Indoleamine-Sensitive Adenylate Cyclase in Rabbit Retina: Characterization and Distribution

Previous histological, electrophysiological, and biochemical reports have addressed the hypothesis that serotonin functions as a neurotransmitter in mammalian retinas. We have tested the effect on the levels of cyclic AMP of the application of exogenous serotonin, 5-methoxytryptamine, melatonin, and 5-methoxydimethyl-tryptamine to isolated, incubated rabbit retinas. All indoleamines tested significantly elevated intracellular levels of cyclic AMP in both light- and dark-adapted, incubated, intact retinas, provided a phosphodiesterase inhibitor was present. In homogenates of rabbit retina, all indoleamines tested also markedly increased adenylate cyclase activity over basal levels. Maximal activity was observed with 50 microM indoleamine; addition of GTP augmented this increase. The increase in enzyme activity persisted in the presence of known antagonists of dopamine and serotonin 5-HT2-receptors, but was blocked by the mixed 5-HT1, 5-HT2-antagonist lysergic acid diethylamide. The retinal locations of this response have also been identified using layer microdissection techniques on freeze-dried samples obtained from rabbit eyecups suprafused with indoleamine plus phosphodiesterase inhibitor. Cyclic AMP levels were measured in discrete retinal layers of both light- and dark-adapted suprafused eyecups, and increased levels were observed primarily in the inner and outer plexiform layers, which contain the synapses of the retinal neurons.     

Tryptophan Availability Modulates Serotonin Release from Rat Hypothalamic Slices

Application of a novel in vitro experimental system has allowed us to describe the relationship between tryptophan availability and serotonin release from rat hypothalamic slices. Superfusing hypothalamic slices with a physiologic medium containing l-tryptophan (1, 2, 5, or 10 microM) caused dose-dependent elevations in tissue tryptophan levels; the magnitude of the elevations produced by supplementing the medium with less than 5 microM tryptophan was within the physiologic range for rat brain tryptophan levels. Slice serotonin levels rose biphasically as the tryptophan concentration in the medium was increased. Superfusing the slices with medium supplemented with a low tryptophan concentration (1 or 2 microM) caused proportionally greater incremental changes in serotonin levels than the increases caused by further elevating the tryptophan concentration (5 or 10 microM). The spontaneous release of serotonin from the slices exhibited a dose-dependent relationship with the tryptophan concentration of the superfusion medium. Electrically evoked serotonin release, which was calcium-dependent and tetrodotoxin-sensitive, also increased in proportion to the medium tryptophan concentration. These data suggest that the rate at which serotonin is released from hypothalamic nerve terminals is coupled to brain tryptophan levels. Accelerations in hypothalamic serotonin synthesis, caused by elevating brain tryptophan levels, result in proportionate increases in the rates of serotonin release during rest and with membrane depolarization.     

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.     

Melatonin and its generating system in vertebrate retina: circadian rhythm, effect of environmental lighting and interaction with dopamine

Retinas of rats, rabbits, chicks and carp possess enzymes, i.e. serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), which convert serotonin (5-HT) to melatonin, NAT activity and melatonin levels, but not HIOMT activity, show distinct circadian rhythms, with peak values occurring during the dark (night) phase of the 12 h light-dark cycle. Exposure of the animals to light at night inhibited the night-stimulated NAT activity. Treatment of rats and rabbits with the dopaminergic agonist, apomorphine, inhibited the retinal NAT activity. Dopamine levels in the rabbit retina showed diurnal variations, with higher contents seen during the light phase of both the 12 h light-dark cycle with lights on between 06:00–18:00, and that with reversed periods of illumination (lights on between 18:00–06:00). Melatonin potently inhibited the electrically-evoked calcium-dependent release of [³H]dopamine from pieces of retina from both albino and pigmented rabbits. Our results indicate that the light-regulated melatonin-generating system does operate in the vertebrate retina. The present data, together with other findings, suggest that in the retina there is an antagonistic interplay between melatonin and dopamine. Thus, melatonin inhibits dopamine synthesis in, and release from, the retinal dopaminergic cells, whilst dopamine inhibits the night (dark)-stimulated melatonin formation by decreasing NAT activity. Since light increases metabolic activity of the retinal dopaminergic cells (it enhances the amine synthesis, levels and release), it seems likely that the retinal dopamine plays a role of a “light” messenger in the inhibition of melatonin synthesis. It is suggested that an interplay between melatonin and dopamine in the retina is responsible for regulation of those retinal events which follow circadian rhythmicity, and/or are dependent on light-dark conditions.     

Serotonin synthesis and its light-dark variation in the rat retina

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.     

Interactions between dopamine and melatonin organize circadian rhythmicity in the retina of the green iguana

Circadian physiology in the vertebrate retina is regulated by several neurotransmitters. In the lateral eyes of the green iguana the circadian rhythm of melatonin content peaks during the night while the rhythm of dopamine peaks during the day. In the present work, the authors explore the interaction of these 2 neurotransmitters during the circadian cycle. They depleted retinal dopamine with intravitreal injections of 6-hydroxydopamine (6-OHDA) and measured ocular melatonin content in vivo throughout 1 circadian cycle. The circadian rhythm of ocular melatonin not only persisted but increased 10-fold in amplitude. This increase was substantially reduced by the intraocular administration of dopamine. 6-OHDA-treated retinas, unlike those from untreated animals, did not express a circadian rhythm of melatonin synthesis in vitro. To deplete retinal melatonin, the authors pinealectomized iguanas and blocked retinal melatonin synthesis by depleting serotonin with intraocular injections of 5,6-dihydroxytryptamine. In animals so treated, they found that the circadian rhythm of retinal dopamine content was abolished, the levels of dopamine were lowered, and the levels of dopamine metabolites were greatly increased. The data suggest that in iguanas, the amplitude of the circadian rhythm of melatonin synthesis in the eye is suppressed by dopamine while the rhythm of dopamine depends, at least in part, on the presence of melatonin.     

Retinal ganglion cells are autonomous circadian oscillators synthesizing N-acetylserotonin during the day

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.     

Circadian proteomics of the mouse retina

The circadian clock in the retina regulates a variety of physiological phenomena such as disc shedding and melatonin release. Although these events are critical for retinal functions, it is almost unknown how the circadian clock controls the physiological rhythmicity. To gain insight into the processes, we performed a proteomic analysis using 2-DE to find proteins whose levels show circadian changes. Among 415 retinal protein spots, 11 protein spots showed circadian rhythmicity in their intensities. We performed MALDI-TOF MS and NanoLC-MS/MS analyses and identified proteins contained in the 11 spots. The proteins were related to vesicular transport, calcium-binding, protein degradation, metabolism, RNA-binding, and protein foldings, suggesting the clock-regulation of neurotransmitter release, transportation of the membrane proteins, calcium-binding capability, and so on. We also found a rhythmic phosphorylation of N-ethylmaleimide-sensitive fusion protein and identified one of the amino acid residues modified by phosphorylation. These findings provide a new perspective on the relationship between the physiological functions of the retina and the circadian clock system.     

MELATONIN CIRCADIAN RHYTHM IN THE RETINA OF MAMMALS

Melatonin has been traditionally considered to be derived principally from the pineal gland. However, several investigations have now demonstrated that melatonin synthesis occurs also in the retina (and in other organs as well) of several vertebrate classes, including mammals. As in the pineal, melatonin synthesis in the retina is elevated at night and reduced during the day. Since melatonin receptors are present in the retina and retinal melatonin does not contribute to the circulating levels, retinal melatonin probably acts locally as a neuromodulator. Melatonin synthesis in the retinas of mammals is under control of a circadian oscillator located within the retina itself, and circadian rhythms in melatonin synthesis and/or release have been described for several species of rodents. These rhythms are present in vivo, persist in vitro, are entrained by light, and are temperature compensated. The recent cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the only enzyme unique to the melatonin synthetic pathway) will facilitate localizing the cellular site of melatonin synthesis in the retina and investigating the molecular mechanism responsible for the generation of retinal melatonin rhythmicity. Melatonin has been implicated in many retinal functions, and the levels of melatonin and dopamine appear to regulate several aspects of retinal physiology that relate to light and dark adaptation. In conclusion, it seems that retinal melatonin is involved in several functions, but its precise role is yet to be understood. (Chronobiology International, 17(5), 599–612, 2000)     

Melatonin circadian rhythm in the retina of mammals

Melatonin has been traditionally considered to be derived principally from the pineal gland. However, several investigations have now demonstrated that melatonin synthesis occurs also in the retina (and in other organs as well) of several vertebrate classes, including mammals. As in the pineal, melatonin synthesis in the retina is elevated at night and reduced during the day. Since melatonin receptors are present in the retina and retinal melatonin does not contribute to the circulating levels, retinal melatonin probably acts locally as a neuromodulator. Melatonin synthesis in the retinas of mammals is under control of a circadian oscillator located within the retina itself, and circadian rhythms in melatonin synthesis and/or release have been described for several species of rodents. These rhythms are present in vivo, persist in vitro, are entrained by light, and are temperature compensated. The recent cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the only enzyme unique to the melatonin synthetic pathway) will facilitate localizing the cellular site of melatonin synthesis in the retina and investigating the molecular mechanism responsible for the generation of retinal melatonin rhythmicity. Melatonin has been implicated in many retinal functions, and the levels of melatonin and dopamine appear to regulate several aspects of retinal physiology that relate to light and dark adaptation. In conclusion, it seems that retinal melatonin is involved in several functions, but its precise role is yet to be understood.     

Circadian rhythm of tryptophan hydroxylase activity in chicken retina

1. Retinal tryptophan hydroxylase activity in chickens (1-4 weeks old and embryos) was estimated by determination of levels of 5-hydroxytryptophan (5HTP) in retinas at defined intervals after inhibition of aromatic L-amino acid decarboxylase with m-hydroxybenzylhydrazine (NSD1015). 2. The relationship of tryptophan hydroxylase activity to photoperiod was explored. In chickens maintained on a 12-hr light: 12-hr dark cycle, a diurnal cycle in tryptophan hydroxylase activity was observed. Activity during middark phase was 4.4 times that seen in midlight phase. Cyclic changes in tryptophan hydroxylase activity persisted in constant darkness with a period of approximately 1 day, indicating regulation of the enzyme by a circadian oscillator. The phase of the tryptophan hydroxylase rhythm was found to be determined by the phase of the light/dark cycle. The relationship of the tryptophan hydroxylase rhythm to the light/dark cycle mirrors previously described rhythms of melatonin synthesis and serotonin N-acetyltransferase (NAT) activity in the retina. 3. Light exposure for 1 hr during dark phase suppressed NAT activity by 82%, while tryptophan hydroxylase activity was suppressed by only 30%. 4. Based on the differential responses of retinal NAT activity and tryptophan hydroxylase activity to acute light exposure during dark phase, it was predicted that exposure to light during dark phase would divert serotonin in the retina from melatonin biosynthesis to oxidation by MAO. In support of this, levels of 5-hydroxyindole acetic acid (5HIAA) in retina were found to be elevated approximately two-fold in chickens exposed to 30 min of light during dark phase. In pargyline-treated chickens, 2 hr of light exposure during dark phase was found to increase retinal serotonin levels by 64% over pargyline-treated controls. 5. Cyclic changes in tryptophan hydroxylase activity and NAT activity persisted for 2-3 days in constant light. Tryptophan hydroxylase activity at mid-night gradually decreased on successive days in constant light; on the first day of constant light, tryptophan hydroxylase activity at mid-night was 70% of activity seen during middark phase of the normal light/dark cycle and decreased further on subsequent days. In contrast, on each of 3 days of constant light, NAT activity at mid-night was approximately 15% of normal middark phase activity. 6. Cycloheximide completely inhibited the nocturnal increase in tryptophan hydroxylase activity when given immediately before light offset. The nocturnal increase in NAT activity was inhibited in a similar fashion. 7. Like the development of the NAT rhythm, cyclic changes of tryptophan hydroxylase activity in the retinas of chickens began on or immediately before the day of hatching. hatching.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential contribution of rod and cone circadian clocks in driving retinal melatonin rhythms in Xenopus

Background

Although an endogenous circadian clock located in the retinal photoreceptor layer governs various physiological events including melatonin rhythms in Xenopus laevis, it remains unknown which of the photoreceptors, rod and/or cone, is responsible for the circadian regulation of melatonin release.

Methodology/Principal Findings

We selectively disrupted circadian clock function in either the rod or cone photoreceptor cells by generating transgenic Xenopus tadpoles expressing a dominant-negative CLOCK (XCLΔQ) under the control of a rod or cone-specific promoter. Eyecup culture and continuous melatonin measurement revealed that circadian rhythms of melatonin release were abolished in a majority of the rod-specific XCLΔQ transgenic tadpoles, although the percentage of arrhythmia was lower than that of transgenic tadpole eyes expressing XCLΔQ in both rods and cones. In contrast, whereas a higher percentage of arrhythmia was observed in the eyes of the cone-specific XCLΔQ transgenic tadpoles compare to wild-type counterparts, the rate was significantly lower than in rod-specific transgenics. The levels of the transgene expression were comparable between these two different types of transgenics. In addition, the average overall melatonin levels were not changed in the arrhythmic eyes, suggesting that CLOCK does not affect absolute levels of melatonin, only its temporal expression pattern.

Conclusions/Significance

These results suggest that although the Xenopus retina is made up of approximately equal numbers of rods and cones, the circadian clocks in the rod cells play a dominant role in driving circadian melatonin rhythmicity in the Xenopus retina, although some contribution of the clock in cone cells cannot be excluded.     

Diurnal patterns of dopamine release in chicken retina

The retinal dopaminergic system appears to play a major role in the regulation of global retinal processes related to light adaptation. Although most reports agree that dopamine release is stimulated by light, some retinal functions that are mediated by dopamine exhibit circadian patterns of activity, suggesting that dopamine release may be controlled by a circadian oscillator as well as by light. Using the accumulation of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) in the vitreous as a measure of dopamine release rates, we have investigated the balance between circadian- and light control over dopamine release. In chickens held under diurnal light:dark conditions, vitreal levels of DOPAC showed daily oscillations with the steady-state levels increasing nine-fold during the light phase. Kinetic analysis of this data indicates that apparent dopamine release rates increased almost four-fold at the onset of light and then remained continuously elevated throughout the 12h light phase. In constant darkness, vitreal levels of DOPAC displayed circadian oscillations, with an almost two-fold increase in dopamine release rates coinciding with subjective dawn/early morning. This circadian rise in vitreal DOPAC could be blocked by intravitreal administration of melatonin (10 nmol), as predicted by the model of the dark-light switch where a circadian fall in melatonin would relieve dopamine release of inhibition and thus be responsible for the slight circadian increase in dopamine release. The increase in vitreal DOPAC in response to light, however, was only partially suppressed by melatonin. The activity of the dopaminergic amacrine cell in the chicken retina thus appears to be dominated by light-activated input.     

Regulation of tryptophan hydroxylase activity in Xenopus laevis photoreceptor cells by cyclic AMP

The aim of this study was to investigate the role of cyclic AMP in the regulation of tryptophan hydroxylase activity localized in retinal photoreceptor cells of Xenopus laevis, where the enzyme plays a key role in circadian melatonin biosynthesis. In photoreceptor-enriched retinas that lack serotonergic neurons, tryptophan hydroxylase activity is markedly stimulated by treatments that increase intracellular levels of cyclic AMP or activate cyclic AMP-dependent protein kinase, including forskolin, phosphodiesterase inhibitors, and cyclic AMP analogues. In contrast, cyclic AMP has no effect on tryptophan hydroxylase mRNA abundance. Experiments using cycloheximide and actinomycin D demonstrate that cyclic AMP exerts its regulatory effect via posttranslational mechanisms mediated by cyclic AMP-dependent protein kinase. The effect of cyclic AMP is independent of the phase of the photoperiod, suggesting that the nucleotide is not a mediator of the circadian rhythm of tryptophan hydroxylase. Cyclic AMP accumulation is higher in darkness than in light, as is tryptophan hydroxylase activity. Furthermore, the stimulatory effect of forskolin and that of darkness are inhibited by H89, an inhibitor of cyclic AMP-dependent protein kinase. In conclusion, cyclic AMP may mediate the acute effects of light and darkness on tryptophan hydroxylase activity of retinal photoreceptor cells.     

Determination of rat pineal gland melatonin content by a radioimmunoassay.

Melatonin content in individual rat pineal glands was measured by radioimmunoassay (RIA). The RIA used can very reliably detect as little as 50 pg of melatonin. The various precursors, analogues, and the metabolite of melatonin (6-hydroxymelatonin) which were tested for cross-reactivity were not recognized by the antibody. The effects on melatonin levels in rat pineal glands following the administration of L-tryptophan, 5-hydroxy-L-tryptophan, serotonin, N-acetylserotonin, melatonin and pargyline are also presented.     

Melatonin and tryptophan as therapeutic agents against the impairment of the sleep-wake cycle and immunosenescence due to aging in Streptopelia risoria

All organisms present circadian rhythm in most of their physiological functions, and among them there stand out sleep, motor activity, immune function, the secretion of melatonin, and the production and release of numerous neurotransmitters, in particular of serotonin because of its relationship with the aforementioned factors. Aging changes these rhythms, altering sleep quality and contributing to immunosenescence. Treatment with exogenously administered melatonin or tryptophan may restore these impaired functions due to aging. In our animal model (Streptopelia risoria), both the hormone and the amino acid acted on the activity-rest rhythms, modulating the circulating levels of melatonin and serotonin, and increased the cell viability and resistance to induced oxidative stress of blood heterophils, at the same time as enhancing the phagocytic function and neutralizing the superoxide anions deriving from this immune function. Also, in the old individuals, the treatments with melatonin and tryptophan at the concentrations and times of administration considered suitable improved nocturnal rest besides reverting the immunosuppressory and oxidative effects accompanying phagocytosis at these advanced ages.     

Hormonal Modulation of Cyclic Melatonin Production in the Pineal Gland of Rats and Syrian Hamsters: Effects of Thyroidectomy or Thyroxine Implant

Night-time pineal levels of tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid and the activities of the two enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase involved in the cyclic production of melatonin were determined in male albino rats and Syrian hamsters that were implanted with thyroxine or thyroidectomized two weeks earlier. Both treatments depressed nocturnal pineal melatonin content in rats and hamsters. The cause of this depression is not known, although minor alterations in the substrates and the enzymes involved in melatonin production were observed. The data suggest that alterations in thyroid hormone levels may increase the release of nocturnal melatonin from the pineal, thereby allowing less to accumulate in the gland.     

High potassium treatment resets the circadian oscillator in Xenopus retinal photoreceptors

In vertebrate retina, light hyperpolarizes the photoreceptor membrane, and this is an essential cellular signal for vision. Cellular signals responsible for photic entrainment of some circadian oscillators appear to be distinct from those for vision, but it is not known whether changes in photoreceptor membrane potential play roles in photic entrainment of the photoreceptor circadian oscillator. The authors show that a depolarizing exposure to high potassium resets the circadian oscillator in cultured Xenopus retinal photoreceptor layers. A 4-h pulse of high [K(+)] (34 mM higher than in normal culture medium) caused phase shifts of the melatonin rhythm. This treatment caused phase delays during the early subjective day and phase advances during the late subjective day. In addition to the phase-shifting effect, high potassium pulses stimulated melatonin release acutely at all times. High [K(+)] therefore mimicked dark in its effects on oscillator phase and melatonin synthesis. These results suggest that membrane potential may play a role in photic entrainment of the photoreceptor circadian oscillator and in regulation of melatonin release.