Ocular Melatonin Levels Research Articles

Immune-pineal-ocular axis in amphibians: unveiling a novel connection?

Melatonin is a hormone known as an endogenous temporal marker signaling the dark phase of the day. Although the eyes seem to be the main site of melatonin production in amphibians, little information is available about the natural variation in the ocular melatonin levels and its modulation following immune stimulation. We investigated the daily variation of plasma and ocular melatonin levels in bullfrogs (Lithobates catesbeianus) and their modulation following an immune stimulation with lipopolysaccharide (LPS) in yellow cururu toads (Rhinella icterica). For the daily variation, bullfrogs were bled and then euthanized for eye collection every 3h over 24h to determine plasma and ocular melatonin levels. We found a positive correlation between ocular and plasma melatonin levels, with maximum values at night (22h) for both plasma and the eyes. For immune stimulation, yellow cururu toads received an intraperitoneal injection of LPS or saline solution during the day (10h) or at night (22h). Two hours after injection, toads were bled and euthanized for eye collection to obtain plasma and ocular melatonin levels. In addition, the liver and bone marrow were collected to investigate local melatonin modulation. Our results demonstrate that retina light-controlled rhythmic melatonin production is suppressed while liver and bone marrow melatonin levels increase during the inflammatory assemblage in anurans. Interestingly, the LPS injection decreased only ocular melatonin levels, reinforcing the central role of the eyes (i.e., retina) as an essential organ of melatonin production, and a similar role to the pineal gland during the inflammatory response in amphibians. Together, these results point to a possible immune-pineal-ocular axis in amphibians, yet to be fully described in this group.

Annual patterns of ocular melatonin level in the female grass puffer, Takifugu alboplumbeus: possible involvement in seasonal reproductive response.

The aim of this study was to investigate the expression patterns of ocular melatonin in the annual reproductive cycle of the female grass puffer. Spawning season of the female grass puffer is from June to July in Jeju, South Korea. Time-resolved fluoroimmunoassay revealed that levels of ocular melatonin, which show an annual change, peaked in May (spawning season). Additionally, expression of reproductive-related genes also showed annual patterns: GnRH1 peaked in August, GnRH2 peaked in February, GnRH3, Kiss2, and LPXRFa peaked in November. These results suggest that ocular melatonin may be related to the annual reproductive cycle in the grass puffer. To better understand the photic regulation of AANAT1a mRNA in the retina, we observed the nocturnal pattern of ocular melatonin levels daily, which shows a nocturnal pattern in both short photoperiod (SD) and long photoperiod (LD) conditions. In the brain, AANAT2 mRNA also shows a nocturnal pattern in both SD and LD; however, the time of peak expression of AANAT2 mRNA was unchanged in both conditions. Following intraperitoneal injection of melatonin for 2weeks, expression of GnRH2 and LPXRFa mRNA in the brain significantly increased, while that of Kiss2 mRNA was decreased, suggesting that melatonin has a reproduction-related effect. Furthermore, under SD and LD conditions for 14weeks, the gonadosomatic index more increased and the maturity of the ovary progressed under LD compared with those under SD, suggesting that the SD photoperiodic signal inactivated ovarian development. These results indicate that the ocular melatonin may have a possible role in the reproductive endocrinology of the grass puffer.

Ocular Melatonin Rhythms in Teleost Fish

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized in the pineal organ and the retina of vertebrates. In some teleost species, ocular melatonin levels can exhibit a circadian periodicity with elevated levels during the dark phase under light-dark (LD) cycles and this periodicity can persist even under constant dark (DD) cycles. However, reversed melatonin profiles and an absence of circadian ocular melatonin rhythms have also been reported. In this study, we investigated the daily rhythms of ocular melatonin in 32 teleost species under LD cycles. The melatonin profiles could be classified into three types: (1) normal profiles, with higher melatonin levels during the dark phase than the light phase; (2) reversed profiles, with higher levels during the light phase than the dark phase; (3) no significant differences in melatonin levels. We also studied whether ocular melatonin exhibits circadian rhythms under DD in selected species. Our results showed that ocular melatonin exhibited circadian rhythms in some but not all of the species examined. These results indicate that ocular melatonin rhythms in teleost fish exhibit species-specific variations as a result of the changes in the regulatory mechanisms during the course of evolution.

Circadian rhythms of ocular melatonin in the wrasse Halichoeres tenuispinnis, a labrid teleost

Using in vivo and in vitro methods we studied the regulation of ocular melatonin rhythms in the wrasse Halichoeres tenuispinnis, by either light or the circadian clock. Rhythmic changes in ocular melatonin levels under light–dark (LD) cycles were persistent under constant darkness (DD), and had a circadian periodicity of approximately 24 h. However, ocular melatonin levels remained low under constant light conditions. When wrasse were exposed to a single 6-h light pulse at three different circadian phases under DD, phase-dependent phase shifts in the circadian rhythms of ocular melatonin were observed. When eyecups were prepared during mid-light periods or at the onset of darkness, and incubated in vitro in either light or dark periods, both time and light conditions affected melatonin release. These results indicate that the melatonin rhythms in the wrasse eye are driven by an ocular circadian clock that is entrained to LD cycles via local photoreceptors.

Influence of light-dark and lunar cycles on the ocular melatonin rhythms in the seagrass rabbitfish, a lunar-synchronized spawner.

The aim of this study was to investigate the effects of light-dark (LD) cycles and lunar phases on ocular melatonin rhythms in the seagrass rabbitfish, Siganus canaliculatus, a lunar-synchronized spawner. Under a natural 24-hr LD (12.00:12.00) cycle, ocular melatonin levels were low during daylight hours. The levels significantly elevated to peak during the mid-dark phase at 24.00 hr and then declined sharply in the early morning around 06.00 hr. These rhythms disappeared under either constant light (LL) or constant dark (DD) conditions. Melatonin levels remained low in LL compared with those in DD condition. These results suggest that ocular melatonin rhythms in the seagrass rabbitfish are suppressed in the presence of light. When fish were exposed to natural moon phases, ocular melatonin concentrations were higher around the new moon than both the first quarter and full moon phases. Exposure to experimental new moon conditions caused a significant increase in melatonin levels while those of the fish exposed to experimental full moon conditions were decreased. These results suggest that the seagrass rabbitfish perceives moonlight through the eye and that moonlight directly causes melatonin suppression.

Daily locomotor activity and melatonin rhythms in Senegal sole ( Solea senegalensis)

The daily locomotor and melatonin rhythms of the Senegal sole, a benthonic species of increasing interest in aquaculture, are still unknown, despite the fact that such knowledge is of prime importance for optimising its production. The aim of the present research was therefore to investigate the daily rhythms of locomotor activity and melatonin in the Senegal sole. For this purpose, the individual locomotor activity rhythms of fish were registered using a photocell. Plasma and ocular melatonin rhythms were studied in animals reared in circular tanks placed in earth under an LD 12:12 light regime and 16–18 °C temperature range (spring equinox). Blood and eye samples were taken every 3 h during a complete 24-h cycle. The impact of a light pulse in the middle of the dark period (MD) on plasma melatonin was also studied. Locomotor activity was mainly nocturnal, with 84.3% of the total activity occurring during darkness. The levels of plasma melatonin were higher at night (55 pg/ml) than during the day (2 pg/ml), while ocular melatonin levels appeared to be arrhythmic. Both weight and melatonin content were found to be significantly higher in the left eye in relation to the right eye. A light pulse in MD provoked a significant decrease in plasma melatonin levels. In summary, photoperiod is a key factor in synchronising locomotor activity and melatonin rhythms in the Senegal sole, whose nocturnal habits should be taken into account for their rearing by aquaculture.

Developmental and diel changes in plasma thyroxine and plasma and ocular melatonin in the larval and juvenile bullfrog, Rana catesbeiana

Diel variation in plasma thyroxine (T 4), and plasma and ocular melatonin was studied in Rana catesbeiana tadpoles and postmetamorphic froglets on 12:12 and 6:18 light/dark (LD) regimens. A progressive rise in plasma T 4 initiates metamorphosis while melatonin can modulate metamorphic progress. Changes in the phase of the rhythms of these two hormones during development might influence the hormonal regulation of metamorphosis. The hormones studied exhibited LD cycle-specific diel fluctuations except in froglet plasma T 4 and all hormones at prometamorphosis on 6L:18D. On 12L:12D, plasma T 4 and ocular melatonin peaked during the scotophase at prometamorphosis and early climax, whereas the plasma melatonin acrophase shifted from the light to the dark at climax. A nocturnal peak of plasma melatonin closely correlated with the onset and offset of dark appeared in the froglet, while the peak of ocular melatonin shifted to the light. Compared to 12L:12D, the peaks of the diel fluctuations on 6L:18D occurred later than on 12L:12D in synchrony with an earlier onset, and increase in length, of the scotophase. The phase of the hormone rhythms changed during metamorphosis in such a way that the peaks of melatonin had a different relationship to the T 4 peaks as development proceeded. On both LD cycles, the 24-h mean of plasma T 4 rose at climax and fell in the froglet whereas plasma melatonin decreased at climax and then rose to a high level in the froglet. After only minor changes during metamorphosis, froglet ocular melatonin levels decreased on 12L:12D and increased on 6L:18D. The findings indicate that the hormonal flux during metamorphosis has circadian aspects, which might explain variations in the response to exogenous hormone treatment at different times of the day and LD cycle-specific timing of development. A fall in plasma melatonin at climax appears to be as much a part of the hormonal changes of metamorphosis as a rise in plasma T 4.

Influence of Cortisol on the Larval Bullfrog Thyroid Axis in Vitro and in Vivo and on Plasma and Ocular Melatonin

Adrenal (interrenal) steroids have an important role in amphibian development, antagonizing the metamorphic changes induced by the thyroid at first and then synergizing with the thyroid hormones as their level rises during metamorphosis. Because most of the studies of corticoids at metamorphosis have focused on peripheral tissues, we investigated the effect of cortisol (hydrocortisone; HC) in vitro and in vivo on the thyroid of Rana catesbeiana (bullfrog) tadpoles on 12:12 light/dark (LD) cycles. Plasma and ocular melatonin, which is altered by changes in thyroxine (T4) levels, were also assayed in some experiments. Thyroids from premetamorphic tadpoles secreted less T4 into culture media when incubated with 10 μg/ml HC and 0.2 μg/ml ovine thyrotropin (TSH) than with TSH alone and when cultured in the absence of TSH following 5 days of 10-μg HC injections, indicating that HC inhibited the thyroid at young stages. The effect of 10 μg/ml HC at older stages was investigated by culturing thyroids and pituitaries separately on the first day in control or HC media and then incubating the thyroids on the second day in homologous pituitary-conditioned media as a bioassay for pituitary TSH. HC had no effect on baseline T4 secretion by the thyroids of prometamorphic or climax tadpoles on the first day but increased T4 secretion over the control on the second day. Thyroids cultured with TSH and HC showed no increase in T4 secretion over the control TSH group on the second day, indicating that, in the previous experiments, HC had enhanced pituitary secretion of TSH, rather than the response of the thyroid to TSH. In vivo, 5 days of injections of 10 μg HC increased plasma T4 at prometamorphosis and decreased it at climax. There was no marked effect of HC on plasma or ocular melatonin levels. The findings showed that the nature of the effect of HC on the thyroid axis changes during metamorphosis from inhibition at early stages to a positive influence at prometamorphosis and finally to a negative effect on the T4 level in the plasma at climax.

Unusual responses to light and darkness of ocular melatonin in European sea bass.

Regulation by light and darkness of melatonin rhythms in the plasma and eye of the European sea bass (Dicentrarchus labrax) was studied. During light-dark cycles, plasma and ocular melatonin exhibited day-night changes with higher levels at mid-dark and at mid-light, respectively. Circulating melatonin levels were low in constant light but high in constant darkness (DD); ocular melatonin levels showed the reverse pattern. Plasma melatonin exhibited circadian rhythm for 1 cycle but the rhythm was no longer apparent on day 2. There was no circadian rhythm in ocular melatonin. Acute light exposure in DD decreased plasma melatonin but increased ocular melatonin. These results suggest that circulating melatonin may be used as a signal for darkness but ocular melatonin is used as a signal for the light phase.

In vivo microdialysis studies of pineal and ocular melatonin rhythms in birds.

Pineal and retinal melatonin has an important role in the control of avian circadian rhythms. In order to study the mechanisms of circadian rhythms of melatonin synthesis in the pineal and in the eye, in vivo microdialysis was applied to these organs. In both pigeons and Japanese quails, pineal and ocular melatonin levels were high during the dark and low during the day under light-dark (LD) cycles. These rhythms persisted under constant dim light (LLdim) conditions indicating the circadian nature of pineal and ocular melatonin release. Light has two effects on melatonin synthesis. One is acute inhibition of melatonin synthesis and the other is entrainment of circadian melatonin rhythms. We have examined photoreceptors mediating these effects in the pigeon. The results have indicated that the eyes are not involved in light-induced suppression and photic entrainment of pineal melatonin release, and pineal photoreceptors themselves are likely to mediate these effects. Concerning ocular melatonin, retinal photoreceptors seem to mediate light-induced suppression and photic entrainment and no evidence supporting mediation of extraretinal photoreceptors was obtained. Because dopamine is implicated in retinal melatonin synthesis, we measured dopamine and melatonin release simultaneously from the eye of pigeon. In contrast to melatonin rhythms, dopamine increased during the day and decreased during the dark. This antiphase relationship between melatonin and dopamine persisted in LLdim, suggesting an interaction between these two rhythms. The results of an intraocular injection of dopamine or melatonin in the phase of melatonin and dopamine rhythms indicated that the interaction is required for maintaining the antiphase relationship between the two rhythms.

Photic and circadian regulations of melatonin rhythms in fishes.

Photic and circadian regulations of melatonin rhythms in the pineal organ and the retina of several teleosts were studied to investigate the regulatory mechanisms of melatonin rhythms in fishes. In the eyecup preparations of the goldfish, Carassius auratus, both time of day and lighting conditions affected melatonin production, with high melatonin production observed only in the dark-treated group incubated during the 'subjective' night. Thus, in the goldfish retina, local photoreceptors and an ocular circadian clock seem to regulate melatonin production, as in the zebrafish retina and in the pineal organ of a number of teleosts, including the goldfish. However, this circadian regulation of melatonin rhythms is not universal among fishes. Although the superfused pineal organ of the masu salmon Oncorhynchus masou secreted melatonin in a rhythmic fashion under light-dark (LD) cycles, the rhythm disappeared under constant darkness (DD), as in the rainbow trout, with a large amount of melatonin released both during the subjective day and the subjective night. These results suggest that all salmonids lack circadian regulation of melatonin rhythms. Furthermore, when ocular melatonin rhythms were compared in two cyprinids, the ugui Tribolodon hakonensis and the oikawa Zacco platypus occupying different ecological niches, ocular melatonin contents exhibited daily variations, with higher values during the dark phase of LD cycles in both species. The rhythmic changes persisted in the ugui under DD, with higher levels at subjective midnight than at subjective midday; however, ocular melatonin levels in the oikawa were consistently high under DD. Thus, the circadian regulation of melatonin rhythms in fishes is influenced not only by phylogeny, but also by the ecological niches of the animals. These results suggest that the physiological functions of melatonin in the circadian and photoperiodic systems differ among fishes.

Effects of melatonin and haloperidol given via vortex vein on the intraocular pressure.

Melatonin injected into the vortex vein of a rabbit eye produced an increase in intraocular pressure (IOP) which lasted for up to 5 h. Injection of haloperidol caused a decrease in IOP; this effect was totally reversed by melatonin. It is probable that these effects are caused by physiological antagonisms. Injection of the mu opiate agonist (D-ala2-n-methyl-ph2-gly5-ol) enkephalin caused a decrease in the IOP of artificially ventilated rabbits and a decrease in melatonin levels in iris, iris root-ciliary body, and retina of the rabbit eye. Melatonin levels did not decrease after anesthesia with rompunketamine. It is probable that the decrease in melatonin levels is specific to certain classes of opioids, and endogenous opioids may play a role in the regulation of ocular melatonin levels and hence IOP.

Effect of environmental temperature and photoperiod on the melatonin levels in the pineal, lateral eye, and plasma of the frog, Rana perezi: Importance of ocular melatonin

Day-night melatonin concentrations were studied in the pineal body, lateral eye, and plasma of the frog Rana perezi in animals maintained in February and July under long (18L:6D) or short (6L:18D) photoperiod and high (25 ± 1°) or low (6 ± 1°) temperature in order to evaluate the influence of these environmental factors. When frogs were kept under short photoperiod and low temperature in February, no melatonin rhythm was observed in the pineal, ocular tissue, and plasma. High temperature at this period of the year induced a day-night rhythm of melatonin levels in the lateral eye and plasma. In July, under long photoperiod and high temperature, animals showed pronounced rhythms of melatonin in the pineal, eye, and plasma. A decrease of environmental temperature in this season abolished the melatonin rhythm. When animals were maintained in August under high (25 ± 1°) temperature and long (18L:6D) or short (6L:18D) photoperiod, the duration of high night time ocular melatonin levels was correlated to the length of the dark phase. In all experiments the high ocular melatonin concentrations and the close parallelism observed between ocular and circulating melatonin profiles suggest that in this species melatonin could be released from the eyes in the general circulation.

Melatonin rhythms in the eyes, pineal bodies, and blood of Japanese quail ( Coturnix coturnix japonica)

Melatonin levels in the eyes, pineal bodies, and blood of Japanese quail exposed to 12L:12D show robust daily rhythms with high levels occurring in the night and low levels occurring during the day. Since melatonin is synthesized in both the eyes and pineal bodies of birds, the relative contribution of these structures to the blood melatonin levels was determined. A rhythm of blood melatonin persisted in 12L:12D in birds blinded by complete orbital enucleation and in pinealectomized birds but the nighttime levels were reduced by 33 and 54%, respectively, as compared to melatonin levels in control quail. Only a small melatonin rhythm (13% of control levels) was detected in the blood of pinealectomized, blinded quail. This “residual” rhythm could indicate either the contribution of extrapineal, extraocular sources of melatonin or melatonin secretion from remnants (if any) of pineal body tissue remaining after pinealectomy. Blinding did not obviously affect pineal melatonin levels nor did pinealectomy affect ocular melatonin levels. It was concluded that (1) daily rhythms in melatonin content occur in the pineal bodies, the eyes, and the blood of quail; (2) the blood rhythm is the result of melatonin secretion from both the pineal body and the eyes; (3) extraretinal photoreceptors can mediate entrainment of the pineal melatonin rhythm; and (4) obvious compensatory changes in melatonin levels do not occur in the eye following pinealectomy or in the pineal body following blinding.