances include the discovery that circadian oscillators are present in the retinas of diverse vertebrate species, and evidence that circadian rhythmicity is generated by the photoreceptor cells. Research on the cellular and molecular mechanisms of photoreceptor circadian rhythms has revealed that expression of a subset of genes associated with photoreception, melatonin synthesis and transcriptional control are regulated by a circadian oscillator. Finally, it has been found that cAMP mimics darkness in resetting the phase of the retinal photoreceptor circadian oscillator, suggesting that it may be a component of a transduction pathway for entrainment of the oscillator.

Circadian melatonin rhythms in cultured zebrafish pineals are not affected by catecholamine receptor agonists.

Catecholamine receptors of multiple classes have been shown to influence pineal melatonin synthesis in a species-specific manner. In these experiments, the effects of catecholamine receptor agonists on circadian melatonin rhythms of zebrafish (Danio rerio) pineal in vitro were examined. Cyclic application of adrenergic receptor agonists (norepinephrine, phenylephrine, clonidine, and isoproterenol) had no effect on zebrafish pineal melatonin release, nor on the circadian oscillator that regulates melatonin rhythms. Pineal melatonin release was partially suppressed by quinpirole, a D2 dopamine receptor agonist, but cyclic application of quinpirole did not reset the pineal circadian oscillator. Pineal melatonin release was unaffected by either dopamine or SKF38393, a D1 receptor agonist, suggesting that the effects of quinpirole were not mediated by dopamine receptors. The regulatory mechanisms underlying pineal melatonin rhythms appear to differ among teleosts.

The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland.

A remarkably constant feature of vertebrate physiology is a daily rhythm of melatonin in the circulation, which serves as the hormonal signal of the daily light/dark cycle: melatonin levels are always elevated at night. The biochemical basis of this hormonal rhythm is one of the enzymes involved in melatonin synthesis in the pineal gland-the melatonin rhythm-generating enzyme-serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, E.C. 2.3.1.87). In all vertebrates, enzyme activity is high at night. This reflects the influences of internal circadian clocks and of light. The dynamics of this enzyme are remarkable. The magnitude of the nocturnal increase in enzyme activity ranges from 7- to 150-fold on a species-to-species basis among vertebrates. In all cases the nocturnal levels of AA-NAT activity decrease very rapidly following exposure to light. A major advance in the study of the molecular basis of these changes was the cloning of cDNA encoding the enzyme. This has resulted in rapid progress in our understanding of the biology and structure of AA-NAT and how it is regulated. Several constant features of this enzyme have become apparent, including structural features, tissue distribution, and a close association of enzyme activity and protein. However, some remarkable differences among species in the molecular mechanisms involved in regulating the enzyme have been discovered. In sheep, AA-NAT mRNA levels show relatively little change over a 24-hour period and changes in AA-NAT activity are primarily regulated at the protein level. In the rat, AA-NAT is also regulated at a protein level; however, in addition, AA-NAT mRNA levels exhibit a 150-fold rhythm, which reflects cyclic AMP-dependent regulation of expression of the AA-NAT gene. In the chicken, cyclic AMP acts primarily at the protein level and a rhythm in AA-NAT mRNA is driven by a noncyclic AMP-dependent mechanism linked to the clock within the pineal gland. Finally, in the trout, AA-NAT mRNA levels show little change and activity is regulated by light acting directly on the pineal gland. The variety of mechanisms that have evolved among vertebrates to achieve the same goal-a rhythm in melatonin-underlines the important role melatonin plays as the hormonal signal of environmental lighting in vertebrates.

Circadian regulation of melatonin production in cultured zebrafish pineal and retina.

Melatonin release was measured from zebrafish pineal organs and retinas maintained in flow-through culture. Pineal organs released melatonin in a strong circadian rhythm through 5 days in constant darkness, and the phase of this rhythm was reset by in vitro exposure to phase-shifted light cycles. In contrast, the retinal melatonin rhythm rapidly damped out in constant darkness, even in the presence of (phase-shifted) light cycles. The zebrafish pineal should be useful for in vitro studies of vertebrate circadian clock mechanisms.

Tryptophan hydroxylase is expressed by photoreceptors in Xenopus laevis retina.

Serotonin has important roles, both as a neurotransmitter and as a precursor for melatonin synthesis. In the vertebrate retina, the role and the localization of serotonin have been controversial. Studies examining serotonin immunoreactivity and uptake of radiolabeled serotonin have localized serotonin to inner retinal neurons, particularly populations of amacrine cells, and have proposed that these cells are the sites of serotonin synthesis. However, other reports identify other cells, such as bipolars and photoreceptors, as serotonergic neurons. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the serotonin synthetic pathway, was recently cloned from Xenopus laevis retina, providing a specific probe for localization of serotonin synthesis. Here we demonstrate that the majority of retinal mRNA encoding TPH is present in photoreceptor cells in Xenopus laevis retina. These cells also contain TPH enzyme activity. Therefore, in addition to being the site of melatonin synthesis, the photoreceptor cells also synthesize serotonin, providing a supply of the substrate needed for the production of melatonin.

Regulation of tryptophan hydroxylase expression by a retinal circadian oscillator in vitro.

Many aspects of retinal physiology are controlled by a circadian clock including at least two steps in the melatonin synthetic pathway: the activity of the enzyme, N-acetyltransferase (NAT), and mRNA levels of the rate-limiting enzyme trytophan hydroxylase (TPH). Light and dopamine (through D2-like dopamine receptors) can phase shift the clock, and can also acutely inhibit NAT activity, resulting in supressed melatonin synthesis. In this paper, we show that eyecups cultured in constant darkness maintain a clock-controlled rhythm in TPH mRNA, with low levels in early day, rising to a peak in early night. Both eyecups and isolated retinas, cultured in light during the day, also exhibit a similar increase in TPH mRNA levels, indicating that this expression is not acutely inhibited by light. Treatment with light or quinpirole (D2 dopamine receptor agonist) in early night, at a time and dose that acutely inhibits NAT activity, does not change levels of TPH mRNA. Addition of eticlopride (D2 dopamine receptor antagonist) during the day, also has no effect on the normal daytime increase in TPH message levels. Therefore, TPH mRNA level is controlled by a circadian clock located within the eye, but acute effects of light or dopamine are not detected.

Circadian clock functions localized in xenopus retinal photoreceptors.

A circadian oscillator that regulates visual function is located somewhere within the vertebrate eye. To determine whether circadian rhythmicity is generated by retinal photoreceptors, we isolated and cultured photoreceptor layers from Xenopus retina. On average, 94% of the viable cells in these preparations were rod or cone photoreceptors. Photoreceptor layers produced melatonin rhythmically, with an average period of 24.3 hr, in constant darkness. The phase of the melatonin rhythm was reset by in vitro exposure of the photoreceptor layers to cycles of either light or quinpirole, a D2 dopamine receptor agonist. These data indicate that other parts of the eye are not necessary for generation or entrainment of retinal circadian melatonin rhythms and suggest that rod and/or cone photoreceptors are circadian clock cells.

Light-sensitive melatonin synthesis by Xenopus photoreceptors after destruction of the inner retina.

Several lines of evidence indicate that retinal photoreceptors produce melatonin. However, there are other potential melatonin sources in the retina, and melatonin synthesis can be regulated by feedback from the inner retina. To analyze cellular mechanisms of melatonin regulation in retinal photoreceptors, we have developed an in vitro method for destruction of the inner retina that preserves functional photoreceptors in contact with the pigment epithelium. Eyecups, which include the neural retina, retinal pigment epithelium, choriod, and sclera were prepared. The vitreal surface of the retina in each eyecup was washed sequentially with 1% Triton X-100, water, and culture medium. This lysed the ganglion cells and neurons and glia of the inner nuclear layer, causing the retina to split apart within the inner nuclear layer. The damaged inner retina was peeled away, leaving photoreceptors attached to the pigment epithelium. The cell density of the inner nuclear layer was reduced 94% by this method, but there was little apparent damage to the photoreceptors. Lesioned eyecups produced normal melatonin levels in darkness at night, and melatonin production was inhibited by light. These results indicate that the inner retina is not necessary for melatonin production nor for regulation of photoreceptor melatonin synthesis by light. The lesion method used in this study may be useful for other physiological and biochemical studies of photoreceptors.

Rhythmic regulation of retinal melatonin: metabolic pathways, neurochemical mechanisms, and the ocular circadian clock.

1. Current knowledge of the mechanisms of circadian and photic regulation of retinal melatonin in vertebrates is reviewed, with a focus on recent progress and unanswered questions. 2. Retinal melatonin synthesis is elevated at night, as a result of acute suppression by light and rhythmic regulation by a circadian oscillator, or clock, which has been localized to the eye in some species. 3. The development of suitable in vitro retinal preparations, particularly the eyecup from the African clawed frog, Xenopus laevis, has enabled identification of neural, cellular, and molecular mechanisms of retinal melatonin regulation. 4. Recent findings indicate that retinal melatonin levels can be regulated at multiple points in indoleamine metabolic pathways, including synthesis and availability of the precursor serotonin, activity of the enzyme serotonin N-acetyltransferase, and a novel pathway for degradation of melatonin within the retina. 5. Retinal dopamine appears to act through D2 receptors as a signal for light in this system, both in the acute suppression of melatonin synthesis and in the entrainment of the ocular circadian oscillator. 6. A recently developed in vitro system that enables high-resolution measurement of retinal circadian rhythmicity for mechanistic analysis of the circadian oscillator is described, along with preliminary results that suggest its potential for elucidating general circadian mechanisms. 7. A model describing hypothesized interactions among circadian, neurochemical, and cellular mechanisms in regulation of retinal melatonin is presented.

Resetting the circadian clock in cultured Xenopus eyecups: regulation of retinal melatonin rhythms by light and D2 dopamine receptors.

A circadian oscillator is located within the eye of Xenopus laevis. This oscillator regulates retinal melatonin synthesis, stimulating it at night. The primary goal of the studies reported here was to define input pathways to this circadian oscillator as a step toward identification of circadian clock mechanisms. A flow-through superfusion culture system was developed to monitor circadian rhythms of melatonin release from individual eyecups. This system was used to determine the effects of light and dopaminergic agents on melatonin production and on the phase of the circadian oscillator. Six hour light pulses suppressed melatonin production and reset the phase of the free-running melatonin rhythm. Light pulses caused phase delays when applied during the early subjective night, phase advances when applied during the late subjective night, and no phase shift when applied during the subjective day. Dopamine receptor agonists mimicked light in suppressing melatonin release and resetting the phase of the circadian rhythm. The phase-response relationship for phase shifts induced by quinpirole, a D2 dopamine receptor agonist, was similar to that for phase shifts induced by light. Pharmacological analysis with selective catecholamine receptor agonists and antagonists indicated that there are pathways to the melatonin-generating system and the circadian oscillator that include D2 dopamine receptors. A D2 receptor antagonist, eticlopride, completely blocked the effects of dopamine on melatonin release and on circadian phase. However, eticlopride did not alter similar effects induced by light, indicating that dopamine-independent pathways exist for light input to these systems. The effects of light and quinpirole on melatonin release and circadian phase were not additive, indicating that the pathways converge. These pathways to the circadian oscillator in the retina present new avenues for pursuit of cellular circadian clock mechanisms.

Melatonin deacetylation: retinal vertebrate class distribution and Xenopus laevis tissue distribution.

Deacetylation is a rapid clearance mechanism for ocular melatonin. We have studied the distribution of retinal melatonin deacetylase activity among vertebrate classes. Exogenous radiolabeled melatonin is metabolized by ocular tissue prepared from the amphibian Xenopus laevis, the reptile Anolis carolinensis, the teleost fish Carassius auratus, and the bird Gallus domesticus. In contrast, we were unable to detect ocular melatonin breakdown in rat or pig. In each species exhibiting ocular melatonin breakdown, melatonin is first deacetylated to 5-methoxytryptamine, which is deaminated, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. Deacetylation of melatonin is inhibited by eserine (physostigmine), causing a reduction in the levels of all 3 metabolites. Deamination of 5-methoxytryptamine is inhibited by the monoamine oxidase inhibitor pargyline, such that 5-methoxyindoleacetic acid and 5-methoxytryptophol levels are decreased while levels of 5-methoxytryptamine are increased. Incubation with the deacetylase inhibitor eserine increases endogenous melatonin levels in Xenopus and Carassius eyecups, indicating that endogenous melatonin is metabolized via the deacetylase. We also studied the tissue distribution of the deacetylase in Xenopus laevis. Melatonin deacetylation occurs in retina, retinal pigment epithelium, and skin, all of which are sites of melatonin action. These results indicate that among non-mammalian vertebrates, deacetylation is a common clearance mechanism for ocular melatonin, and may degrade melatonin at other sites of action as well. Melatonin deacetylation may help regulate local melatonin concentration, and generates other biologically active methoxyindoles.

Circadian regulation of melatonin in the retina of Xenopus laevis: limitation by serotonin availability.

Treatments expected to increase retinal serotonin levels were found to stimulate melatonin production by cultured eyecups from Xenopus laevis. The monoamine oxidase inhibitor pargyline (100 microM) caused a sixfold increase in melatonin release, and the serotonin precursor 5-hydroxy-L-tryptophan (100 microM) caused a 70-fold increase. Both acted synergistically with eserine, an inhibitor of melatonin deacetylation in the retina. The effect of 5-hydroxytryptophan was dose dependent, with effects increasing from 1 to 100 microM. Increasing the tryptophan level in the culture medium had no effect on melatonin release. These results indicate that the rate-limiting step in retinal melatonin synthesis is 5-hydroxylation of tryptophan. Melatonin released from individual eyecups in superfusion culture in constant darkness with and without added 5-hydroxy-L-tryptophan was monitored over a 5-day period. Control eyecups released low levels of melatonin, with circadian rhythmicity persisting for 1-3 days. With 5-hydroxy-L-tryptophan added, melatonin levels were elevated 10-20-fold at all times, and rhythmicity was apparent for as long as five cycles. This provides a model system for studies of the circadian clock in the eye.