Serotonin N-acetyltransferase Activity Research Articles

Clonidine in vivo mimics the acute suppressive but not the phase‐shifting effects of light on circadian rhythm of serotonin N‐acetyltransferase activity in chick pineal gland

Comparative in vivo studies on effects of pulses of light and clonidine, a selective agonist of alpha 2-adrenergic receptors, on the circadian rhythm of serotonin N-acetyltransferase (NAT) activity in chick pineal gland were performed. Six-hour pulses of white light caused an acute suppression of the nocturnal NAT activity and phase-dependent phase shifts of the circadian rhythm of the enzyme activity relative to controls. Systemic administration of clonidine acutely suppressed NAT activity of chick pineal gland, but did not affect the phase of subsequent cycles in constant darkness. These results give further support to the concept based on in vitro studies that alpha 2-adrenergic receptors are involved in regulation of melatonin biosynthesis in chick pineal gland by a mechanism distal to the pacemaker that generates the circadian melatonin rhythm.

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.

Dopamine receptor regulating serotonin N-acetyltransferase activity in chick retina represents a D 4-like subtype: Pharmacological characterization

The dopamine (DA) receptor regulating serotonin N-acetyltransferase (NAT) activity in chick retina was characterized pharmacologically. Intraocular (i.o.) administration of DA significantly decreased the nighttime NAT activity of chick retina. The effect of DA was antagonized by blockers of the D 2 family of DA receptors, spiroperidol, YM-09151-2 and clozapine, and it was not affected by SCH 23390, a selective antagonist of D 1 DA receptor. Several agonists of D 2 family of DA receptors given i.o. suppressed the nighttime NAT activity of the chick retina with quinpirole (D 3/D 4 receptor selective) and bromocriptine (D 2/D 3 receptor selective) being the most and the least potent drugs, respectively. The rank-order potency of antagonists of D 2 family of DA receptors to block the inhibitory effect of quinpirole on the enzyme activity (with clozapine being relatively very potent, and (+)-butaclamol, raclopride and remoxipride-ineffective) match the characteristics of the D 4 DA receptor. Moreover, although sulpiride effectively prevented the quinpirole-induced decline in the nighttime NAT activity of the chick retina, there was no marked stereoselectivity in its action. It is suggested that DA receptor regulating NAT activity in chick retina represents a D 4-like subtype.

The locus controlling pineal serotonin N-acetyltransferase activity ( Nat-2) is located on mouse chromosome 11

Melatonin is synthesized from serotonin by the enzymes serotonin N-acetyltransferase (SNAT) and hydroxyindole- O-methyltransferase (HIOMT). We have previously reported that C57BL/6 mice do not have SNAT activity because of a mutation in an autosomal gene which is responsible for the absence of normal SNAT activity. In the present study, we have tried to map the loci of Nat-2 (the locus controlling SNAT activity) on chromosomes using a set of the BxH recombinant inbred strains which were derived from an initial cross between C3H/He with SNAT and C57BL/6 without the enzyme. Based on strain distribution patterns (SDPs), a close linkage on chromosome 11 was found between Nat-2, Es-3 (esterase-3), Glk (the locus controlling galactokinase activity) and Myla (myosin alkali light chains expressed in cardiac atrial muscle). The linkage between Nat-2 and Es-3 was confirmed by a conventional linkage test and the recombination frequency between these loci was estimated to be 16.1 ± 3.6% (mean ± S.E.M.).

Does D 4 dopamine receptor mediate the inhibitory effect of light on melatonin biosynthesis in chick retina?

The dopamine (DA) receptor mediating the inhibitory effect of light on melatonin formation in the chick retina was characterized pharmacologically. Nighttime serotonin N-acetyltransferase (NAT) activity was significantly decreased by either light exposure or by intraocular (i.oc.) administration of DA and quinpirole (a predominant D 3/D 4 DA receptor agonist). Several D 2-like DA receptor antagonists, i.e. clozapine, haloperidol, spiroperidol, sulpiride, (+)-UH-232 and YM-09151-2, given i.oc. to light-adapted chicks markedly elevated retinal NAT activity. In contrast, raclopride (a D 2/D 3 DA receptor antagonist) and remoxipride (a D 2-selective DA receptor antagonist) were ineffective. Spiroperidol, clozapine, haloperidol and sulpiride significantly increased melatonin content in the light-exposed retina, but had no effect on the activity of hydroxyindole- O-methyltransferase. None of D 2-like DA receptor blockers tested modified the nighttime NAT activity in the chick retina. Our results indicate that the light-evoked inhibition of the nocturnal increase in melatonin biosynthesis in chick retina may involve stimulation of the D 4 subtype DA receptor.

Intravitreal application of membrane-permeable analogs of cyclic GMP increases serotonin N-acetyltransferase (NAT) activity in retinas of light-exposed chicks: Comparison with the effect of cyclic AMP analogs and darkness

Intravitreal administration of membrane-permeable analogs of cGMP and cAMP markedly enhanced serotonin N-acetyltransferase (NAT) activity in retinas of light-exposed chicks. The effects of cGMP analogs were usually weaker than the actions of cAMP analogs, however, they were more pronounced than the effects of non-cyclic derivative of GMP (i.e., 8-bromo-GMP). Combined treatment with dibutyryl-derivatives of cGMP and cAMP yielded NAT values that were similar to the values produced by the compounds administered separately, whereas NAT activity in chicks receiving a combination of db-cGMP and aminophylline was higher than the enzyme activity produced by the drugs individually. Neither db-cGMP nor aminophylline affected hydroxyindole- O-methyltransferase (HIOMT) activity in retinas of light-exposed chicks. It is hypothesized that cGMP analogs enhance the retinal NAT activity indirectly, via cAMP.

Periodic mother deprivation during the light period reversed the phase of serotonin N-acetyltransferase activity rhythm of the pineal gland in rat pups

It has been reported that nursing mother rats can postnatally entrain the circadian rhythms of blinded rat pups, such as locomotor, drinking, and corticosterone rhythms. To gain more insight in the mechanism of the postnatal entrainment of such pups' circadian rhythms, we examined the serotonin N-acetyltransferase (NAT) activity rhythm in blinded rat pups subjected to periodic mother deprivation (PMD) in which mothers were periodically deprived of their pups during either half of a day. We found that only PMD during the light period shifted the phase of NAT activity rhythm in the pups. To cause a reversal of the NAT activity rhythm, it was necessary to repeat PMD for more than 6 days. PMD for 6 h each day also shifted the phase of the blinded rat pups, but it did not reverse the NAT rhythm, even when it was repeated for 10 days. In 9-h deprivation for 10 days, however, deprivation during the first 9 h of the light period reversed the phase, although the latter 9 h failed to cause reversal of the phase. On the other hand, restricted feeding of the mother took more than 11 days to reverse the phase and a foster mother in the cross-fostering experiment failed to affect the phase of pup's rhythm, when the rhythm was determined on the 11th postnatal day. These facts indicate that PMD during the light period is a potent entrainer of the pups' circadian NAT rhythms and provide a useful method of exploring the underlying mechanism of the entrainment of the pups' rhythm by the mother.

Serotonin N-Acetyltransferase (NAT) Activity and Melatonin Levels in the Frog Retina Are Not Correlated during the Seasonal Cycle

The seasonal pattern of daily serotonin N-acetyltransferase (NAT) and melatonin rhythms in the lateral eye of Rana perezi has been characterized to assess any seasonal relationship between enzymatic activity and melatonin production and to evaluate the photothermal responses of such rhythms. In winter there was no daily rhythm either in NAT activity or in melatonin production, although the highest values of NAT activity and lowest melatonin concentrations were measured at this time. Ocular melatonin showed a high-amplitude diurnal rhythm during the summer, whereas a low-amplitude rhythm was observed in spring and autumn. Diurnal melatonin levels were temperature-independent; however, nocturnal melatonin production significantly increased with temperature. Ocular NAT was always higher at lower than at higher temperatures (two- to threefold), both at night and during the day. As suggested for pineal melatonin production in other poikilotherm species, photoperiod controls the duration of the melatonin nocturnal rise, whereas temperature regulates the amplitude of the daily melatonin rhythm in the frog retina. The lack of correlation between daily NAT and melatonin rhythms in the frog retina may result from the high thermal sensitivity of the ocular NAT and a temperature-dependent inhibition substrate of NAT activity.

Regulation of melatonin and dopamine biosynthesis in chick retina: the role of GABA.

Melatonin biosynthesis in chick retina occurs as a circadian rhythm. Biosynthesis of the neurohormone is highest at night in darkness, and is suppressed by light. The role of gamma-aminobutyric acid (GABA) in the nocturnal regulation of melatonin synthesis was examined. Systemic or intravitreal administration of muscimol, a GABA-A receptor agonist, to light-exposed chicks at the beginning of the dark phase of the light/dark cycle increased retinal melatonin levels and the activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme of the melatonin biosynthetic pathway. Baclofen, a GABA-B receptor agonist, also increased NAT activity of light-exposed retinas, but muscimol was approximately 40-fold more potent than baclofen. Effects of both muscimol and baclofen on NAT activity were inhibited by GABA-A antagonists, bicuculline and picrotoxin, and the effect of baclofen was unaffected by the GABA-B selective antagonist, CGP 35348. Thus, activation of GABA-A receptors appears to be associated with increased melatonin biosynthesis. The GABA-uptake inhibitor, nipecotic acid, and the GABA-transaminase inhibitor, aminooxyacetic acid, also increased NAT activity of light-exposed retinas. The high levels of NAT activity associated with exposure to darkness were unaffected by either muscimol or baclofen, but picrotoxin and bicuculline significantly inhibited retinal NAT activity in darkness. The rate of dopamine synthesis, estimated from in situ tyrosine hydroxylase activity, was higher in light-exposed retinas than in darkness. Muscimol inhibited dopamine synthesis in light, and picrotoxin stimulated dopamine synthesis in darkness. The stimulation of melatonin synthesis by muscimol in light-exposed retinas appears to be related to inhibition of retinal dopamine neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of melatonin production by pineal photoreceptor cells: role of cyclic nucleotides in the trout (Oncorhynchus mykiss).

The light/dark cycle influences the rhythmic production of melatonin by the trout pineal organ through a modulation of the serotonin N-acetyltransferase (NAT) activity. In static organ culture, cyclic AMP (cAMP) levels (in darkness) and NAT activity (in darkness or light) were stimulated in the presence of forskolin, isobutylmethylxanthine, or theophylline. Analogues of cAMP, but not of cyclic GMP, induced an increase in NAT activity. Light, applied after dark adaptation, inhibited NAT activity. This inhibitory effect was partially prevented in the presence of drugs stimulating cAMP accumulation. In addition, cAMP accumulation and NAT activity increase, induced by forskolin, were temperature dependent. Finally, melatonin release, determined in superfused organs under normal conditions of illumination, was stimulated during the light period of a light/dark cycle by adding an analogue of cAMP or a phosphodiesterase inhibitor. However, no further increase in melatonin release was observed during the dark phase of this cycle in the presence of the drugs. This report shows for the first time that cAMP is a candidate as intracellular second messenger participating in the control of NAT activity and melatonin production by light and temperature.

Chick retina and pineal gland differentially respond to constant light and darkness: in vivo studies on serotonin N-acetyltransferase (NAT) activity and melatonin content

Oscillations in serotonin N-acetyltransferase (NAT) activity and melatonin content were investigated in retina and pineal gland of chicks kept for 5 days in constant darkness (DD) or continuous light (LL). Under DD the rhythm of the pineal melatonin biosynthesis resembled that found under diurnal illumination (LD), whereas in the retina DD resulted in significant elevations of NAT activity and melatonin level during subjective light. A low-amplitude rhythm of retinal NAT activity continued under LL with a period close to 24 h. In the pineal gland, light exposure suppressed the level of NAT activity and melatonin content (an effect being substantially weaker than that observed in retina), delayed the first peak of NAT activity by 3 h, and prolonged the rhythm's period to about 26 h. Our data suggest the existence of some differencies in the activity of circadian oscillators that regulate the melatonin generating system in the retina and pineal gland of chick.

Does the Circadian Pacemaker Act through Cyclic AMP to Drive the Melatonin Rhythm in Chick Pineal Cells?

Cyclic AMP is a key regulator of melatonin production in the chick pineal gland. Agents that raise cyclic AMP levels (such as forskolin), or cyclic AMP analogues (such as 8-bromocyclic AMP), increase melatonin synthesis and release, whereas agents that lower cyclic AMP levels (including light) decrease melatonin synthesis and release. A circadian oscillator in these cells also raises and lowers melatonin output. We have been investigating the relationships between cyclic AMP and the circadian pacemaker in the regulation of melatonin production. In the chick pineal (unlike certain neuronal systems), the weight of the evidence indicates that cyclic AMP is not on an entrainment pathway to the circadian pacemaker. Instead, cyclic AMP appears to act downstream from the pacemaker. The pacemaker might itself act directly through cyclic AMP, regulating melatonin content by raising and lowering cyclic AMP levels. If this were the case, and if the effects of cyclic AMP levels on melatonin output are saturable (as they must be), then, in the face of such saturating levels of cyclic AMP, the pacemaker should no longer raise or lower melatonin output. To test this prediction, maximally effective concentrations of forskolin and 8-bromocyclic AMP were determined. Both agents markedly increased melatonin output. After 36 hr, cells were refractory to additional stimulation of melatonin output by addition of both agents together, or by higher concentrations of forskolin (although cyclic AMP levels could still be raised further). Nonetheless, the circadian pacemaker continued to raise and lower melatonin output: The rhythm persisted in the face of saturating levels of cyclic AMP. It is therefore suggested that the circadian pacemaker in chick pineal cells acts with, not through, cyclic AMP to regulate melatonin synthesis. Cyclic AMP and the pacemaker act synergistically to regulate serotonin N-acetyltransferase activity and the melatonin rhythm, with cyclic AMP mediating acute effects and amplitude regulation.

Melatonin increases serotonin N-acetyltransferase activity and decreases dopamine synthesis in light-exposed chick retina: in vivo evidence supporting melatonin-dopamine interaction in retina.

The administration of melatonin, either peripherally (0.01-10 mg/kg) or intraocularly (0.001-10 mumol/eye), to light-exposed chicks dose-dependently increased serotonin N-acetyltransferase (NAT) activity in retina but not in pineal gland. The effect of melatonin was slightly but significantly reduced by luzindole (2-benzyl-N-acetyltryptamine), and not affected by two other purported melatonin antagonists, N-acetyltryptamine and N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23). The elevation of the enzyme activity induced by melatonin was substantially stronger than that evoked by 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, or 5-methoxytryptamine. The melatonin-evoked rise in the retinal NAT activity was counteracted by two dopamine D2 receptor agonists, quinpirole and apomorphine, and prevented by the dopamine D2 receptor blocker spiroperidol, and by an inhibitor of dopamine synthesis, alpha-methyl-p-tyrosine. Melatonin (0.1-10 mg/kg i.p.) dose-dependently decreased the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), as well as the DOPAC/dopamine ratio, in chick retina but not in forebrain. The results obtained (1) indicate that melatonin in vivo potently inhibits dopamine synthesis selectively in retina, and (2) suggest that the increase in retinal NAT activity evoked by melatonin in light-exposed chicks is an indirect action of the compound, and results from the disinhibition of the NAT induction process from the dopaminergic (inhibitory) signal. The results provide in vivo evidence supporting the idea (derived on the basis of in vitro findings) that a mutually antagonistic interaction between melatonin and dopamine operates in retinas of living animals.

In vitro effects of putative neurotransmitters on synaptic ribbon numbers and N-acetyltransferase activity in the rat pineal gland.

The pineal contains a large number of classical transmitters and neuropeptides. Some of these neurochemicals are involved in the regulation of serotonin N-acetyltransferase (NAT) activity and hence in melatonin synthesis. Synaptic ribbons present in the pineal gland also exhibit a numerical day/night rhythm parallel to that of NAT activity. There is scarcity of information regarding the regulation of synaptic ribbon (SR) numbers. In the present study, we have investigated in vitro effects of a number of classical neurotransmitters and neuropeptides. NAT activity was used to monitor melatonin synthesis under the experimental conditions used. Norepinephrine (NE), Delta sleep-inducing peptide (DSIP), vasoactive intestinal polypeptide (VIP), adenosine and N-acetyl-asp-glu (NAAG) significantly increased NAT activity in rat pineal. DSIP and VIP also increase the stimulatory effect of NE on NAT activity. These neurochemicals had no effect on SR numbers. Gamma aminobutyric acid (GABA), serotonin and taurine affected neither NAT activity nor SR. Somatostatin increased SR numbers significantly, without having any effect on NAT activity. The effect of somatostatin is regarded to be pharmacologic, since rather high dosages (10(-4) M) were required to obtain a significant effect. Although somatostatin is present in the pineal and may change rhythmically, the inconsistency of the day/night rhythmicity and the lack of such a rhythm in female rats and male gerbils speaks against an important physiological role of somatostatin in regulating SR numbers.

Calcium channel blockers in vivo inhibit serotonin N‐acetyltransferase (NAT) activity in chicken retina stimulated by darkness and not by agents elevating intracellular cyclic AMP level

The molecular mechanism underlying the role of calcium influx in the regulation of retinal serotonin N-acetyltransferase (NAT) activity was studied in vivo in chickens. Systemic administration of organic antagonists of voltage-sensitive calcium channels (VSCC), i.e., nimodipine and nifedipine, resulted in a marked suppression of the nocturnal increase of NAT activity in chicken retina. In contrast, NAT activity stimulated by nonhydrolysable analogs of cyclic AMP (dibutyryl-cyclic AMP and 8-bromo-cyclic AMP), forskolin, a direct activator of adenylate cyclase, and by phosphodiesterase inhibitors (aminophylline and 3-isobutyl-1-methylxanthine), was not significantly affected by various tested VSCC antagonists. The inhibitory effect of nimodipine on the dark-dependent increase in NAT activity of chicken retina was abolished by Bay K 8644, a selective VSCC agonist. The results presented in this paper indicate an important role of calcium influx through L-type of VSCC in the induction of NAT activity in chicken retina, and suggest that a requirement of calcium ions in the process of NAT induction in the retina may be primarily at the level of cyclic AMP production.

Thermal sensitivity and effect of temperature acclimation on ocular serotonin N-acetyltransferase activity in Rana perezi

The thermal sensitivity and the response to thermal acclimation of the serotonin N-acetyltransferase (NAT) activity in frog ocular tissue were studied. The ocular NAT shows a positive thermal modulation for both cosubstrates (tryptamine and acetyl-CoA). The higher NAT activity in the cold-acclimated group (4°C) with respect to the warm-acclimated one (24°C) implies a partial thermal compensation of the enzyme. The present results suggest that frog ocular NAT response to temperature entails a modulation of thermal sensitivity of the enzyme rather than changes in enzyme concentration.

Development of MEKA (phosducin), Gβ, Gγ and S-antigen in the rat pineal gland and retina

Pinealocytes and retinal photoreceptor cells contain an unusual cytoplasmic complex composed of the Gβγ dimer of GTP-binding regulatory proteins (G-proteins) tightly bound to an acidic 33 kDa phosphoprotein termed MEKA or phosducin; MEKA is a substrate of cyclic AMP-dependent protein kinase. This study characterized the developmental appearance of these and two related proteins, Gγ and S-antigen, in pineal and retinal tissue. MEKA was absent in the pineal gland prior to birth, at a time when it was possible to detect Gβ in pineal cytoplasm, indicating that the appearance of Gβ in the cytoplasm precedes that of MEKA and does not appear to require the presence of MEKA. The absence of MEKA at this time indicates that the cyclic AMP stimulation of pineal serotonin N-acetyltransferase activity is not mediated by MEKA, which has been considered as a possible role of MEKA. After postnatal day 7, pineal MEKA and cytoplasmic Gβ increased in a parallel manner, with peak values occurring at about postnatal day 21. Thereafter, both proteins in the pineal gland decreased in a parallel fashion to 10 and 35% of their peak values, respectively; in contrast, the cytoplasmic protein S-antigen and membrane associated Gβ remained at maximal levels after this time. Whereas both MEKA and Gβ decreased late in development in the pineal gland, these proteins either increased or remained constant in the retina. These tissue-specific patterns were found to differ from those of another cytosolic protein found exclusively in the pineal gland and retina, S-antigen, which remained constant after 21 in the pineal gland but decreased in the retina late in life.

Melatonin synthesis in chicken retina: effect of kainic acid-induced lesions on the diurnal rhythm and D2-dopamine receptor-mediated regulation of serotonin N-acetyltransferase activity.

The effect of kainic acid (KA)-induced lesions of retinal neurons on regulation of serotonin N-acetyltransferase (NAT) activity in chicken retina was investigated. Although NAT activity was higher in KA-lesioned retinas than in controls, the pattern of diurnal variation of enzyme activity throughout 36 h of constant darkness was similar for both tissues. Quinpirole, a selective D2-dopamine receptor agonist, inhibited the nocturnal increase of NAT activity in both control and KA-treated retinas. Quinpirole was significantly more potent in KA-treated retinas than in controls; the ED50 value for quinpirole was 3 times lower in KA-treated retinas than in control tissues. KA treatment markedly reduced retinal levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). We conclude that: (1) NAT activity in retina is localized primarily to KA-insensitive cells, presumably photoreceptors; (2) KA-sensitive inner retinal neurons are not essential to the maintenance of the circadian rhythm of NAT activity; and (3) KA-induced lesions of retinal cells result in supersensitivity of D2-dopamine receptors regulating NAT activity in a mechanism that involves adaptive changes following a decline in retinal dopamine neurotransmission.

Calcium channel drugs affect nocturnal serotonin N-acetyltransferase (NAT) activity in rat pineal gland

The effects of different organic compounds that block and increase Ca2+ influx through the voltage-sensitive calcium channels (VSCC) on the nocturnal serotonin N-acetyltransferase (NAT) activity was investigated in vivo in rats. Systemic administration of VSCC antagonists, i.e. nimodipine, nifedipine, verapamil and diltiazem, resulted in a marked suppression of the nighttime pineal NAT activity. Bay K 8644, a VSCC agonist, injected to rats before the time of the light offset of the light-dark cycle significantly enhanced the nocturnal increase of the pineal NAT activity. Although Bay K 8644 given during the dark phase of an imposed illumination cycle had little effect on the nocturnal pineal NAT activity, it antagonized the nimodipine- and verapamil-induced decrease in the enzyme activity. These results support the role of Ca2+ influx through the VSCC in the nocturnal increase of NAT activity in the pineal gland of rat.

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.