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.     

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 Kd value of melatonin for its receptors in rats and European hamsters (about 70 pM). In contrast, the Bmax 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.     

持续光照或摘除松果腺对仓鼠视交叉上核神经元褪黑素敏感性的影响

为探讨视交叉上核 (SCN)神经元对褪黑素敏感性的昼夜节律改变的机制 ,先对仓鼠进行持续光照或摘除松果腺 ,然后制成下丘脑薄片 ,记录昼夜周期中 SCN神经元的自发单位放电 ,并观察其对褪黑素的反应。结果表明 ,取自正常光照动物的脑薄片 ,SCN对外源性褪黑素产生抑制反应的单位数量有昼多夜少的节律性。取自持续光照条件下或摘除松果腺动物的 SCN,对外源性褪黑素反应的昼夜节律性消失。持续光照条件下 ,起抑制反应的单位数量增加 ,引起反应的阈值无明显改变 ;在摘除松果腺后 ,起抑制反应的单位数量减少 ,引起反应的阈值升高。实验结果提示 ,SCN神经元对外源性褪黑素敏感性 ,与内源性褪黑素水平和褪黑素受体的适应性调制有关 ,还可能与松果腺和内源性褪黑素的其他神经生化作用有关。     

Photic regulation of mt1 melatonin receptors in the Siberian hamster pars tuberalis and suprachiasmatic nuclei: involvement of the circadian clock and intergeniculate leaflet

In the Siberian hamster suprachiasmatic nuclei and pars tuberalis of the pituitary, high affinity mt1 melatonin receptors are present. We have previously shown that night applied light pulse induced an increase in mt1 mRNA expression in the suprachiasmatic nuclei of this species, independently of the endogenous melatonin. Here, we report the photic regulation of melatonin receptor density and mRNA expression in the suprachiasmatic nuclei and pars tuberalis of pinealectomized Siberian hamsters and the implication in this control of either the circadian clock or the intergeniculate leaflet. The results show that: (1) A 1-h light pulse, delivered during the night, induces a transitory increase in mt1 mRNA expression in the suprachiasmatic nuclei and pars tuberalis. After 3 h this increase has totally disappeared (suprachiasmatic nuclei) or is greatly reduced (pars tuberalis). (2) The melatonin receptor density, in the suprachiasmatic nuclei, is not affected by 1 or 3 h of light, while it is strongly increased in the pars tuberalis. (3) In hamsters kept in constant darkness, the mt1 mRNA rise is gated to the subjective night in the suprachiasmatic nuclei and pars tuberalis. In contrast, the light-induced increase in melatonin binding is also observed in the subjective day in the pars tuberalis. (4) intergeniculate leaflet lesion totally inhibits the mt1 mRNA expression rise in the suprachiasmatic nuclei, while it has no effect on the light-induced increase in mt1 mRNA in the pars tuberalis. However, the light-induced increase in melatonin receptor density is totally prevented by the intergeniculate leaflet lesion in the pars tuberalis. These results show that: (1) the photic regulations of mt1 mRNA expression and receptor density are independent of each other in both the suprachiasmatic nuclei and pars tuberalis; and (2) the circadian clock and the intergeniculate leaflet are implicated in the photic regulation of melatonin receptors but their level of action differs totally between the suprachiasmatic nuclei and pars tuberalis.     

Maternal pinealectomy increases depressive-like responses in Siberian hamster offspring

This study investigated the effect of maternal pinealectomy and postnatal pinealectomy on affective responses. Siberian hamsters were born to either pinealectomized or sham-operated dams and then underwent pinealectomy or a sham operation. Maternal pinealectomy increased depressive-like responses of offspring in the forced swim test. Maternal pinealectomy increased rearing behaviour and postnatal pinealectomy increased locomotor behaviour in the open field test. These results suggest that prenatal melatonin organizes adult affective responses.     

Pinealectomy aggravates and melatonin administration attenuates brain damage in focal ischemia.

Large infarcts develop in pinealectomized rats subjected to middle cerebral artery occlusion, which was attributed to loss of antioxidant action of melatonin. However, melatonin also has vascular actions, and pinealectomy may induce hypertension. The authors investigated (1) whether hemodynamic factors contribute to infarct development in pinealectomized rats, (2) whether melatonin administration can reverse the unfavorable effect of pinealectomy on infarct formation, and (3) whether melatonin can reduce the infarct volume in nonpinealectomized rats subjected to focal transient ischemia (2 hours middle cerebral artery occlusion, 22 hours reperfusion). Rats were pinealectomized 3 months before ischemia to eliminate any possible action of pinealectomy-induced hypertension on stroke. Blood pressure and regional CBF values during ischemia and reperfusion were not significantly different between pinealectomized and sham-operated rats, suggesting that pinealectomy-induced increase in infarct was not related to hemodynamic factors. The infarct volume resumed to the level of sham-operated rats on melatonin administration. Injection of melatonin (4 mg/kg) before both ischemia and reperfusion reduced infarct volume by 40% and significantly improved neurologic deficit scores in pinealectomized as well as sham-operated rats subjected to middle cerebral artery occlusion. These data suggest that physiologic melatonin release as well as exogenously given melatonin has a neuroprotective action in focal cerebral ischemia.     

Brain and Pituitary Melatonin Receptors in Male Rat during Post-Natal and Pubertal Development and the Effect of Pinealectomy and Testosterone Manipulation

Using quantitative autoradiography, melatonin receptors have been studied during post-natal and pubertal development of the rat in 2 brain and 2 pituitary structures. In the pars distalis of anterior pituitary, melatonin receptors decrease gradually in density after birth and disappear in 30 day-old animals. In contrast melatonin binding is only expressed in the paraventricular nuclei of the thalamus at the age of 21–23 days and is always present in adult animals. In the suprachiasmatic nuclei and in the pars tuberalis of the pituitary, melatonin receptor density decreases after birth, remains stable for approximately 1 month and increases again at puberty to reach the birth values in the adult. This increase was absent in pinealectomized and in castrated animals but present in castrated animals receiving testosterone suggesting that it depends upon circulating testosterone and melatonin levels. These results show that melatonin receptors are differentially regulated during post-natal development in each of the 4 structures studied, and that melatonin and testosterone are 2 factors which could be involved in the regulation of melatonin receptor density in the suprachiasmatic nuclei and pars tuberalis.     

Pinealectomy stimulates and exogenous melatonin inhibits harmful effects of epileptiform activity during pregnancy in the hippocampus of newborn rats: an immunohistochemical study

Objectives Epilepsy during the pregnancy is an important problem in clinical practice for newborn individuals. Recently, it has been demonstrated that mothers’ epileptic seizures have some harmful effects on newborns, but present data concerning the effects of epileptic phenomena in pregnant mothers on newborn pups are still limited. The current study was undertaken to investigate the morphological changes in the hippocampus of newborn pups of pinealectomized rats subjected to experimental epilepsy during pregnancy.Methods In this study, rats were randomly divided into four groups (ten animals each): intact control group, epilepsy control group, surgical pinealectomy + epilepsy group, and group with melatonin treatment following pinealectomy procedure. The animals in surgical pinealectomy + epilepsy and melatonin treatment groups underwent a surgical intervention consisting of pineal gland removal. At 1 month after surgical pinealectomy, an acute grand mal epileptic seizure was induced by 400 IU penicillin G administration into their hippocampal CA3 region on the 13th day of their pregnancy in all animals except the intact control animals. On the first neonatal day, the hippocampi were removed and processed for microscopic examination. Nestin expression was analysed in the developing hippocampal tissue.Results Normal migration and hippocampal maturation were determined in the postnatal rat hippocampus in intact control group, but the morphological structure of the hippocampus in the epilepsy control group corresponded to the early embryonal period. It was found that experimental epilepsy and pinealectomy enhanced nestin immunoreactivity, whereas exogenous melatonin treatment (30 μg/100 g body weight, intraperitoneal) inhibited pinealectomy-stimulated nestin expression in CA1 region of the hippocampus.Conclusion These findings suggest that epileptic seizures during pregnancy may cause an impaired hippocampal neurogenesis and neuronal maturation in the newborn, and the negative effects in the postnatal rat hippocampus are more dramatic after pinealectomy of the mother; conversely, melatonin administration suppresses these negative changes. This is the first report investigating the effects of maternal epilepsy during pregnancy in pinealectomized rats on nestin immunoexpression in the newborn rat hippocampus.Presented in part at the 4th Asian-Pacific International Congress of Anatomists (APICA), Kuşadası, Turkey, 7-10 September 2005.     

Both pineal and lateral eyes are needed to sustain daily circulating melatonin rhythms in sea bass

This study investigates the role of the pineal organ and lateral eyes (the two most important sources of melatonin in vertebrate species) on daily melatonin rhythms of sea bass, a fish exhibiting reversed melatonin profiles, as well as their contribution to circulating melatonin levels. To this aim, the pineal and/or the eyes were surgically removed (Exp. 1), the optic nerve sectioned and retinal dopaminergic neurons damaged with injections of 6-hydroxydopamine (Exp. 2), and the pineal or the eyes covered with aluminium foil (Exp. 3). The results show that plasma and ocular melatonin display opposing profiles. In Experiment 1, pinealectomized fish displayed lower nightly plasma melatonin levels (66+/-22 pg/ml) than intact or sham-operated groups (131+/-14 pg/ml), as it occurred in ophthalmectomized fish (64+/-12 pg/ml). Fish that were both pinealectomized and ophthalmectomized showed a further decrease in plasma melatonin levels (1.4+/-0.4 pg/ml), which approached daytime levels. In Experiment 2, plasma melatonin levels in both optic nerve-sectioned and ophthalmectomized fish were lower than control levels, while injection of 6-hydroxydopamine did not modify plasma melatonin concentrations. In Experiment 3, covering only the pineal made melatonin drop after a light pulse at MD, and covering only the eyes had a similar effect. In conclusion, these findings suggest that even though sea bass eyes do not directly contribute to plasma melatonin, the pineal organ, which unlike that of mammals is a direct photoreceptor in fish, requires light information from the lateral eyes to normally secrete melatonin into the bloodstream.     

Possible melatonin involvement in the hypotensive effect of MAO inhibitors

Summary Effect of selective inhibitors of MAO-A and B isoenzymes on pineal melatonin (and related indoles), arterial blood pressure and brain MAO-A and B activities has been evaluated in intact, pinealectomized and shamoperated rats.Selective inhibition of MAO-A but not MAO-B activity stimulated pineal melatonin synthesis and decreased arterial blood pressure in intact and sham-operated animals. Pinealectomy attenuated the hypotensive effect of MAO-A inhibition.The possible melatonin contribution to both antidepressive and hypotensive effects of MAO inhibitors is discussed.     

Circadian photic regulation of melatonin receptor density in rat suprachiasmatic nuclei: Comparison with light induction of fos-related protein

High-affinity melatonin receptors are present in rat suprachiasmatic nuclei (SCN), and their density exhibits a daily rhythm regulated by the light/dark cycle. In this study we demonstrate that the light regulation of these receptors depends on a circadian mechanism. Pinealectomized rats kept in constant darkness were subjected to 1-hr light pulses delivered across the circadian cycle. The density of melatonin receptors was significantly increased when photic exposure was performed during subjective night, and not different from control animals kept in darkness when the light pulse was applied during subjective day. The protein product (Fos) of the immediate early gene c-fos studied in the same paradigm showed globally the same circadian sensitivity phase. These results clearly show that, although the rhythmic appearance of melatonin receptor density in SCN follows and is directly regulated by the standard light/dark cycle, this light regulation is not passive. As is the case with Fos-like protein, it is only during a precise phase of the circadian cycle that light is able to regulate the density of melatonin receptors in SCN. © 1996 Wiley-Liss,Inc.     

Beneficial effects of melatonin on morphological changes in postnatal cerebellar tissue owing to epileptiform activity during pregnancy in rats: light and immunohistochemical study

Although it has been demonstrated that maternal epilepsy has some harmful effects on newborn individuals, current data concerning the effects of epileptic phenomena in pregnant mothers on newborn pups are still limited. This study was undertaken to investigate the changes in the cerebellum of newborns of pinealectomized rats subjected to experimental epilepsy during pregnancy. In our study, the rats were randomly divided into six groups: intact control group, anesthesia control group, epilepsy group, melatonin-treated epileptic group, surgical pinealectomy group, and group of melatonin treatment following pinealectomy procedure. At 1 month after pinealectomy, an acute grand mal epileptic seizure was induced by 400 IU penicillin-G administration into their intrahippocampal CA3 region during the 13th day of their pregnancy in all animals except intact control group. On the neonatal first day, pups were perfused transcardially and the cerebellums removed were processed for light microscopic and immunohistochemical studies. Normal migration and maturation were determined in the postnatal rat cerebellum in both intact control and anesthesia (ketamine-xylazine) control groups, but the morphological structure of cerebellum in the epilepsy control group corresponded to the early embryonal period. It was found that experimental epilepsy or pinealectomy procedure enhanced nestin immunoreactivity, but exogenous melatonin treatment (30 microg/100 g body weight, i.p.) following pinealectomy inhibited increased nestin expression induced by melatonin deprival in vermis region of newborn rat cerebellum (P < 0.001). Our results confirm that epileptic seizures during pregnancy may impair neurogenesis and neuronal maturation in newborns, which are more dramatic in the presence of melatonin deficiency during pregnancy, explaining more harmful effects of epileptic seizures to embryos of aged mothers. To the best of our knowledge, this is the first study reporting the effects of maternal epilepsy during pregnancy in pinealectomized rats on nestin immunoexpression in the newborn rat cerebellum.     

The effects of the intraamygdalar melatonin injections on the anxiety like behavior and the spatial memory performance in male Wistar rats

In the present study, the effects of intraamygdalar administrations of melatonin (1 and 100 μg/kg), saline and diazepam on the anxiety-like behavior and spatial memory performance in pinealectomized and sham-pinealectomized Wistar rats were investigated. The animals were tested by open field and elevated plus maze tests for anxiety-like behavior, and Morris water maze test for spatial memory. In open field, (a) diazepam was more effective in reducing the anxiety, (b) control subjects were more mobile than pinealectomized subjects and (c) 100 μg/kg melatonin administrations reduced the velocity of the animals. In elevated plus maze, (a) 100 μg/kg melatonin administrations increased the distance totally travelled and (b) enhanced the time spent in open arms, however, after the pinealectomy, 1 μg/kg melatonin administrations decreased it and (c) control animals were less mobile than pinealectomized ones. In Morris water maze, (a) diazepam group travelled more distance than the others in control condition whereas, in pinealectomy condition high dose of melatonin and saline groups travelled more distance than the others, (b) in pinealectomy condition subjects who received 100 μg/kg melatonin also travelled more distance than those who received 1 μg/kg melatonin and diazepam, (c) the subjects who received 1 μg/kg spent less time than those who received other treatments, and (d) in control condition subjects who received 100 μg/kg melatonin were slower than those who received the other treatments. In conclusion, melatonin administration to amygdala decreased the anxiety; however, spatial memory performance of the rats was impaired by the pinealectomy and melatonin administrations.     

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.     

褪黑素治疗可纠正去松果体大鼠齿状回颗粒下区Bax和Bcl-2蛋白表达的比例异常

以Morris水迷宫和嗅觉记忆实验测定去松果体大鼠平均逃避潜伏期较假损伤大鼠明显延长,褪黑素治疗后平均逃避潜伏期较假损伤大鼠稍延长,但比去松果体大鼠明显缩短。免疫组织化学和TUNEL染色结果显示,褪黑素治疗去松果体大鼠海马齿状回颗粒下区Bax阳性细胞数、TUNEL阳性细胞数均显著低于去松果体大鼠,Bcl-2阳性细胞数高于去松果体大鼠,褪黑素治疗去松果体大鼠各项指标均接近假损伤大鼠。提示褪黑素治疗可纠正去松果体大鼠海马齿状回颗粒下区促凋亡基因Bax和抗凋亡基因Bcl-2蛋白表达水平比例的异常,抑制细胞凋亡,进而改善其空间学习记忆能力。     

Investigations of the regulation of specific 2-[I]iodomelatonin binding sites in Siberian hamsters by endogenous and exogenous melatonin

These studies used quantitative in vitro autoradiography to investigate whether endogenous or exogenous melatonin modulate specific 2-[¹²⁵I]iodomelatonin binding sites in the pars tuberalis or suprachiasmatic nuclei of Siberian hamsters. Saturation analyses were conducted on tissue sections from hamsters that were pinealectomized, exposed to constant illumination (72 h), or injected with melatonin, and from hamsters that were treated as controls. High affinity (K_d approximately 20–75 pM) specific 2-[¹²⁵I]iodomelatonin binding sites were detected in the suprachiasmatic nuclei and pars tuberalis of all animals. Neither pinealectomy nor constant illumination significantly affected either the affinity or the density of the specific 2-[¹²⁵I]iodomelatonin binding sites in either region. Melatonin injections led to a decrease in specific 2-[¹²⁵I]iodomelatonin binding to the pars tuberalis at 3–4 h after the last injection. However, washing the sections before incubation with 2-[¹²⁵I]iodomelatonin eliminated this effect, suggesting that melatonin was occupying the binding sites rather than decreasing their actual number. Furthermore, when hamsters were sacrificed 18 h after the last melatonin injection, no effect of melatonin on either the affinity or density of specific 2-[¹²⁵I]iodomelatonin sites was observed. These data suggest that 2-[¹²⁵I]iodomelatonin binding sites in Siberian hamsters are not regulated by changes in circulating melatonin levels.     

Opposite effects of pinealectomy and melatonin administration on brain damage following cerebral focal ischemia in rat

The endocrine system has been recognized as an important factor that may contribute to the outcome of stroke. We tested in rats the hypothesis that the pineal gland and/or its hormone melatonin may affect the outcome of a transient cerebral arteries occlusion (CerAO). Reversible 90 min focal ischemia was produced using a three-vessel occlusion method. Surgically or sham pinealectomized rats were exposed to CerAO 15 days after surgery. Melatonin (4 x 2.5 mg/kg: 30 min prior to onset of CerAO, immediately after recirculation was established, and 1 and 2 hr later) or its vehicle were administered intraperitoneally. The outcome of CerAO was assessed by quantitative assay of DNA damage or by Nissl staining and measurement of the infarct volume. Pinealectomy increased both the extent of DNA damage and the infarct volume; administration of melatonin to pinealectomized rats reduced both these markers of brain injury. We propose that the pineal endocrine system may influence the outcome of stroke. The mechanism of action and the pathophysiological role of this system, e.g., in aging, should be further characterized.     

The regional and cellular expression profile of the melatonin receptor MT1 in the central dopaminergic system

The physiological effects of pineal melatonin are primarily mediated by melatonin receptors located in the brain and periphery. Even though there are a number of studies demonstrating the regulatory role of melatonin in the development of dopaminergic behaviors, such as psychostimulant-induced diurnal locomotor sensitization or drug seeking, little is known about the contribution of melatonin receptors (i.e., MT1) to this role. Therefore, as a first step in understanding the functional role of melatonin receptors in dopaminergic behaviors, we focused on determining the expression pattern of MT1 receptors in the dopaminergic system of the human and rodent brain. Regional (e.g., nucleus accumbens shell) and cellular (e.g., tyrosine hydroxylase immunopositive cells) expression of MT1 mRNA was characterized by applying the immuno-laser capture microdissection (immuno-LCM) technique coupled with nested RT-PCR. Moreover, employing quantitative Western immunoblotting and RT-PCR, we found that the mouse MT1 receptor expression presents diurnal variations (i.e., low mRNA and high protein levels at night, ZT21). The dopaminergic system-based presence of MT1 receptor proteins was not limited to rodents; we found these receptors in postmortem human brain as well. Further research is needed to understand the regional/cellular functional role of melatonin receptors in the regulation of dopaminergic behaviors, using models such as melatonin receptor knockout mice.     

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.