Density Of Melatonin Receptors Research Articles

Daily Melatonin Injections Induce Cytological Changes in Pars Tuberalis‐Specific Cells Similar to Short Photoperiod

Hypophyseal pars tuberalis (PT)-specific cells are known to exhibit remarkable seasonal changes in morphology especially in photoperiodic animals like the Djungarian hamster Phodopus sungorus. Their high density of melatonin-receptors leads to the supposition that fluctuations in circulating melatonin levels are a crucial factor for the morphological alterations induced by photoperiodic signals. To prove this hypothesis the nocturnal elevation of melatonin in long photoperiods was prolonged by late afternoon administration of melatonin. We investigated whether this treatment induces cytological changes usually observable under short photoperiod. Electron microscopy revealed that in contrast to hamsters maintained in long photoperiods PT-specific cells of hamsters injected with melatonin or those kept in short photoperiods appear inactive, containing a relatively high number of secretory granules, sparse endoplasmatic reticulum, irregularly outlined and invaginated cell nuclei and a high amount of glycogen. Furthermore immunoreactivity for the common alpha-chain of glycoprotein hormones and beta-TSH was significantly weaker in hamsters kept in short photoperiods or daily injected with melatonin than untreated or vehicle injected controls in long photoperiod. These results demonstrate that an exogenous prolongation of the elevated nocturnal melatonin levels causes a similar morphological appearance of PT-specific cells as observed in short photoperiods. It is tempting to speculate that the melatonin signal is a direct 'Zeitgeber' for the transduction of photoperiodic information to the secretory activity in this cell type.

Influence of maternal melatonin on melatonin receptors in rat offspring.

Using quantitative autoradiography, we have studied the influence of maternal plasma melatonin on the expression and density of melatonin receptors in the brain and pituitary of rat offspring. At birth, the same structures displayed melatonin receptors whether the rats were born to and reared by intact or pinealectomized dams. The receptor density was, however, about 20% lower in the group born to pinealectomized dams. At postnatal day 9, when the pups of both groups synthetize rhythmically their own melatonin, this difference was suppressed. These results indicate that melatonin does not appear to be a requirement for the expression of its receptors, but seems to play a stimulatory role in their synthesis.

Seasonal regulation of melatonin receptors in rodent pars tuberalis: correlation with reproductive state.

Using quantitative autoradiography, we have studied the relationship between melatonin receptor density in the pars tuberalis (PT) and photoperiodic changes in sexual activity in a photoperiodic (Syrian hamster), and a non photoperiodic (rat) rodent. Syrian hamsters exposed to short photoperiod (SP) for 13 weeks or kept in long photoperiod (LP) with daily melatonin injections for 8 weeks, had both gonadal regression and a significant decrease in 2-125I-melatonin binding site density in the PT when compared to controls. In contrast, when the animals were sexually active, photorefractory Syrian hamsters exposed to SP for 27 weeks, or rats kept for 13 weeks in SP, the PT melatonin receptor density was similar to that of control animals kept in LP. These results show clearly that a correlation exists between gonadal status and density of melatonin receptors in the PT and suggest that the PT could be the site where melatonin mediates its effects on seasonal function.

The density of melatonin receptors is dependent upon the prevailing photoperiod in the Japanese quail ( Coturnix japonica)

Two groups of Japanese quail ( Coturnix japonica) were exposed to two different photoperiods (short and long-days: LD 8:16 and LD 16:8, respectively), and their brains examined for the presence and distribution of melatonin receptors by means of quantitative in vitro autoradiography. Animals belonging to the LD 8:16 group expressed a significantly higher melatonin receptor density in the optic tectum and nucleus triangularis, while the LD 16:8 animals had a higher density of receptors in the hyperstriatum and nucleus preopticus dorsalis. These data demonstrate an apparent influence of the photoperiod on the density of melatonin receptors, especially in nuclei of the tectofugal pathway, related to the control of visual pattern and intensity discrimination, localization and orientation.

The NMDA antagonist MK-801 blocks light effect on melatonin receptor density in rat suprachiasmatic nuclei

In pinealectomized rats, melatonin receptor density in the suprachiasmatic nuclei was recently shown to be strongly decreased during the dark period. Furthermore, a one-hour long light pulse is able to reverse this dark-induced decrease. We demonstrate here that this effect of a light-pulse during night time is blocked by prior application of the NMDA receptor antagonist MK-801. These results show that the light effect on melatonin receptor density in the SCN is mediated by activation of NMDA-type receptors.

Daily variations in melatonin receptor density of rat pars tuberalis and suprachiasmatic nuclei are distinctly regulated

Suprachiasmatic nuclei (SCN) and pars tuberalis (PT) are two structures in the rat exhibiting high affinity receptors for melatonin. Melatonin receptor density in these two structures was previously shown to be inversely related to endogenous ligand concentration, thus elevated at daytime. We now demonstrate that, in the PT, these daily variations are directly induced by the circadian rhythm of plasma melatonin concentration. Variations persist in constant darkness and can only be blocked by pinealectomy. Thus, autoregulation loop of melatonin receptors determines the circadian rhythm in PT melatonin receptor de density. However, this process of densensitization does not determine the daily variations in SCN melatonin receptor density. Indeed, in the SCN, the light/dark cycle is the regulatory factor: melatonin receptor density was shown to be specifically reduced during the night even in pinealectomized animals, while one h light was shown to reverse this nocturnal decrease in the SCN. Moreover, this darkness-induced down-regulation of SCN melatonin receptors has a masking effect on the earlier shown ligand-dependent desensitization process in this structure. This explain why, in constant darkness, SCN melatonin receptor density did not show any variation throughout the 24 h subjective day and night, although the circadian rhythm of melatonin persisted. These results thus clearly show that although daily rhythms in the density of melatonin receptors are identical in the PT and in the SCN, their regulation is totally different in each of these two structures.

Differential regulation of melatonin receptors by short- versus long-term pinealectomy in the rat suprachiasmatic nuclei and pars tuberalis.

We have investigated the effects of short- and long-term pinealectomy on the density of melatonin receptors in both the suprachiasmatic nuclei (SCN) and pars tuberalis (PT) of the rat. Short-term pinealectomy (conducted 3-5 days before sacrifice) induced a significant increase in the density of melatonin receptors in both structures. This increase can be interpreted as an up-regulation of melatonin receptors following the absence of plasma melatonin. In contrast, 7 days after pinealectomy, the receptor density began to decrease, reaching a low point 1.5 months after pinealectomy. At this time, the decrease represents a 40-45% reduction in binding sites as compared with values measured 3-5 days after pinealectomy. This slow decrease could be related to a slowdown of melatonin receptor synthesis. These results suggest that melatonin has a stimulatory effect on its own receptor synthesis. However, at 1.5 months after pinealectomy a novel equilibrium between synthesis and hydrolysis seems to be reached. Melatonin does not appear to be indispensable for the synthesis of its receptors.

Seasonality and melatonin receptors in the pars tuberalis in some long day breeders.

The density of high-affinity melatonin receptors in the pars tuberalis of different long day breeders exhibits a seasonal variation, with high values in spring-summer and low values in autumn-winter. In all the studies, this decrease in receptor density was always associated with both a short photoperiod and sexual inactivity. Moreover, during the hibernation period, the reduction in receptor number was more marked in hypothermic than in normothermic animals. These seasonal changes in melatonin receptors were restricted to the pars tuberalis, which suggests that this structure represents an important target site for mediation of the photoperiodic effect of melatonin on seasonal functions. The mechanism of melatonin action at this level is discussed.

Differential Seasonal Regulation of Melatonin Receptor Density in the Pars Tuberalis and the Suprachiasmatic Nuclei: A Study in the Hedgehog (Erinaceus europaeus, L.)

Using quantitative autoradiography, we have studied the seasonal changes of high affinity melatonin receptor density in both the SCN and PT of the hedgehog, a seasonal breeder and an hibernator. Animals in 3 different physiological states were studied: sexually active animals, and sexually inactive animals during the hibernation period, being then either euthermic or hypothermic. In sexually active animals, Bmax were 75.8 +/- 7.1 fmol/mg protein in PT and 9.1 +/- 0.5 fmol/mg protein in SCN; and Kd values were: 94 +/- 22 pM in the PT and 101 +/- 15 pM in the SCN. This specific binding was strongly decreased in the PT of sexually inactive animals. Moreover, this decrease was significantly stronger in hypothermic than in euthermic hedgehogs. Saturation studies and Scatchard analysis revealed that the observed decrease in the PT resulted from change in the Bmax but not in the Kd, Bmax values being respectively 56.4 +/- 5.9 and 29.5 +/- 1.9 fmol/mg protein in euthermic and hypothermic sexually at rest animals. In none of the different physiological states, did the density of melatonin receptors of the SCN show any changes, Bmax values being respectively 9.8 +/- 0.5 and 9.8 +/- 0.4 fmol/mg protein in euthermic and hypothermic sexually at rest animals. This shows for the first time a tissue-specific regulation of melatonin receptor density occurring in the PT but not in the SCN. Furthermore, this decrease of binding in the PT is correlated with both sexual inactivity and hibernation period. This strongly suggests that the mediation of the photoperiodic effect on seasonal functions like seasonal hypothermia and reproduction involves an effect of melatonin on the PT rather than on the SCN.

Effect of pinealectomy and the light/dark cycle on 2-[125I]iodomelatonin binding in the chick optic tectum.

1. These studies investigated the regulation of melatonin receptor sites within the chick brain by the light/dark cycle and by endogenous melatonin using quantitative autoradiography. Saturation analyses performed in coronal brain sections of the optic tectum revealed a single class of high-affinity binding sites. 2. For diurnal rhythm studies, chicks were maintained on a 14:10 L:D cycle (light on 0400) and sacrificed at 4-h intervals. No significant variation was observed in either the affinity (KD = 67.8-76.4 pM) or the density (BMAX = 88.9-118.9 fmol/mg protein) of 2-[125I]iodomelatonin binding sites as a function of time of day. 3. In pinealectomized or age-matched sham-operated chicks, no changes were found in either the affinity (KD = 66.7-89.1 pM) or the density (BMAX = 112.8-180.4 fmol/mg protein) of 2-[125I]iodomelatonin binding sites at either 1, 7, or 14 days following surgery. These data suggest that endogenous pineal melatonin may not play a role in the regulation of melatonin receptor site affinity or density in the chick optic tectum.

Melatonin receptor density is regulated in rat pars tuberalis and suprachiasmatic nuclei by melatonin itself

The pineal hormone melatonin interacts with the neuroendocrine system via high affinity binding sites. The density of these receptors is increased after 72 h constant light or pinealectomy in both rat pars tuberalis and suprachiasmatic nuclei. The present results show that, in rat PT and SCN, this increase can be inversed by a single melatonin injection (50 μg s.c. in 0.1 ml NaCl, 4 h before sacrifice). At the time of sacrifice, plasma melatonin concentrations of injected animals were no longer significantly different when compared to control values. Thus, this observed effect is not a result of a receptor occupation, but demonstrates a direct regulation of melatonin receptors by melatonin itself.

Asymmetric distribution of melatonin receptors in the brain of the lizard Anolis carolinensis

The pineal hormone melatonin may regulate seasonal reproduction and entrainment of circadian rhythms by binding to specific brain receptors. An analysis of melatonin receptor distribution in the lizard brain revealed an asymmetry of melatonin binding in the diencephalon. A high degree of melatonin binding was present in the left habenular nucleus, but no binding was observed in the habenulum of the right brain hemisphere. It is intriguing that the left habenular nucleus, in contrast to the right habenulum, both possesses a high density of melatonin receptors and receives primary photic input from the parietal eye. Similarly, the optic tectum, which receives primary visual input from the retina, is also rich in melatonin receptors. These observations suggest that the left habenulum is under dual control (neuronal and hormonal) of the parietal eye/pineal complex, and that melatonin may play a significant role in neural processing of visual information.

Effect of Constant Light, Pinealectomy and Guanosine Triphosphate Gamma‐S on the Density of Melatonin Receptors in the Rat Suprachiasmatic Nucleus: A Possible Implication of Melatonin Action

We report here the effects of pinealectomy and exposition to constant light on the density of melatonin receptors in the suprachiasmatic nuclei of the rat using quantitative autoradiography. The B(max) values were significantly increased when the animals were maintained in constant light for 3 days (8.22 ± 0.95 fmol/mg protein versus 4.55±0.14 fmol/mg protein in control group, 12 h light/12 h dark cycle (12L/12D), n = 6). A similar increase was also observed in rats pinealectomized 3 days before sacrifice and then maintained either under 12L/12D (B(max) 7.56±0.80 fmol/mg protein) or in constant light (B(max) 7.85±1.02 fmol/mg protein), while K(d) values failed to show any variations after constant light and/or pinealectomy. The effect of GTPγS on the density of rnelatonin binding sites was also investigated in control animals and after 3 days of constant light. In 12L/12D animals, the B(max) shifted from 5.94 ± 0.14 fmol/mg protein in the absence of GTPγS to 3.97±0.22 fmol/ mg protein in the presence of 50μiM GTPγS. In animals maintained for 3 days in constant light, a similar decrease in the B(max) value was observed (8.95 ± 0.25 fmol/mg protein in absence and 5.95 ± 0.22 fmol/mg protein in presence of 50 μ GTPγS). In both cases, K(d) values were not affected by GTPγS. Pinealectomy and constant light exposition are known to induce a suppression of the nocturnal peak of plasma rnelatonin and to keep plasma rnelatonin concentrations at a very low level. These results could suggest a regulatory effect of rnelatonin on the density of its own receptors which are shown here to be also coupled with a G-protein.

Melatonin binding to the anteroventral and anterodorsal thalamic nuclei in the rat.

Pineal melatonin is a putative humoral coordinator of various circadian body rhythms. Rather few loci in the brain contain such a density of melatonin receptors that specific binding can be demonstrated by autoradiography. In this study we measured, using in vitro receptor autoradiography, displaceable binding of iodo-melatonin to the anteroventral and anterodorsal thalamic nuclei of Wistar rats (0.6-1.4 fmol/mg protein at 100 pM ligand concentration). These nuclei of the limbic system have direct connections with the retina and they participate in learning and memory functions.

Pinealectomy and constant illumination increase the density of melatonin binding sites in the pars tuberalis of rodents

We report here the effects of pinealectomy and light exposure on the melatonin receptor density in the pars tuberalis of the rat and the European hamster using quantitative autoradiography. Scatchard analysis revealed that 24 and 72 h of constant light exposure (LL) before sacrifice did not modify the K d value of melatonin for its receptors in rats and European hamsters (about 70 pM). In contrast, the B max value was significantly increased in both species when the animals were kept in constant illumination for 72 h before sacrifice (50%–70% compared with the controls). A similar increase was also observed in rats pinealectomized 3 days before sacrifice and then kept in either constant illumination or in 12L/12D. Pinealectomy or constant light exposure are known to result in a clear decrease in the concentration of circulating melatonin. We demonstrate here that they also result in an increase in the density of melatonin receptors. This could suggest a direct effect of melatonin on its own receptors.

Melatonin binding sites in brain of the 2-day-old chicken: An autoradiographic localisation

Melatonin, released in a circadian manner from the avian pineal gland into the circulation, is concentrated in distinct brain areas, possibly by receptor-mediated uptake mechanisms. For anatomical localisation of putative melatonin receptors in birds, cryostat-cut, coronal 15 microns brain sections of 2-day-old chicken were investigated by means of in vitro receptorautoradiography, using 125I-melatonin as a ligand. Binding occurred in brain structures receiving or mediating visual or auditory sensory input. Binding characteristics were investigated in homogenates of a brain region labelled by 125I-melatonin and showed to be specific and saturable (Kd: 87.2 pM; Bmax: 16.1 fmol/mg protein). Results confirm that a wealth of structures in chicken brain are equipped with putative melatonin receptors, including the visual suprachiasmatic nucleus. However, as results show that fewer structures were labelled in the brain of 2-day-old chicken as compared to a similar investigation conducted with 12-day-old chicken, developmental changes in distribution and/or density of putative melatonin receptors might be present.

Impact of circulating testosterone on iodomelatonin binding sites in the male rat brain

The effects of castration and subsequent testosterone and estradiol treatment and of a single injection of ethylene-1,2-dimethanesulphonate (EDS) on the distribution of [2- 125I]iodomelatonin ([ 125I]melatonin) binding sites in the male rat brain were investigated. Castration produced a marked testosterone-reversible decrease in [ 125I]melatonin binding in the male rat brain, particularly in the hypothalamus and hippocampus. In contrast, [ 125I]melatonin binding in the parietal cortex, medulla-pons and cerebellum was generally unaffected by castration. Estradiol did not reverse the effect of castration on [ 125I]melatonin binding. A single injection of EDS which causes the destruction of Leydig cells led to a marked decrease in [ 125I]melatonin binding in the brain of the rats between 3 and 7 days after treatment. This decrease correlated with the decline in serum concentrations of testosterone. Specific [ 125I]melatonin binding and serum concentrations of testosterone subsequently increased to control levels within 37 days after treatment in accord with the repopulation of the Leydig cells. The results clearly show that testosterone regulates the density of melatonin receptors in the hypothalamus and hippocampus of the male rat.

Regulation of melatonin's activity in the female rat brain by estradiol: effects on neurotransmitter release and on iodomelatonin binding sites.

The effects of ovariectomy and of 17 beta-estradiol (estradiol) treatment in vivo and in vitro on the ability of melatonin to inhibit dopamine release from the female rat hypothalamus and on [125I]-iodomelatonin binding sites in the brains and hypothalami of female rats were investigated. In long-term (2-4 weeks) ovariectomized (OVX) female rats, the inhibitory effect of melatonin in vitro on dopamine release from the hypothalami was abolished. After implantation of estradiol capsules, the ability of melatonin to inhibit dopamine release from the hypothalamus was reinstated and resembled that observed in intact female rats at estrus and in females exhibiting spontaneous persisting estrus. Similar reinstatement of the responsiveness to melatonin was observed in hypothalami of OVX rats shortly (2 h) after a single subcutaneous injection of estradiol (200 micrograms/animal). Incubation of hypothalami of OVX rats in vitro for 50-90 min with estradiol (0.1-1 nM) resulted in a partial (up to 50%), time and steroid concentration-dependent reinstatement of the ability of melatonin to inhibit the induced dopamine release. Conversely, incubations with estradiol in vitro reduced the ability of melatonin to inhibit dopamine release from the hypothalami of intact rats at proestrus. Such incubations had no effect on the release of dopamine from hypothalami of rats at estrus or of short-term OVX (3 days) rats. The changes in the responsiveness of the hypothalamus to melatonin were accompanied by profound changes in the binding of [125I]-iodomelatonin, to synaptosomes isolated from whole brains and from hypothalami of the OVX female rats. In OVX rats, the densities of the binding sites in the brains and particularly in the hypothalami decreased to 18 and 24% respectively, of the values observed in control females at estrus. The apparent dissociation constant of the remaining sites was significantly lower (ca. 90 nM) than that observed in the intact controls (ca. 300 nM). An almost complete reinstatement of the [125I]-iodomelatonin binding sites was observed in synaptosomes prepared from the hypothalami of OVX rats shortly (2 h) after a single subcutaneous injection of estradiol, or after incubation with estradiol in vitro. The estradiol-mediated reinstatement of [125I]-iodomelatonin binding sites was less pronounced in synaptosomes prepared from whole brains. The results clearly show that estradiol directly modulates the responses of the dopaminergic neurosecretory system in the hypothalamus to melatonin. This phenomenon may be primarily associated with the estradiol-induced changes in the density and function of melatonin receptors in the hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Diurnal changes in melatonin binding sites of hamster and rat brains. Correlation with neuroendocrine responsiveness to melatonin

A diurnal variation in neuroendocrine sensitivity to melatonin is known to occur in hamsters and rats. The present experiments were carried out to examine the possibility that affinity and/or number of melatonin binding sites in brain could change accordingly at the two times when exogenous melatonin is known to be ineffective or effective to produce neuroendocrine changes, i.e., at 07 : 00 or 20 : 00 h (lights on from 07 : 00 to 21 : 00 h daily). The number of melatonin binding sites in hamster and rat brains was at 20 : 00 h 34–56% higher than at 07 : 00 h, without changing their affinity towards [ 3H]melatonin (hamster: K d = 53 nM; rat: K d = 73–77 nM). These alterations in melatonin receptor density may play a role in daily changes in sensitivity to the exogenous methoxyindole.

Neural regulation of circadian rhythms.

Neural regulation of circadian rhythms.