Melatonin Biosynthetic Pathway Research Articles

Pineal arylalkylamine N-acetyltransferase (Aanat) gene expression as a target of inflammatory mediators in the chicken

Previously, we demonstrated that experimental peritonitis in chickens was attenuated by treatment with exogenous melatonin, while the developing inflammation decreased pineal AANAT activity. This suggested the existence of a bidirectional relationship between the activated immune system and pineal gland function. The aim of the present study was to identify the step(s) in the chicken pineal melatonin biosynthetic pathway that are affected by inflammation. Peritonitis was evoked by i.p. injection of thioglycollate solution, either 2h after the start, or 2h before the end of the light period, and the animals were sacrificed 4h later. The effect of inflammation on the expression of genes encoding enzymes participating in melatonin biosynthesis in the pineal gland, i.e. tryptophan hydroxylase 1 (Tph1), dopa decarboxylase (Ddc), arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), was evaluated by qPCR. The pineal and serum melatonin concentration as well as the content of its precursors in the pineal gland were measured, along with the activity of the relevant biosynthetic enzymes. Developing peritonitis caused an increase in the pineal levels of the Tph1 mRNA during the night and the Asmt mRNA during the day, while nocturnal Aanat transcription was reduced. Both the pineal and serum melatonin level and the pineal content of N-acetylserotonin (NAS) were decreased during the night in birds with peritonitis. The amount and activity of pineal AANAT were significantly reduced, while the activity of HIOMT was increased under these experimental conditions. These results indicate that the observed decrease in MEL biosynthesis in chickens with developing inflammation is a result of transcriptional downregulation of the Aanat gene, followed by reduced synthesis and activity of the encoded enzyme.

Comparative evaluation of the antioxidant activity of melatonin and related indoles

Recent scientific evidence suggests that melatonin is present in foods and beverages. Therefore, its antioxidant activity needs to be evaluated and compared with that of other bioactive compounds such as polyphenols. This paper presents a systematic study of the antioxidant activity of melatonin and other compounds involved in the melatonin biosynthetic pathway and chemically related substances. The methods selected were the ferric reducing antioxidant power (FRAP) assay, the oxygen radical absorbance capacity (ORAC) assay and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The reactions depend on the mechanisms involved (electron transfer or hydrogen atom transfer). The kinetic parameters (stoichiometric factors, rate constants) and antioxidant activity show considerable differences within the indole compounds: some compounds do not react at all, melatonin presents a discrete in vitro antioxidant activity while the highest values are shown by 5-hydroxy compounds. These compounds have a clear relation between structure and activity when an electron transfer mechanism is involved (FRAP and DPPH assays). Melatonin and indole-3-acetic acid react better through the hydrogen atom transfer mechanism. The radical scavenging ability towards the DPPH was studied and EC50, TEC50 and antioxidant efficacy (AE) were calculated: the 5-hydroxy-indoles present values that are similar to those of ascorbic acid or kaempferol.

Daily Oscillation in Melatonin Synthesis in The Turkey Pineal Gland and Retina: Diurnal and Circadian Rhythms

The aim of the present study was to examine arylalkylamine N‐acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light‐dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night‐time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high‐amplitude melatonin rhythms in the turkey.

Diurnal and circadian rhythms in melatonin synthesis in the turkey pineal gland and retina

The pineal gland and retina of the turkey rhythmically produce melatonin. In birds kept under a daily light–dark (LD) illumination cycle melatonin concentrations in the pineal gland and retina were low during the light phase and high during the dark phase. A similar melatonin rhythm with high night-time values was also observed in the plasma. The pineal and retinal melatonin rhythms mirror oscillations in the activity of serotonin N-acetyltransferase (AANAT; the penultimate enzyme in the melatonin biosynthetic pathway). In contrast, in both the pineal gland and retina the activity of the enzyme hydroxyindole- O-methyltransferase (HIOMT) did not exhibit significant changes throughout the 24-h period. Acute exposure of turkeys to light at night dramatically decreased melatonin levels in the pineal gland, retina and plasma. The rhythms in AANAT activity and melatonin concentrations in the turkey pineal gland and retina were circadian in nature as they persisted under conditions of constant darkness (DD). Under DD, however, the amplitudes of AANAT and melatonin rhythms were significantly lower (by 50–80%) than those found under the LD cycle. The findings indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The rapid dampening of the rhythms under DD suggests that of these two regulatory factors, environmental light may be the primary stimulus in the maintenance of the high amplitude melatonin rhythms in the turkey.

UV‐A light regulation of arylalkylamine N‐acetyltransferase activity in the chick pineal gland: role of cAMP and proteasomal proteolysis

Acute exposure of dark-adapted, cultured chick pineal glands to UV-A light significantly decreased the tissue cAMP concentration and the activity of arylalkylamine N-acetyltransferase (AANAT), the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. The magnitude of these changes was dependent on the duration of UV-A exposure. The UV-A light-evoked decline in pineal AANAT activity was blocked by cAMP protagonists (forskolin and dibutyryl-cAMP) and by inhibitors of the proteasomal degradation pathway (MG-132, proteasome inhibitor I, and lactacystin). These results indicate that the chick pineal gland is directly sensitive to UV-A light. By analogy to white light, the suppressive action of UV-A radiation on AANAT activity in the chick pineal gland involves changes in the tissue cAMP level and enhanced proteasomal proteolysis.

Diurnal rhythms of tryptophan hydroxylase 1 and 2 mRNA expression in the rat retina.

Tryptophan hydroxylase is the first of four enzymes in the melatonin biosynthetic pathway. Recent studies have shown that there are two genes, Tph1 and Tph2, that encode tryptophan hydroxylase in mammals. In this study, we investigated which of the two genes is expressed in the rat retina. To that end, we measured Tph1 (classical Tph) and Tph2 mRNA levels using real-time quantitative RT-PCR in the retina. Our data demonstrate that Tph1 mRNA is the prevalent form expressed in the retina; Tph2 mRNA is also present but the level is very low. We also measured Tph1 expression levels in the outer nuclear layer, inner nuclear layer, and ganglion cell layer by combining laser capture microdissection and real-time RT-PCR. Tph1 mRNA is more abundant in the photoreceptors of the outer nuclear layer than in the inner nuclear layer or ganglion cell layer. Tph1 and Tph2 transcripts showed robust diurnal rhythms of abundance, with highest levels at night. Our results support the hypothesis that Tph1 is involved in melatonin synthesis in retinal photoreceptor cells.

Retinal illumination phase shifts the circadian rhythm of serotonin N-acetyltransferase activity in the chicken pineal gland

The pineal gland of birds, in contrast to its mammalian counterpart, is a directly photosensitive organ. It has recently been demonstrated that light also acting via the retina acutely suppresses melatonin synthesis in the chicken pineal gland. The present study was aimed to investigate whether retinal illumination alone was capable of resetting the biological oscillator generating the circadian rhythm of pineal serotonin N-acetyltransferase (AA-NAT) activity in the chicken. Ocular exposure of chickens to 6 h low intensity white light (4 lux) potently suppressed AA-NAT activity (the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway) in the pineal gland. In addition, this light pulse produced phase-dependent shifts in the circadian AA-NAT rhythm. Exposure to light early in the subjective night (circadian time (CT) 12–18) caused a phase delay in the circadian rhythm of pineal AA-NAT activity by 3.5±0.4 h compared to non-exposed controls. When the light pulse was applied during the second half of the subjective night (CT18–24), it produced a large phase advance of the circadian rhythm of pineal AA-NAT activity by 10.9±0.4 h. The advancing effect of light was more pronounced than the phase-delaying effect. Our results suggest that in the chicken retinally perceived light provides a powerful and important signal for synchronization of circadian rhythmicity in the pineal gland.

Localization of Aa-nat mRNA in the rat retina by fluorescence in situ hybridization and laser capture microdissection

Arylalkylamine N-acetyltransferase (AA-NAT) is the key regulatory enzyme in the melatonin biosynthetic pathway. Previous investigations have reported that Aa-nat mRNA in rat is only detected in a sub-population of photoreceptor cells that resemble cones in shape and size. In the present study, we investigated Aa-nat expression in the rat retina by using in situ hybridization and laser capture microdissection combined with the reverse transcription/polymerase chain reaction technique. Our results demonstrate that, contrary to previous reports, Aa-nat transcripts are present not only in the photoreceptor cells, but also in the inner nuclear layer and in the ganglion cell layer. However, the rhythmic expression of Aa-nat mRNA was observed only in photoreceptor cells.

Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells

Arylalkylamine N-acetyltransferase (AANAT) is the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. In chicken retina in vivo, AANAT is expressed in a circadian fashion, primarily in photoreceptor cells. AANAT activity is high at night in darkness, low during the daytime, and suppressed by light exposure at night. In the present study, we investigated the circadian and photic regulation of adenosine 3′,5′-monophosphate (cAMP) in cultured retinal cells entrained to a daily light–dark (LD) cycle, as well as the role of Ca 2+ and cAMP in the regulation of AANAT activity. Similar to AANAT activity, cAMP levels fluctuate in a daily fashion, with high levels at night in darkness and low levels during the day in light. This daily fluctuation continued with reduced amplitude in constant (24 h/day) darkness (DD). These changes in cAMP appear to be causally related to control of AANAT activity. Adenylyl cyclase and protein kinase A inhibitors suppress the noctural increase of AANAT in DD, while 8Br-cAMP augments it. The nocturnal increase of AANAT activity also involves Ca 2+ influx, as it is inhibited by nitrendipine, an inhibitor of L-type voltage-gated channels, and augmented by Bay K 8644, a Ca 2+ channel agonist. The effect of Bay K 8644 was antagonized by the adenylyl cyclase inhibitor MDL 12330A, suggesting a link between Ca 2+ influx, cAMP formation, and AANAT activity in retinal cells. Light exposure at night, which rapidly suppresses AANAT activity, also suppressed cAMP levels. The effect of light on AANAT activity was reversed by Bay K 8644, 8Br-cAMP, and the proteasome inhibitor lactacystin. These results indicate a dynamic interplay of circadian oscillators and light in the regulation of cAMP levels and AANAT activity in photoreceptor cells.

Daily variation in the concentration of melatonin and 5-methoxytryptophol in the goose pineal gland, retina, and plasma

The goose pineal gland rhythmically produces two 5-methoxyindole compounds, namely melatonin and 5-methoxytryptophol. Melatonin concentrations were high at night and low during the day, while in contrast 5-methoxytryptophol levels were markedly higher during the day compared to the night-time values. Rhythmic oscillations in melatonin content, with high night-time values, have also been found in plasma and the retina of goose. The pineal and retinal melatonin rhythm mirrored oscillations in the activity of serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway). Acute exposure of geese to light at night markedly decreased melatonin levels in the pineal, plasma, and retina. In addition, this light exposure resulted in a significant increase in pineal 5-methoxytryptophol content. Our results demonstrate, for the first time, the ability of the goose pineal gland and retina to synthesise melatonin and 5-methoxytryptophol in a rhythmic manner.

Induction of photosensitivity in cultured rat pineal affects Aa-nat regulation

In a previous investigation we have demonstrated that neonatal rat pineal may become photosensitive if removed from the animal at post-natal day 1 and cultured for 7 days. In the present study we demonstrated that Aa-nat mRNA, the key enzyme in melatonin biosynthetic pathway, is affected by illumination in photosensitive pineal, and Aa-nat transcription in photosensitive pineal, but not in adult or non photosensitive pineals, is up-regulated by BMAL1:CLOCK.

Photic and circadian regulation of retinal melatonin in mammals.

Several studies have established that melatonin synthesis occurs in the retina of vertebrates, including mammals. In mammals, a subpopulation of photoreceptors (probably the cones) synthesize melatonin. Melatonin synthesis in the retina is elevated at night and reduced during the day in a fashion similar to events in the pineal gland. Both the MT1 and MT2 melatonin receptors are present in the retina and retinal melatonin does not contribute to circulating levels, suggesting that retinal melatonin acts locally as a neurohormone and/or neuromodulator. Melatonin synthesis in the retina of mammals is under the control of a circadian oscillator, and circadian rhythms in melatonin synthesis and/or release have been described for several species of mammals. These rhythms are present in vivo, persist in vitro, are entrained by light and are temperature compensated. The cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the key enzyme in the melatonin biosynthetic pathway) has allowed studies of the molecular mechanisms responsible for the generation of retinal melatonin rhythmicity. The present review focuses on the cellular and molecular mechanisms that regulate melatonin synthesis. In particular, we discuss how the photic environment and the circadian clock interact in determining melatonin levels, in addition to the role that melatonin plays in retinal physiology.

Daily variation in the concentration of 5-methoxytryptophol and melatonin in the duck pineal gland and plasma.

The duck pineal gland rhythmically produces two 5-methoxyindole compounds, i.e. 5-methoxytryptophol and melatonin. 5-Methoxytryptophol levels are low at night and high during the day, while melatonin concentrations are high at night and low during the day. The melatonin rhythm reflects oscillations in the activity of serotonin N-acetyltransferase (AA-NAT; a penultimate and key regulatory enzyme in the melatonin biosynthetic pathway). The activity of hydroxyindole-O-methyltransferase (HIOMT; an enzyme involved in the synthesis of both 5-methoxytryptophol and melatonin) does not exhibit any significant rhythmic changes throughout the 24-hr period. Plasma levels of melatonin exhibited daily changes that were parallel to fluctuations in pineal melatonin content. Although plasma concentrations of 5-methoxytryptophol were low in ducks, they showed daily variations. The mean 5-methoxytryptophol concentration between zeitgeber time 9 (ZT9) and ZT15 was 2.4-times higher than the mean value for samples collected between ZT18 and ZT3. These findings indicate that in the duck the pineal production of 5-methoxytryptophol and melatonin may be inversely correlated.

Dopamine Mediates Circadian Rhythms of Rod–Cone Dominance in the Japanese Quail Retina

A circadian clock modulates the functional organization of the Japanese quail retina. Under conditions of constant darkness, rods dominate electroretinogram (ERG) b-wave responses at night, and cones dominate them during the day, yielding a circadian rhythm in retinal sensitivity and rod-cone dominance. The activity of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, also exhibits a circadian rhythm in the retina with approximately threefold higher levels during the day than at night. The rhythm of tyrosine hydroxylase activity is opposite in phase to the circadian activity of tryptophan hydroxylase, the first enzyme in the melatonin biosynthetic pathway. We tested whether dopamine may be related to the physiological rhythms of the retina by examining the actions of pharmacological agents that effect dopamine receptors. We found that blocking dopamine D2 receptors in the retina during the day mimics the nighttime state by increasing the amplitude of the b-wave and shifting the retina to rod dominance. Conversely, activating D2 receptors at night mimics the daytime state by decreasing the amplitude of the b-wave and shifting the retina to cone dominance. A selective antagonist for D1 dopamine receptors has no effect on retinal sensitivity or rod-cone dominance. Reducing retinal dopamine partially abolishes rhythms in sensitivity and yields a rod-dominated retina regardless of the time of day. These results suggest that dopamine, under the control of a circadian oscillator, has a key role in modulating sensitivity and rod-cone dominance in the Japanese quail retina.

Elevation of melatonin in chicken retina by 5-hydroxytryptophan: differential light/dark responses.

Melatonin is synthesized in the chicken retina under the influence of a circadian clock, which also regulates the expression of tryptophan hydroxylase (TPH) and serotonin N-acetyltransferase (AA-NAT). In order to examine the role of substrate supply in the rhythmic synthesis of melatonin in chicken retina, tryptophan and 5-hydroxytryptophan were administered day and night in light or darkness. When administered systemically at night in darkness, 5-hydroxytryptophan, but not tryptophan, dramatically stimulates melatonin levels in the chick retina in a dose-dependent manner. Intraocular administration of 5-hydroxytryptophan also increases melatonin levels locally, indicating a retinal site of action of the serotonin precursor. The effect of 5-hydroxytryptophan is much greater at night, when TPH and AA-NAT activities are high, than during the day, when the enzyme activities are low. Similarly, unexpected light exposure at night, which inactivates AA-NAT, significantly reduces the ability of 5-hydroxytryptophan to increase retinal melatonin levels. The results suggest that TPH, but not AA-NAT or other enzymes in the melatonin biosynthetic pathway, is saturated with substrate in situ. The rate of melatonin production appears to be a function of the concentration of serotonin, which is regulated by TPH, and by the level of activity of AA-NAT.

Hydroxyindole- O-methyltransferase activity in the silkworm ( Bombyx mori)

Hydroxyindole- O-methyltransferase (HIOMT; EC 2.1.1.4) catalyzes the final step in the melatonin ( N-acetyl-5-methoxytryptamine) biosynthetic pathway. HIOMT-like activity was detected in the head of fifth last-instar larvae of the silkworm ( Bombyx mori), and the optimum pH for this activity was 7.9. The apparent Michaelis constants ( K m) for S-adenosyl- l-methionine and N-acetylserotonin were 87.6 μM and 96.6 μM, respectively. When the silkworms were entrained to a 12 h light/12 h dark lighting schedule, the HIOMT-like activity showed a significant diurnal variation with high levels during the dark period. The diurnal variation in the activity persisted in constant darkness, but was suppressed by constant light. These findings demonstrate that HIOMT-like activity in the silkworm head occurs as a circadian rhythm and that the rhythm entrains to environmental light/dark cycles. The melatonin rhythm in the silkworm head may therefore be regulated not only by serotonin N-acetyltransferase (EC 2.3.1.87), but also by HIOMT.

Hydroxyindole- O-methyltransferase activity assay using high-performance liquid chromatography with fluorometric detection: determination of melatonin enzymatically formed from N-acetylserotonin and S-adenosyl- l-methionine

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole- O-methyltransferase (HIOMT; S-adenosyl- l-methionine: N-acetylserotonin- O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin ( N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyl- l-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.

Clozapine and other neuroleptic drugs antagonize the light-evoked suppression of melatonin biosynthesis in chick retina: involvement of the D4-like dopamine receptor.

The subtype of dopamine receptor mediating the suppressive effect of light on melatonin biosynthesis in chick retina was characterized pharmacologically. Acute exposure of animals to light during the dark phase of the light-dark cycle dramatically decreased melatonin levels and activity of serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthetic pathway). Various antagonists of dopamine receptors were tested for their ability to block this action of light on the retinal melatonin formation. Intraocular (i. oc.) pretreatment of chicks with neuroleptic drugs--blockers of the D2-family of dopamine receptors, i.e., clotiapine, clozapine (an atypical neuroleptic with high affinity for a D4-subtype dopamine receptor), haloperidol, spiroperidol, sulpiride, and YM-09151-2, significantly antagonized the light-evoked suppression of the nighttime NAT activity of the chick retina in a dose-dependent manner. In contrast, remoxipride (a D2-selective dopamine antagonist), raclopride and (+)-UH-232 (D2/D3-dopamine receptor antagonists), as well as SCH 23390, a blocker of the D1-family of dopamine receptors, were ineffective. Clozapine, haloperidol, spiroperidol and sulpiride also potently antagonized the suppressive action of light on melatonin content of the chick retina. It is suggested that the dopamine receptor mediating the inhibitory effect of light stimulation on the nighttime melatonin biosynthesis in the retina of chick represents a D4-like subtype.

Tryptophan Hydroxylase Expression Is Regulated by a Circadian Clock in Xenopus laevis Retina

A circadian clock has been localized to the photoreceptor layer in the Xenopus laevis retina. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin synthesis in Xenopus laevis retina is tryptophan hydroxylase. A cDNA clone coding for Xenopus tryptophan hydroxylase was isolated, characterized, and used as a probe for analysis of tryptophan hydroxylase mRNA expression. Northern blot analyses of total retinal RNA show that the tryptophan hydroxylase message levels are low in the day and higher at night. The expression of tryptophan hydroxylase mRNA is under circadian control because rhythmic changes are also seen in constant darkness, with elevated levels during the subjective night. Nuclear run-on analysis during the first subjective day in constant darkness revealed that transcription initiation is low early in the day and increases throughout the day. Our observations suggest that the circadian clock modulates tryptophan hydroxylase gene expression. An understanding of how the circadian clock controls tryptophan hydroxylase expression may lead to a clearer understanding of the melatonin biosynthetic pathway, and possibly the clock itself.

Immunocytochemical localisation of hydroxyindole‐O‐methyltransferase in pineal photoreceptor cells of several fish species

Melatonin is an internal "Zeitgeber," involved in the timing and control of a number of rhythmic functions and behaviours. Its synthesising cells remain to be identified in the fish pineal. The last step in the melatonin biosynthetic pathway is catalysed by the enzyme hydroxyindole-O-methyltransferase. An affinity-purified antibody, directed against chicken pineal hydroxyindole-O-methyltransferase, was used in the present study to identify the melatonin synthesising cells in four fish species: a primitive chondrostean (sturgeon), a saltwater teleost (dorado), and two freshwater teleosts (pike, trout). Western blot immunolabeling of pike and trout pineal proteins revealed a single band at 38 KDa, which corresponds to the known molecular weight of the enzyme in bovine, rat, and chicken pineal. Regardless of the species, a specific immunocytochemical labeling, visualised by means of the peroxidase-antiperoxidase method, was exclusively associated with the photoreceptor cells. These results provide evidence that photoreceptors of the fish pineal are responsible for the biosynthesis of 5-methoxyindoles, including melatonin. In the pike, reactions were less intense in the distal portion of the pineal vesicle than in the other regions of the organ. It is questioned whether this might be related to the existence of a germinative zone, generating new photoreceptor cells in this distal portion. Hydroxyindole-O-methyltransferase has been previously demonstrated in mammalian pinealocytes, and modified photoreceptors of the avian pineal. It is now demonstrated in pineal photoreceptors of a primitive fish and of more evolved saltwater and freshwater fish. The results strengthen the view that these cells are related through phylogeny and that their well conserved melatoninergic function appears early in the course of evolution.