The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

Melatonin MT1 receptors as a target for the psychopharmacology of bipolar disorder: A translational study.

The treatment of bipolar disorder (BD) still remains a challenge. Melatonin (MLT), acting through its two receptors MT1 and MT2, plays a key role in regulating circadian rhythms which are dysfunctional in BD. Using a translational approach, we examined the implication and potential of MT1 receptors in the pathophysiology and psychopharmacology of BD. We employed a murine model of the manic phase of BD (Clock mutant (ClockΔ19) mice) to study the activation of MT1 receptors by UCM871, a selective partial agonist, in behavioral pharmacology tests and in-vivo electrophysiology. We then performed a high-resolution Nuclear Magnetic Resonance study on isolated membranes to characterize the molecular mechanism of interaction of UCM871. Finally, in a cohort of BD patients, we investigated the link between clinical measures of BD and genetic variants located in the MT1 receptor and CLOCK genes. We demonstrated that: 1) UCM871 can revert behavioral and electrophysiological abnormalities of ClockΔ19 mice; 2) UCM871 promotes the activation state of MT1 receptors; 3) there is a significant association between the number of severe manic episodes and MLT levels, depending on the genetic configuration of the MT1 rs2165666 variant. Overall, this work lends support to the potentiality of MT1 receptors as target for the treatment of BD.

Melatonin suppresses the metastatic potential of osteoblastic prostate cancers by inhibiting integrin α2 ß1 expression.

Advanced prostate cancer often develops into bone metastasis, which is characterized by aberrant bone formation with chronic pain and lower chances of survival. No treatment exists as yet for osteoblastic bone metastasis in prostate cancer. The indolamine melatonin (N-acetyl-5-methoxytryptamine) is a major regulator of the circadian rhythm. Melatonin has shown antiproliferative and antimetastatic activities but has not yet been shown to be active in osteoblastic bone lesions of prostate cancer. Our study investigations reveal that melatonin concentration-dependently decreases the migratory and invasive abilities of two osteoblastic prostate cancer cell lines by inhibiting FAK, c-Src, and NF-κB transcriptional activity via the melatonin MT1 receptor, which effectively inhibits integrin α2 ß1 expression. Melatonin therapy appears to offer therapeutic possibilities for reducing osteoblastic bone lesions in prostate cancer.

Melatonin impedes prostate cancer metastasis by suppressing MMP-13 expression.

Prostate cancer has high metastatic potential. Men with higher urinary levels of the sleep hormone melatonin are much less likely to develop advanced prostate cancer compared with men with lower levels of melatonin. Melatonin has shown anticancer activity in experimental investigations. Nevertheless, the therapeutic effect of melatonin in metastatic prostate cancer has largely remained a mystery. Analyses of Gene Expression Omnibus data and human tissue samples indicated that levels of matrix metallopeptidase 13 (MMP-13) expression are higher in prostate cancer patients than in healthy cancer-free individuals. Mechanistic investigations revealed that melatonin inhibits MMP-13 expression and the migratory and invasive capacities of prostate cancer cells via the MT1 receptor and the phospholipase C, p38, and c-Jun signaling cascades. Importantly, tumor growth rate and metastasis to distant organs were suppressed by melatonin in an orthotopic prostate cancer model. This is the first demonstration showing that melatonin impedes metastasis of prostate cancer by suppressing MMP-13 expression in both in vitro and in vivo models. Thus, melatonin is promising in the management of prostate cancer metastasis and deserves to undergo clinical investigations.

Nociceptive responses in melatonin MT2 receptor knockout mice compared to MT1 and double MT1 /MT2 receptor knockout mice.

Melatonin, a neurohormone that binds to two G protein-coupled receptors MT1 and MT2, is involved in pain regulation, but the distinct role of each receptor has yet to be defined. We characterized the nociceptive responses of mice with genetic inactivation of melatonin MT1 (MT1 -/- ), or MT2 (MT2 -/- ), or both MT1 /MT2 (MT1 -/- /MT2 -/- ) receptors in the hot plate test (HPT), and the formalin test (FT). In HPT and FT, MT1 -/- display no differences compared to their wild-type littermates (CTL), whereas both MT2 -/- and MT1 -/- /MT2 -/- mice showed a reduced thermal sensitivity and a decreased tonic nocifensive behavior during phase 2 of the FT in the light phase. The MT2 partial agonist UCM924 induced an antinociceptive effect in MT1 -/- but not in MT2 -/- and MT1 -/- /MT2 -/- mice. Also, the competitive opioid antagonist naloxone had no effects in CTL, whereas it induced a decrease of nociceptive thresholds in MT2 -/- mice. Our results show that the genetic inactivation of melatonin MT2 , but not MT1 receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT2 receptor subtype is selectively involved in the regulation of pain responses.

Melatonin MT1 receptor as a novel target in neuropsychopharmacology: MT1 ligands, pathophysiological and therapeutic implications, and perspectives.

Melatonin (MLT), a neuromodulator mainly acting through two G-protein coupled receptors MT1 and MT2, regulates many brain functions, including circadian rhythms, mood, pain and sleep. MLT and non-selective MT1/MT2 receptor agonists are clinically used in neuropsychiatric and/or sleep disorders. However, the selective roles of the MT1 and MT2 receptors need to be clarified. Here, we review the role of the MT1 receptor in neuropsychopharmacology, describe the anatomical localization of MT1 receptors in the brain, discuss the medicinal chemistry, biochemistry and molecular aspects of the receptor, and explore the findings linking MT1 receptors to psychiatric and neurological disorders. MT1 receptors are localized in brain regions which regulate circadian rhythms, sleep, and mood, such as the suprachiasmatic nucleus, cortex, hippocampus, dorsal raphe nucleus and lateral hypothalamus. Their activation modulates intracellular signaling pathways also targeted by psychoactive drugs, including antidepressants and mood stabilizers. MT1 receptor knockout mice display increased anxiety, a depressive-like phenotype, increased propensity to reward and addiction, and reduced Rapid-Eye-Movement sleep. These behavioral dysfunctions are associated with altered serotonergic and noradrenergic neurotransmissions. Several studies indicate that the MT1, rather than MT2, receptor is implicated in circadian rhythm regulation. The involvement of MT1 receptors in Alzheimer's and Huntington diseases has also been proposed. Postmortem studies in depressed patients have further confirmed the possible involvement of MT1 receptors in depression. Overall, there is substantial evidence indicating a role for MT1 receptor in modulating brain function and mood. Consequently, this MLT receptor subtype deserves to be further examined as a novel target for neuropsychopharmacological drug development.

Sleep well. Untangling the role of melatonin MT1 and MT2 receptors in sleep.

The pharmacological potential of targeting selectively melatonin MT1 or MT2 receptors has not yet been exploited in medicine. Research using selective MT1/MT2 receptor ligands and MT1/MT2 receptor knockout mice has indicated that the activation of MT2 receptors selectively increases non-rapid eye movement (NREM) sleep whereas MT1 receptors seem mostly implicated in the regulation of REM sleep. Moreover, MT1 knockout mice show an increase in NREM sleep, while MT2 knockout a decrease, suggesting an opposite role of these two receptors. A recent paper in mice by Sharma et al (J Pineal Res, 2018, e12498) found that MT1 but not MT2 receptors are expressed on orexin neurons in the perifornical lateral hypothalamus (PFH). Moreover, after injecting melatonin or luzindole into the mouse PFH, the authors suggest that melatonin promotes NREM sleep because activates PFH MT1 receptors, which in turn inhibit orexin neurons that are important in promoting arousal and maintaining wakefulness. In this commentary, we have critically commented on some of these findings on the bases of previous literature. In addition, we highlighted the fact that no conclusions could be drawn on the melatonin receptor subtype mediating the effects of melatonin on sleep because the authors used the non-selective MT1/MT2 receptors antagonist luzindole. More solid research should further characterize the pharmacological function of these two melatonin receptors in sleep.

Melatonin promotes sleep in mice by inhibiting orexin neurons in the perifornical lateral hypothalamus.

Melatonin promotes sleep. However, the underlying mechanisms are unknown. Orexin neurons in the perifornical lateral hypothalamus (PFH) are pivotal for wake promotion. Does melatonin promote sleep by inhibiting orexin neurons? We used C57BL/6J mice and designed 4 experiments to address this question. Experiment 1 used double-labeled immunofluorescence and examined the presence of melatonin receptors on orexin neurons. Second, mice, implanted with bilateral guides targeted toward PFH and sleep-recording electrodes, were infused with melatonin (500 pmole/50 nL/side) at dark onset (onset of active period), and spontaneous bouts of sleep-wakefulness were examined. Third, mice, implanted with bilateral guides into the PFH, were infused with melatonin (500 pmole/50 nL/side) at dark onset and euthanized 2 hours later, to examine the activation of orexin neurons using c-Fos expression in orexin neurons. Fourth, mice, implanted with PFH bilateral guides and sleep-recording electrodes, were infused with melatonin receptor antagonist, luzindole (10 pmol/50 nL/side), at light onset (onset of sleep period), and spontaneous bouts of sleep-wakefulness were examined. Our results suggest that orexin neurons express MT1, but not MT2 receptors. Melatonin infusion into the PFH, at dark onset, site-specifically and significantly increased NREM sleep (43.7%, P = .003) and reduced wakefulness (12.3%, P = .013). Local melatonin infusion at dark onset inhibited orexin neurons as evident by a significant reduction (66%, P = .0004) in the number of orexin neurons expressing c-Fos. Finally, luzindole infusion-induced blockade of melatonin receptors in PFH at sleep onset significantly increased wakefulness (44.1%, P = .015). Based on these results, we suggest that melatonin may act via the MT1 receptors to inhibit orexin neurons and promote sleep.

Genetic deletion of MT1 melatonin receptors alters spontaneous behavioral rhythms in male and female C57BL/6 mice.

Behaviors vary over the 24h light/dark cycle and these temporal patterns reflect in part modulation by circadian neural circuits and hormones, such as melatonin. The goal of this study was to investigate the involvement of MT1 melatonin receptors in behavioral regulation by comparing male and female C57 wild type (WT) mice with C57 mice with genetic deletion of the MT1 receptor (MT1KO). A comprehensive array of fifteen distinct spontaneous behaviors was recorded continuously in the homecage over multiple days using the HomeCageScan system. Behaviors assessed were activity-like (i.e. come down, hang, jump, walk), exploration-like (i.e. dig, groom, rear up, sniff, stretch), resting-like (i.e. awake, remain low, rest, twitch) and ingestion-like (i.e. drink, eat). Phenotypic array and temporal distribution analysis revealed distinct behavioral rhythms that differed between WT and MT1KO mice. The rhythms were consistent from day to day in males and varied with the estrous cycle in females. We also studied the role of MT1 receptors on depressive and anxiety-like behaviors. Genetic deletion of MT1 receptors increased immobility time in the forced swim test and decreased the number of marbles buried in the marble burying test in both male and female C57 mice. We conclude that MT1 melatonin receptors are involved in neural pathways modulating diurnal rhythms of spontaneous behavior in the homecage as well as pathways regulating depressive and anxiolytic-like behaviors.

Melatonin MT1 receptor-induced transcriptional up-regulation of p27(Kip1) in prostate cancer antiproliferation is mediated via inhibition of constitutively active nuclear factor kappa B (NF-κB): potential implications on prostate cancer chemoprevention and therapy.

Our laboratory has recently demonstrated a melatonin MT1 receptor-mediated antiproliferative signaling mechanism in androgen receptor (AR)-positive prostate epithelial cells which involves up-regulation of p27(Kip1) through dual activation of Gα(s)/protein kinase A (PKA) and Gα(q)/protein kinase C (PKC) in parallel, and down-regulation of activated AR signaling via PKC stimulation. The aim of the present investigation was to identify the transcription factor that mediates melatonin's up-regulatory effect on p27(Kip1) in LNCaP and 22Rv1 prostate cancer cells. Deletion mapping and reporter assays of the p27(Kip1) promoter revealed that the putative melatonin-responsive transcription factor binds to a 116 base-pair region of the promoter sequence, which contains a potential nuclear factor kappa B (NF-κB) binding site. When the NF-κB binding site was abolished by site-directed mutagenesis, the stimulatory effect of melatonin on p27(Kip1) promoter activity was mitigated. Notably, melatonin inhibited the DNA binding of activated NF-κB via MT1 receptor-induced PKA and PKC stimulation. Furthermore, melatonin's up-regulatory effect on p27(Kip1) transcription and consequent cell antiproliferation were abrogated by NF-κB activator but mimicked by NF-κB inhibitor. The results indicate that inhibition of constitutively active NF-κB via melatonin MT1 receptor-induced dual activation of (Gα(s)) PKA and (Gα(q)) PKC can de-repress the p27(Kip1) promoter leading to transcriptional up-regulation of p27(Kip1). MT1 receptor-mediated inhibition of activated NF-κB signaling provides a novel mechanism supporting the use of melatonin in prostate cancer chemoprevention and therapy.

Agomelatine decreases cocaine-induced locomotor sensitisation and dopamine release in rats.

BACKGROUND: Agomelatine is a melatoninergic antidepressant approved to treat the major depressive disorder. Agomelatine exerts its behavioural, pharmacological, and physiological effects through the activation of MT1 and MT2 melatonin receptors and the blockade of 5-HT2B and 5-HT2C serotonin receptors. Some studies have reported that the activation of the MT1 and MT2 melatonin receptors decreased cocaine-induced locomotor activity and cocaine self-administration. These findings from another study showed that agomelatine decreased alcohol consumption. This study aimed to evaluate the effects of agomelatine administration on cocaine-induced behavioural (cocaine-induced locomotor activity and cocaine-induced locomotor sensitisation) and neurochemical (dopamine levels) effects. METHODS: Male Wistar rats (250-280 g) received cocaine (10 mg/kg) during the induction and expression of locomotor sensitisation. Agomelatine (10 mg/kg) was administered 30 minutes before cocaine. After each treatment, locomotor activity was recorded for 30 minutes. Dopamine levels were determined in the ventral striatum, the prefrontal cortex (PFC), and the ventral tegmental area (VTA) by high-performance liquid chromatographic (HPLC) in animals treated with agomelatine and cocaine. Luzindole (30 mg/kg) was administered to block the agomelatine effect. RESULTS: In this study, we found that agomelatine decreased cocaine-induced locomotor activity and the induction and expression of locomotor sensitisation. In addition, agomelatine decreased cocaine-induced dopamine levels. Luzindole blocked the agomelatine-induced decrease in the expression of locomotor sensitisation in rats. CONCLUSION: Our results suggest (1) that agomelatine showed efficacy in decreasing cocaine psychostimulant effects and (2) that agomelatine can be a useful therapeutic agent to reduce cocaine abuse.

Melatonin effective to reduce the microscopic symptoms of polycystic ovary syndrome-related infertility: An experimental study.

Polycystic ovary syndrome (PCOS) is an endocrine disorder seen in women of reproductive age and has been gradually increasing over the years. The mechanism of the syndrome has still not been clearly understood. In this study, the possible effects of exogenously administrated melatonin on melatonin (MT1) receptor, Growth Differentiation Factor-9 (GDF9), and Bone Morphogenetic Protein-15 (BMP15) in experimental PCOS were investigated. Thirty-two 6-8-week-old Sprague-Dawley rats were randomly divided into four groups (n = 8 in each) as Sham control (Group 1), Melatonin (Group 2), PCOS (Group 3), and PCOS + Melatonin (Group 4) groups. At the end of the 21st day, the experiment was terminated, the ovary tissues were taken, and Hematoxylin-Eosin staining, MT1, GDF9, BMP15 immunohistochemical labeling, western blot, and quantitative real-time polymerase chain reaction (qPCR) analyses were performed. Serum Luteinizing Hormone (LH)/Follicle Stimulating Hormone (FSH) levels and colpo-cytological examinations were also carried out. The results revealed that melatonin administration increased the expression levels of the MT1 receptor, GDF9, and BMP15 in PCOS at protein and mRNA levels. It was determined that melatonin administration reduced the microscopic symptoms of PCOS. Melatonin was found to be effective via the MT1 receptor in the pathogenesis of PCOS, and it suppressed the transport pathways of GDF9 to granulosa cells in antral follicles.

Melatonin enhances cell death and suppresses the metastatic capacity of ovarian cancer cells by attenuating the signaling of multiple kinases.

BACKGROUND: Ovarian cancer is a highly aggressive disease that is frequently diagnosed in advanced stages. Melatonin, with its numerous antitumor properties, holds great promise in cancer treatment. Herein, we investigated the effects of melatonin on apoptosis, cell migration, and kinase levels in human ovarian carcinoma SKOV-3 cells and determined whether these effects are mediated by the activation of the MT1 receptor. METHODS: SKOV-3 cells were exposed to different concentrations of melatonin based on the presence of MT1 receptor, and we also performed specific silencing of the melatonin receptor gene MTNR1A. RESULTS: Our findings revealed that melatonin reduced cell viability as shown by the MTT assay, and flow cytometry analysis showed increased rates of apoptosis and necrosis in all melatonin-treated cells. Melatonin significantly decreased the migratory and invasive capacities of the cells. Propidium iodide labeling indicated that melatonin induced cell cycle arrest by reducing DNA content in the S and G2/M phases in SKOV-3 cells. Additionally, the levels of AKT, ERK1/2, JNK, CREB, p70S6K, STAT3/5, and p38 MAP kinase involved in cell survival, proliferation, motility, and stress responses were depressed by melatonin and further reduced after MT1 knockdown. These molecules were found to be associated with lower overall survival in ovarian cancer patients. CONCLUSIONS: Melatonin had obvious oncostatic actions on ovarian cancer cells, and MT1 receptor knockdown intensified its antitumor effect. The inhibition of the MT1 receptor resulted in a substantial reduction in the migratory and invasive capacities of the cells, suggesting its potential as a therapeutic target for the treatment of ovarian cancer.

Melatonin Attenuates Cyclophosphamide-Induced Primordial Follicle Loss by Interaction with MT1 Receptor and Modulation of PTEN/Akt/FOXO3a Proteins in the Mouse Ovary.

This study evaluated the protective effect of melatonin before cyclophosphamide administration on ovarian function and its potential mechanism in a mouse model. Two studies were performed. In the first, mice were pretreated with melatonin (10, 20, or 30 mg/kg body weight, i.p.) once daily for 3 days, followed by injection with a single dose of cyclophosphamide (200 mg/kg body weight, i.p.) 30 min after the last melatonin injection. The second study analyzed whether melatonin type 1 and/or 2 receptors mediate the effects of melatonin on the ovary through administration of non-selective MT1/MT2 antagonist (luzindole) or selective MT2 antagonist (4-PPDOT) before the treatment with melatonin plus cyclophosphamide. After treatment groups, the ovaries were harvested and destined to histology, immunohistochemistry, and fluorescence analyses. Lastly, we examined the p-PTEN, p-Akt, and p-FOXO3a participation in the protective effect of melatonin in cyclophosphamide-induced ovarian damage. Results demonstrated that pretreatment with 20 mg/kg melatonin before cyclophosphamide administration showed more morphologically normal follicles, attenuated primordial follicle loss, decreased growing follicle atresia and mitochondrial damage, and increased GSH concentrations. Furthermore, treatment with luzindole blocked the protective effects of melatonin against the damage caused by cyclophosphamide. Additionally, pretreatment with 20 mg/kg melatonin regulated the PTEN/Akt/FOXO3a signaling pathway components after cyclophosphamide treatment. In conclusion, pretreatment with 20 mg/kg melatonin prevented primordial follicle loss and reduced apoptosis and oxidative damage in the mouse ovary during experimental chemotherapy with cyclophosphamide. Furthermore, the MT1 receptor and PTEN/Akt/FOXO3a proteins mediated these cytoprotective effects.

MT1 Melatonin Receptor Reconstitution in Nanodiscs.

A way to study G protein-coupled receptors in a minimal system is to reconstruct artificial membrane mimics, made of detergent and/or of lipids in which the purified receptor is maintained. In particular, it is now possible to generate lipid nanoparticles, such as nanodiscs, in which a single receptor molecule is included. Such objects offer the invaluable potential of studying an isolated receptor stabilized in a finely controlled membrane-like environment to evaluate its pharmacology, its function, and its structure at the molecular level. In this chapter, we detail the different steps from the extraction and isolation of a recombinant MT1 melatonin receptor in detergent, down to its reconstitution into nanodiscs. A G protein activation test is further described in order to exemplify how the functionality of such particles may be investigated.

GTPγS Binding Assay for Melatonin Receptors in Mouse Brain Tissue.

The [35S]GTPγS assay is a method that measures the level of G protein activation by determining the binding of [35S]GTPγS, a non-hydrolyzable and radioactively labeled GTP analog, to Gα subunit of heterotrimeric G protein upon activation of G protein-coupled receptors (GPCR). The power of this assay lies in the fact that it measures an early event of GPCR signaling in cells expressing recombinant receptors and cells and tissues expressing endogenous receptors. The present protocol describes a sensitive method for studying G protein activation by melatonin receptors MT1 and MT2, in membranes prepared from mouse brain. Immunoprecipitation of [35S]GTPγS-labeled G proteins with Gα subunit specific antibodies (Gi, Gq, etc.) allows to determine the activation of specific G proteins. The assay can be easily applied to other tissues.

Melatonin decreases cocaine-induced locomotor sensitization and cocaine-conditioned place preference in rats.

Melatonin is a hormone that produces behavioral, pharmacological, and physiological effects through the activation of MT1 and MT2 melatonin receptors. Melatonin receptors participate in the modulation of the reinforcing effects of cocaine. Some studies report that dosing of melatonin decreases cocaine-induced locomotor activity and cocaine self-administration and that luzindole, an MT1, and MT2 melatonin receptor antagonist, blocks the melatonin-dependent decrease in cocaine-induced locomotor activity. The objective of this study was to evaluate the effect of acute or chronic dosing of melatonin on the induction and expression of cocaine-induced locomotor sensitization and cocaine-CPP in rats. Male Wistar rats received cocaine during the induction and expression of locomotor sensitization. Melatonin was administered 30 min before cocaine. After each treatment, locomotor activity was recorded for 30 min. Additionally, dopamine levels were determined in the ventral striatum, the prefrontal cortex (PFc), and the ventral tegmental area (VTA) by HPLC in animals treated with melatonin and cocaine. Melatonin decreased cocaine-induced locomotor sensitization and intracellular dopamine levels, as well as cocaine-CPP. Luzindole blocked the melatonin-induced decrease in the expression of locomotor sensitization in rats. These data suggest that melatonin may be a useful therapeutic agent to reduce cocaine abuse; additionally, they suggest that MT1 and MT2 receptors could be therapeutic targets, useful for the treatment of drug abuse disorder.

Differential Function of Melatonin MT1 and MT2 Receptors in REM and NREM Sleep.

The pathophysiological function of the G-protein coupled melatonin MT1 and MT2 receptors has not yet been well-clarified. Recent advancements using selective MT1/ MT2 receptor ligands and MT1/MT2 receptor knockout mice have suggested that the activation of the MT1 receptors are mainly implicated in the regulation of rapid eye movement (REM) sleep, whereas the MT2 receptors selectively increase non-REM (NREM) sleep. Studies in mutant mice show that MT1 knockout mice have an increase in NREM sleep and a decrease in REM sleep, while MT2 knockout mice a decrease in NREM sleep. The localization of MT1 receptors is also distinct from MT2 receptors; for example, MT2 receptors are located in the reticular thalamus (NREM area), while the MT1 receptors in the Locus Coeruleus and lateral hypothalamus (REM areas). Altogether, these findings suggest that these two receptors not only have a very specialized function in sleep, but that they may also modulate opposing effects. These data also suggest that mixed MT1-MT2 receptors ligands are not clinically recommended given their opposite roles in physiological functions, confirmed by the modest effects of melatonin or MT1/MT2 non-selective agonists when used in both preclinical and clinical studies as hypnotic drugs. In sum, MT1 and MT2 receptors have specific roles in the modulation of sleep, and consequently, selective ligands with agonist, antagonist, or partial agonist properties could have therapeutic potential for sleep; while the MT2 agonists or partial agonists might be indicated for NREM-related sleep and/or anxiety disorders, the MT1 agonists or partial agonists might be so for REM-related sleep disorders. Furthermore, MT1 but not MT2 receptors seem involved in the regulation of the circadian rhythm. Future research will help further develop MT1 and/or MT2 receptors as targets for neuropsychopharmacology drug development.

Epigenetic induction of melatonin MT1 receptors by valproate: Neurotherapeutic implications.

We have reported that the anticonvulsant/mood stabilizer and histone deacetylase (HDAC) inhibitor valproate (VPA) induces expression of melatonin receptors both in vitro and in vivo, but the mechanisms involved were not known. Here we show that pharmacological inhibition of CREB, PKC, PI3K, or GSK3ß signaling pathways, which are known targets for VPA, do not prevent its upregulation of melatonin MT1 receptors in rat C6 glioma cells. M344, an HDAC inhibitor unrelated to VPA, mimics the effects of VPA on MT1 expression, whereas valpromide, a VPA derivative lacking HDAC inhibitory activity, does not. Furthermore, VPA, at a concentration which upregulates the MT1 receptor, induces histone H3 hyperacetylation along the length of the MT1 receptor promoter. These results show that an epigenetic mechanism involving histone acetylation underlies induction of MT1 receptor expression by VPA. Given the neuropsychiatric effects of melatonin coupled with evidence that VPA upregulates melatonin receptors in the rat brain, these findings suggest that the melatonergic system contributes to the psychotropic effects of VPA.

Altered melatonin MT1 receptor expression in the ventral midbrain following 6-hydroxydopamine lesions in the rat medial forebrain bundle.

The indoleamine hormone melatonin protects dopamine neurons in the rat nigrostriatal pathway following 6-hydroxydopamine lesioning, and an increase in striatal melatonin levels has been detected in this model of Parkinson's disease. Melatonin induces the expression of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in the ventral midbrain, where G protein-coupled melatonin receptors are present. Based on the interaction between the melatonergic and dopaminergic systems, we hypothesized that 6-hydroxydopamine-induced degeneration of dopamine neurons would affect the expression of melatonin receptors in the nigrostriatal pathway. Following unilateral injection of 6-hydroxydopamine into the rat striatum or medial forebrain bundle, there was a significant increase in apomorphine-induced contralateral rotations in lesioned animals as compared to sham controls. A loss of tyrosine hydroxylase immunoreactivity and/or immunofluorescence in the striatum and substantia nigra was seen in animals lesioned in either the striatum or medial forebrain bundle, indicating degeneration of dopamine neurons. There were no significant differences in melatonin MT1 receptor protein expression in the striatum or substantia nigra, between intrastriatally lesioned animals and sham controls. In contrast, lesions in the medial forebrain bundle caused a significant increase in MT1 receptor mRNA expression (p<0.03) on the lesioned side of the ventral midbrain, as compared with the contralateral side. Given the presence of MT1 receptors on neurons in the ventral midbrain, these results suggest that a compensatory increase in MT1 transcription occurs to maintain expression of this receptor and neuroprotective melatonergic signaling in the injured brain.