Iodomelatonin Research Articles

Differential Expression of Melatonin Receptor Subtypes MelIa, MelIb and MelIc in Relation to Melatonin Binding in the Male Songbird Brain

Previous autoradiography studies illustrated that several areas of the avian brain can bind the pineal hormone melatonin. In birds, there are three melatonin receptor (MelR) subtypes: MelIa, MelIb and MelIc. To date, their brain distribution has not been studied in any passerine bird. Therefore, we investigated mRNA distribution of MelR subtypes in adjacent sections of the brain of two songbirds, the blackcap and the zebra finch, in parallel with that of 2-[¹²⁵I]-iodomelatonin (IMEL) binding sites in the same brains. The general pattern of receptor expression shown by in situ hybridization of species-specific probes matched well with that of IMEL binding. However, the expression of the three subtypes was area specific with similar patterns in the two species. Some brain areas expressed only one receptor subtype, most brain regions co-expressed either MelIa with MelIb or MelIa with MelIc, whereas few areas expressed MelIb and MelIc or all three receptor subtypes. Since many sensory areas, most thalamic areas and subareas of the neopallium, a cortex analogue, express MelR, it is likely that most sensory motor integration functions are melatonin sensitive. Further, the area-specific expression patterns suggest that the regulatory role of melatonin differs among different brain areas. Since subareas of well-defined neural circuits, such as the visual system or the song control system, are equipped with different receptor types, we hypothesize a diversity of functions for melatonin in the control of sensory integration and behavior.

Down-regulation of mt 1 melatonin receptors in rat ovary following estrogen exposure

In this study, we have demonstrated that 2-[ 125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (K D=83 pM; B max=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt 1 melatonin receptor antibody, we have also detected mt 1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d × 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[ 125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[ 125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a B max=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a B max=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt 1 melatonin receptors in rat ovary were down-regulated (∼95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[ 125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[ 125I]-iodomelatonin binding occurred with total 2-[ 125I]-iodomelatonin binding =5.5 fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.

Photoresponsive Fischer 344 Rats are reproductively inhibited by melatonin and differ in 2-[125I] lodomelatonin binding from nonphotoresponsive Sprague-Dawley rats.

Many temperate-zone species use photoperiod as an environmental cue to regulate reproductive timing. Strains of laboratory rats differ in their responsiveness to photoperiod, with the Fischer 344 (F344) strain being the most responsive known. F344 rats and closely related strains that differ in photoresponsiveness may be useful models to study the mechanisms and genetic basis for photoresponsiveness. We tested two hypotheses: (i) that melatonin mediates photoresponsiveness in F344 rats, as is the case in all other mammals tested, and (ii) that the location, abundance, or affinity of melatonin receptors, as estimated by the amount and location of binding of the radioligand 2-[125I]-iodomelatonin (IMEL) in the brain, might cause variation in photoresponsiveness among rat strains. Melatonin injections 1 h before lights off in a stimulatory photoperiod (L14 : D10) induced reproductive inhibition and reduced weight gain in a manner similar to short days of L8 : D16, while injections of ethanolic saline vehicle did not. Interestingly, melatonin injections administered during an inhibitory photoperiod (L10 : D14) caused greater inhibition of both reproduction and weight gain than short photoperiod alone. Pinealectomized F344 rats implanted subcutaneously with melatonin in a silastic capsule did not differ in testis size or body weight from controls with blank implants. The brains and pars tuberalis of the pituitary from photoresponsive F344 rats and nonphotoresponsive Harlan Sprague-Dawley (HSD) rats were processed for autoradiography using IMEL. We found significantly higher specific IMEL binding in the anterior and posterior regions of the paraventricular nucleus of the thalamus (PVNt) and reuniens nucleus of the thalamus of F344 rats than in the same areas in HSD rats. There were no differences between strains in specific IMEL binding in the medial PVNt, anteroventral and anterodorsal nucleus of the thalamus, suprachiasmatic nucleus, or the pars tuberalis. These results indicate that melatonin mediates photoresponsiveness in F344 rats. In addition, they provide support for the hypothesis that F344 rats may be photoresponsive due to differences from other strains in the location, density, or affinity of melatonin receptors.

Differences in hypothalamic 2-[ [formula omitted]]iodomelatonin binding in photoresponsive and non-photoresponsive white-footed mice, Peromyscus leucopus

Photoperiod is an environmental cue used by many temperate-zone species to regulate their reproductive timing. Within species, the degree of reproductive photoresponsiveness can vary widely both among and within populations. The neuroendocrine mechanisms causing this individual variation in photoresponsiveness are unknown. Using selected lines from a population of white-footed mice known to vary genetically in reproductive photoresponsiveness, we tested the hypothesis that variation in the number and/or location of melatonin receptors is the basis for individual differences in reproductive photoresponsiveness. The brains and pars tuberalis of the pituitary from sixteen mice, (eight mice from each of two lines selected for two generations to respond strongly or weakly to photoperiod), were processed for autoradiography using the radioligand 2-[ 125 I ]-iodomelatonin (IMEL). We found significantly higher specific IMEL binding in the medial preoptic area and the bed nucleus of the stria terminalis of non-responsive mice than responsive mice. There were no differences between groups in specific IMEL binding in the suprachiasmatic and dorsomedial nuclei of the hypothalamus, pars tuberalis, or paraventricular nucleus of the thalamus. These results provide support for the hypothesis that individual variation in photoresponsiveness is due in part to differences in the density or affinity of melatonin receptors.

Localization and Partial Characterization of Melatonin Receptors in Amphioxus, Hagfish, Lamprey, and Skate

Through its secretion of melatonin, the pineal complex of vertebrates exerts a range of physiological effects including regulation of circadian rhythms, seasonal reproduction, metamorphosis, and body color change. Little is known about phylogenetic differences in the distribution and characteristics of melatonin binding sites in fishes. We usedin vitroautoradiography to examine binding of [2-125I]iodomelatonin (IMEL) in 20-μm frozen sections of amphioxus (Branchiostoma lanceolatum), Atlantic hagfish (Myxine glutinosa), larval and adult lamprey (Petromyzon marinus), little skate (Raja erinacea), and rainbow trout (Oncorhynchus mykiss). Tissue was incubated with IMEL in the presence or absence of unlabeled melatonin (1 μM, in order to assess nonspecific binding). A concentration of 32 pM IMEL was used for single point assays and competition studies. No specific binding was found in hagfish or amphioxus, which lack a pineal complex. In the optic tecta of lamprey, skate, and trout, IMEL binding is highly specific (melatonin⪢N-acetylserotonin>5- methoxytryptophol⪢serotonin). Scatchard analysis revealed that the tectal binding sites are of high affinity (Kd=36, 38, and 50 pM) and low capacity (Bmax=8.1, 19.8, and 21.8 fmol/mg protein) in lamprey, skate, and trout, respectively. In adult lampreys, intense specific IMEL binding is found in the optic tectum (layer I>II>III) and preoptic nucleus (pars parvocellularis>magnocellularis). Binding was less intense and consistent in the same areas of ammocoete brain. In skates and trout, intense specific binding is found in optic tectum, lateral geniculate body, diencephalic preoptic and suprachiasmatic nuclei, basal hypothalamus, and the medial pallium. These results indicate that specific melatonin binding sites are present in all craniate taxa examined except in hagfish. Although we cannot rule out the possibility that melatonin receptors are secondarily lost in hagfish, their absence in amphioxus makes this unlikely. We speculate that melatonin actions in early vertebrates may have included regulation of visual and endocrine responses to light.

Melatonin Binding in the House Sparrow Song Control System: Sexual Dimorphism and the Effect of Photoperiod

Avian song is a sexually dimorphic behavior which is regulated seasonally. This regulation involves the construction and growth of song control structures: the high vocal center (HVC),nucleus robustus archistrialis(RA),nucleus magnocellularis anterior(MAN), and Area X. Song behavior and its neural correlates are controlled by steroid-dependent and independent processes. The avian circadian system is known to be involved in both daily processes and seasonal reproduction. A major part of this system is the circadian secretion of melatonin by the pineal gland. To determine possible interactions of the circadian and song control systems, the distribution and density of 2-[125I]iodomelatonin (IMEL) binding, an indicator of melatonin sensitivity, were determined in male and female house sparrow brains. Specific binding was found in visual system centers of both genders, but binding in HVC, RA, and Area X was present only in males. Binding in MAN was present in both sexes. Although the effects of short and long photoperiods on male house sparrow IMEL binding in song structures revealed no systematic changes, there were significant differences in binding under different photoperiods in HVC and RA. IMEL binding in the tectofugalnucleus rotundus,however, was consistently highest under short day conditions. IMEL binding in song control nuclei was independent of testicular influence, since castration did not affect it significantly. The data point to a role for the circadian system of house sparrows in song control, but a specific role for melatonin in the daily or seasonal regulation of the song control system in birds, could not be determined.

Are nuclear receptors involved in pituitary responsiveness to melatonin?

Evidence suggests that the pineal hormone melatonin modulates prolactin (PRL) secretion in part through direct effects on the anterior pituitary. However, high-affinity membrane receptors for melatonin are only found in the pars tuberalis (PT) of the anterior pituitary, whereas lactotrophs are confined to the pars distalis (PD). This study therefore sought to determine whether melatonin might have direct effects on the PD, through alternate pathways. Such a possibility had been suggested by recent reports of melatonin binding to members of the retinoid-related orphan nuclear receptor family (ROR/RZR). Expression of RORα/RZRα isoforms was observed by reverse transcription PCR in the ovine PT and PD. Correspondingly unidentified nuclear proteins from these tissues showed binding to consensus DNA response elements for members of the ROR/RZR family. In contrast nuclear 2-[ 125I]iodomelatonin (IMEL) binding was not detectable in PD or PT extracts even at high ligand and tissue concentrations. Nevertheless, the conditions used allowed membrane and nuclear IMEL binding to be observed in PT and liver extracts, respectively. Overall these findings do not support the possibility of direct effects of melatonin on the adult PD, and by implication they reinforce the view that the melatonin-responsive PT is an intermediary in the control of PRL secretion.

Distribution of 2-

Using in vitro autoradiography, the distribution of 2-[125I]iodomelatonin (IMEL) binding sites in the brain of the pied flycatcher and the zebra finch was examined. The results show IMEL binding in retinofugal, tectofugal and thalamofugal brain areas of the visual system. Additionally, IMEL binding sites were detected in the ectostriatum, the thalamus, the mesencephalon and the limbic system. No IMEL binding could be demonstrated in the pineal gland, the hippocampus, the nucleus suprachiasmaticus, the visual wulst or the pituitary.

The Pineal Gland: Photoreception and Coupling of Behavioral, Metabolic, and Cardiovascular Circadian Outputs

Although removal of the pineal gland has been shown to have very little effect on the mammalian circadian system in constant darkness (DD), several recent reports have suggested that the mammalian pineal gland may be more important for circadian organization in nocturnal rodents than was previously believed. Removal of the pineal gland (PINX) facilitates the disruptive effects of constant bright light on wheel-running rhythmicity. This suggests at least two possibilities for the role of the pineal gland in the mammalian circadian system. First, pinealectomized rats may perceive ambient light intensity to be brighter than do sham-operated (SHAM) rats. Second, the pineal gland, probably via its secretion of melatonin, may also be involved in coupling components of the circadian system. Coupling, as we see it, may occur at several levels of organization: (1) between retinohypothalamic afferents and suprachiasmatic nuclei (SCN) oscillatory neurons, (2) among multiple SCN oscillators, (3) between the SCN and their multiple outputs, and/or (4) among the multiple circadian outputs themselves. In this study we show that PINX rats free-run with a longer period in four different light intensities than do SHAM rats. Moreover, the rate of increase of tau is greater among PINX rats than among SHAM rats. This supports the first hypothesis. We also show that in PINX rats the circadian rhythms of wheel running, general activity, body temperature, and heart rate are all more disrupted in constant bright light than are those of SHAM rats, and each rhythmic output is disrupted in parallel. This supports the second hypothesis. Melatonin is probably not involved in coupling presynaptic elements of SCN afferents in the retinohypothalamic tract to pacemaking cells within the SCN, since enucleation has no effect on SCN 2-[125I]iodomelatonin (IMEL) binding. Together the data do not discount either of the two hypotheses but do restrict the possible levels at which the pineal gland is involved in coupling. These data also further support a growing body of literature indicating that the pineal gland and its hormone melatonin play a role in mammalian circadian organization.

Regional Distribution of Iodomelatonin Binding Sites within the Suprachiasmatic Nucleus of the Syrian Hamster and the Siberian Hamster

The pineal hormone melatonin is a potent regulator of seasonal and circadian rhythms in vertebrates. In order to characterize potential target tissues of melatonin, the distribution of iodomelatonin (IMEL)-binding sites was examined within neurochemically and anatomically defined subdivisions of the suprachiasmatic nucleus (SCN), a structure necessary for seasonal and circadian rhythms in mammals. Studies were carried out in both the adult Syrian (Mesocricetus auratus) and Siberian (Phodopus sungorus) hamster. The retinoreceptive zone of the SCN was identified anatomically by immunocytochemical (ICC) visualization of cholera toxin B subunit tracer (ChTB-ir) following its intra-ocular injection. Photically-responsive SCN cells were identified by immunostaining for the protein product of the immediate-early gene c-fos (Fos-ir) following exposure of the animal to light. The non-photoresponsive zone of the SCN was identified using in situ hybridization (ISH) for arginine vasopressin (AVP) mRNA, whilst sites of IMEL-binding in the SCN were identified by in vitro film autoradiography using the specific ligand 2-[125I]-iodomelatonin. To compare directly the distribution of IMEL-binding sites and one of the functional zones of the nucleus, alternate serial coronal sections through the SCN were processed for autoradiography for IMEL and one of the following: ICC for ChTB-ir or Fos-ir, or ISH for AVP mRNA. Overall, the regional distribution of the various markers within the SCN was comparable in the two species. The retinorecipient (ChTB-ir) and photically-responsive (Fos-ir) zones of the SCN mapped together to the middle and caudal thirds of the nucleus, predominantly in its ventro-lateral division.(ABSTRACT TRUNCATED AT 250 WORDS)

Melatonin binding sites in sciurid and hystricomorph rodents: studies on ground squirrels and guinea pigs

Little is known about the distribution of binding sites for the pineal hormone melatonin in non-myomorph rodents. We used 2-[ 125I]iodomelatonin (IMEL) to analyze the distribution, affinity, and specificity of binding sites in the golden-mantled ground squirrel, a sciurid rodent that reportedly lacks IMEL binding sites in the brain. Specific binding was found not only in the pars tuberalis, but also in several telencephalic areas including the hypothalamic suprachiasmatic region. The affinity and specificity of IMEL binding are comparable to those reported in other rodents. IMEL binding studies in a hystricomorph rodent, the guinea pig, revealed high concentrations of receptor in the nucleus accumbens and dorsolateral thalamus. Central melatonin binding sites have now been demonstrated in species of all three rodent families. The heterogeneous distribution of melatonin receptors appears similar in the species studied, and no evidence is found to link IMEL binding sites at any particular locus to photoperiodic, circannual, or non-seasonal breeding patterns.

Daily melatonin administration synchronizes circadian patterns of brain metabolism and behavior in pinealectomized house sparrows,Passer domesticus

Recent research in our laboratory has indicated that in sparrows the visual suprachiasmatic nucleus (vSCN) is metabolically rhythmic such that 2-deoxy[14C]glucose (2DG) uptake and specific binding of 2[125I]iodomelatonin (IMEL) are high during subjective day for up to 10 circadian cycles in constant darkness (DD). These rhythms damp to arrhythmicity in pinealectomized birds (PINX). The present study was designed to test the hypothesis that exogenous melatonin rhythmically applied can restore disrupted behavioral and cerebral rhythmicity. Pinealectomized house sparrows were placed in constant dim light and allowed to become arrhythmic. Experimental birds received 0.86 mM melatonin in 0.01% ethanol (ETOH) to drink for 12 of every 24 h for 14 days. Control birds received 0.01% ETOH only. Behavioral rhythmicity was restored by melatonin but not by ETOH. Birds were injected with 2DG 6 or 18 h following the beginning of melatonin (for experimental birds: MT06 and MT18 respectively) or ETOH (for control birds: ET06 and ET18 respectively) administration, allowed to survive 1 h and killed for 2DG and IMEL autoradiography. The data indicated 2DG rhythmicity such that uptake was high at MT18 in vSCN and several visual, auditory and limbic system structures in birds receiving melatonin but not in birds receiving ETOH. Similarly, IMEL binding rhythms were restored in vSCN and other visual, auditory and limbic system structures in birds receiving melatonin but not in those receiving ETOH. These data indicate that melatonin cycles are responsible for generating and/or driving a wide array of cerebral metabolic rhythms and that this influence is inhibitory.

Pineal regulation of circadian rhythms of 2-deoxy[14C]glucose uptake and 2[125I]iodomelatonin binding in the visual system of the house sparrow,Passer domesticus

The pineal gland and its hormone melatonin are crucial for the generation of circadian rhythms in several species of passerine birds. The sites and mechanisms by which they influence avian behavior are therefore of particular interest. Recent research employing several brain imaging techniques has indicated that the sites of melatonin action within the avian brain are wide-spread within the 4 major visual pathways. In this study, we have investigated whether the avian homologue of the mammalian suprachiasmatic nucleus, the visual suprachiasmatic nucleus (vSCN), and other visually sensitive structures express circadian rhythms of 2-deoxy[14C]glucose (2DG) uptake and 2[125I]iodomelatonin (IMEL) binding in house sparrows,Passer domesticus, under constant environmental conditions in the presence or absence of the pineal gland. The results indicate that 2DG uptake in the vSCN is oscillatory in sham-operated sparrows but damps to arrhythmicity in pinealectomized birds, suggesting this structure contains a damped circadian oscillator independent of pineal input. We have also asked whether IMEL binding is rhythmic under these conditions in the same brains. These results indicate IMEL binding is rhythmic in several structures in the circadian, tectofugal, thalamofugal visual pathways and that pinealectomy increases the level of IMEL binding 2–4 fold suggesting that IMEL binding is down regulated by endogenous melatonin. However, the circadian rhythm of this binding is only gradually abolished, suggesting it too is regulated by a non-pineal circadian clock. These data are discussed in the context of the behavioral neurobiology of avian circadian systems and the neuroendocrine loop model.

Daily and circadian regulation of 2-[125I]iodomelatonin binding in the chick brain.

The pineal gland and its hormone melatonin are important in the control of circadian rhythms in birds. Recent research has indicated the presence of high affinity melatonin-binding sites in a wide array of avian cerebral structures. In this study melatonin-binding sites were localized and characterized in the brains of 2-week-old chicks using in vitro autoradiography and image analysis of 2-[125I]iodomelatonin (IMEL) binding. The IMEL binding predominated in the major components of the chick's visual and auditory systems. These brain binding sites showed a daily variation in IMEL binding, with a higher density of binding during the daytime. A significant peak in IMEL binding was observed in the late afternoon, Zeitgeber time 10. This rhythm of IMEL binding continued in constant darkness, with the peak at circadian time 10. The amplitude of the peak was increased in constant darkness in all structures, with the exception of the ectostriatum and neostriatum. Scatchard analysis of the binding revealed an average increase of 45 +/- 5% in the number of binding sites in the daytime vs. nighttime samples, with little change in the binding affinities, indicating that the rhythm in binding is caused by an increase in the total number of binding sites available in the daytime tissue, rather than an increase in the affinity of the binding sites. The data suggest that a rhythm of melatonin sensitivity in a diverse set of cerebral structures may regulate a temporal control of their function.

Daily and circadian regulation of 2-[125I]iodomelatonin binding in the chick brain

The pineal gland and its hormone melatonin are important in the control of circadian rhythms in birds. Recent research has indicated the presence of high affinity melatonin-binding sites in a wide array of avian cerebral structures. In this study melatonin-binding sites were localized and characterized in the brains of 2-week-old chicks using in vitro autoradiography and image analysis of 2-[125I]iodomelatonin (IMEL) binding. The IMEL binding predominated in the major components of the chick's visual and auditory systems. These brain binding sites showed a daily variation in IMEL binding, with a higher density of binding during the daytime. A significant peak in IMEL binding was observed in the late afternoon, Zeitgeber time 10. This rhythm of IMEL binding continued in constant darkness, with the peak at circadian time 10. The amplitude of the peak was increased in constant darkness in all structures, with the exception of the ectostriatum and neostriatum. Scatchard analysis of the binding revealed an average increase of 45 +/- 5% in the number of binding sites in the daytime vs. nighttime samples, with little change in the binding affinities, indicating that the rhythm in binding is caused by an increase in the total number of binding sites available in the daytime tissue, rather than an increase in the affinity of the binding sites. The data suggest that a rhythm of melatonin sensitivity in a diverse set of cerebral structures may regulate a temporal control of their function.

Sites of melatonin action in the brain of the house sparrow, Passer domesticus

AbstractThe pineal gland and its hormone melatonin are important for the generation of circadian rhythms by passerine birds such as the house sparrow, Passer domesticus. The sites of melatonin action within the brain of this species were determined by employing two techniques. First, the distribution of 2[125I]iodomelatonin (IMEL) binding was determined by in vitro incubation in IMEL, autoradiography, and computer image analysis. Data from these experiments indicated reversible IMEL binding in a wide array of cerebral structures primarily associated with vision. Second, the effects of exogenous melatonin on cerebral uptake of the metabolic marker 2‐deoxy[14C]glucose (2DG) were determined. Many of the same structures that bind IMEL also exhibited decreased 2DG uptake in response to melatonin administration, whereas structures that did not bind IMEL were unaffected by the hormone. The data suggest that much of the house sparrow's visual world is modulated on a circadian basis via the circadian secretion of melatonin. These observations are discussed in the context of avian circadian organization and the role it may play in the behavioral physiology of the bird.

The Distribution of Melatonin Binding Sites in Neuroendocrine Tissues of the Ewe1

Specific high-affinity binding of 2-[125I]iodomelatonin (IMEL) was examined in 20-micrometer sections prepared from intact Suffolk ewes killed during late anestrus or the breeding season. The pars tuberalis contained by far the highest concentration of IMEL binding sites of all areas studied. Within the telencephalon, intense labeling was found in the mediolateral septum, the ventrolateral septal and septohypothalamic nuclei, the entorhinal cortex, the subiculum, and the inner and outer molecular layers of the hippocampus adjacent to the dentate gyrus. Melatonin binding in the medial preoptic nucleus, bed nucleus of the stria terminalis, and medial preoptic area was less striking but still distinct. Among diencephalic regions, melatonin binding sites existed in low concentrations in the anterior hypothalamus, the tuberal medial basal hypothalamus, and the paraventricular thalamic and supramammillary nuclei. Little binding was evident in the suprachiasmatic or ventromedial nuclei. In the midbrain, significant binding was restricted to the ventral raphe complex and the inferior colliculus. Little specific binding was found in the pars distalis or the pineal gland. The distribution of melatonin binding in the sheep brain is discussed in the context of the influence of this pineal hormone upon seasonal changes in neuroendocrine function.