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.

Dysfunction of serotonergic activity and emotional responses across the light-dark cycle in mice lacking melatonin MT2 receptors.

Melatonin (MLT) levels fluctuate according to the external light/dark cycle in both diurnal and nocturnal mammals. We previously demonstrated that melatonin MT2 receptor knockout (MT2 -/- ) mice show a decreased nonrapid eye movement sleep over 24 hours and increased wakefulness during the inactive (light) phase. Here, we investigated the role of MT2 receptors in physiological light/dark cycle fluctuations in the activity of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons and anxiety- and depression-like behavior. We found that the 5-HT burst-firing activity was tonically reduced across the whole 24 hours in MT2 -/- mice compared with MT2 +/+ mice.  Importantly, the physiological changes in the spontaneous firing activity of DRN 5-HT neurons during the light/dark cycle were nullified in MT2 -/- mice, with a higher DRN 5-HT neural firing activity during the light phase in MT2 -/- than in MT2 +/+  mice. The role of MT2 receptors over DRN 5-HT neurons was confirmed by acute pharmacological studies in which the selective MT2 receptors agonist UCM1014 dose dependently inhibited DRN 5-HT activity, mostly during the dark phase. Compared with MT2 +/+ , MT2 -/- mice displayed an anxiety-like phenotype in the novelty-suppressed feeding and in the light/dark box tests; while anxiety levels in the light/dark box test were lower during the dark than during the light phase in MT2 +/+ mice, the opposite was seen in MT2 -/- mice. No differences between MT2 +/+ and MT2 -/- mice were observed for depression-like behavior in the forced swim and in the sucrose preference tests. These results suggest that MT2 receptor genetic inactivation impacts 5-HT neurotransmission and interferes with anxiety levels by perturbing the physiologic light/dark pattern.

Virtual discovery of melatonin receptor ligands to modulate circadian rhythms.

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT1 and MT2. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle1-4. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep5,6 and depression1-4,7-9. Despite their importance, few in vivo active MT1-selective ligands have been reported2,8,10-12, hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT1 inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT1-selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT1- but not in MT2-knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT1-selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.

MTNR1B loss promotes chordoma recurrence by abrogating melatonin-mediated ß-catenin signaling repression.

Chordoma is an extremely rare malignant bone tumor with a high rate of relapse. While cancer stem cells (CSCs) are closely associated with tumor recurrence, which depend on its capacity to self-renew and induce chemo-/radioresistance, whether and how CSCs participate in chordoma recurrence remains unclear. The current study found that tumor cells in recurrent chordoma displayed more dedifferentiated CSC-like properties than those in corresponding primary tumor tissues. Meanwhile, MTNR1B deletion along with melatonin receptor 1B (MTNR1B) down-regulation was observed in recurrent chordoma. Further investigation revealed that activation of Gαi2 by MTNR1B upon melatonin stimulation could inhibit SRC kinase activity via recruiting CSK and SRC, increasing SRC Y530 phosphorylation, and decreasing SRC Y419 phosphorylation. This subsequently suppressed ß-catenin signaling and stemness via decreasing ß-catenin p-Y86/Y333/Y654. However, MTNR1B loss in chordoma mediated increased CSC properties, chemoresistance, and tumor progression by releasing melatonin's repression of ß-catenin signaling. Clinically, MTNR1B deletion was found to correlate with patients' survival. Together, our study establishes a novel convergence between melatonin and ß-catenin signaling pathways and reveals the significance of this cross talk in chordoma recurrence. Besides, we propose that MTNR1B is a potential biomarker for prediction of chordoma prognosis and selection of treatment options, and chordoma patients might benefit from targeting MTNR1B/Gαi2/SRC/ß-catenin axis.

Food-induced reinforcement is abrogated by the genetic deletion of the MT1 or MT2 melatonin receptor in C3H/HeN mice.

Palatable food is known for its ability to enhance reinforcing responses. Studies have suggested a circadian variation in both drug and natural reinforcement, with each following its own time course. The goal of this study was to determine the role of the MT1 and MT2 melatonin receptors in palatable snack food-induced reinforcement, as measured by the conditioned place preference (CPP) paradigm during the light and dark phases. C3H/HeN wild-type mice were trained for snack food-induced CPP at either ZT 6 - 8 (ZT: Zeitgeber time; ZT 0 = lights on), when endogenous melatonin levels are low, or ZT 19 - 21, when melatonin levels are high. These time points also correspond to the high and low points for expression of the circadian gene Period1, respectively. The amount of snack food (chow, Cheetos®, Froot Loops® and Oreos®) consumed was of similar magnitude at both times, however only C3H/HeN mice conditioned to snack food at ZT 6 - 8 developed a place preference. C3H/HeN mice with a genetic deletion of either the MT1 (MT1KO) or MT2 (MT2KO) receptor tested at ZT 6 - 8 did not develop a place preference for snack food. Although the MT2KO mice showed a similar amount of snack food consumed when compared to wild-type mice, the MT1KO mice consumed significantly less than either genotype. We conclude that in our mouse model snack food-induced CPP is dependent on time of day and the presence of the MT1 or MT2 receptors, suggesting a role for melatonin and its receptors in snack food-induced reinforcement.

Melatonin signaling affects the timing in the daily rhythm of phagocytic activity by the retinal pigment epithelium.

Earlier studies in Xenopus have indicated a role for melatonin in the regulation of retinal disk shedding, but the role of melatonin in the regulation of daily rhythm in mammalian disk shedding and phagocytosis is still unclear. We recently produced a series of transgenic mice lacking melatonin receptor type 1 (MT1) or type 2 (MT2) in a melatonin-proficient background and have shown that removal of MT1 and MT2 receptors induces significant effects on daily and circadian regulation of the electroretinogram as well as on the viability of photoreceptor cells during aging. In this study we investigated the daily rhythm of phagocytic activity by the retinal pigment epithelium in MT1 and MT2 knock-out mice. Our data indicate that in MT1 and MT2 knock-out mice the peak of phagocytosis is advanced by 3 h with respect to wild-type mice and occurred in dark rather than after the onset of light, albeit the mean phagocytic activity over the 24-h period did not change among the three genotypes. Nevertheless, this small change in the profile of daily phagocytic rhythms may produce a significant effect on retinal health since MT1 and MT2 knock-out mice showed a significant increase in lipofuscin accumulation in the retinal pigment epithelium.

The Role of the Melatoninergic System in Light-Entrained Behavior of Mice.

The role of endogenous melatonin for the control of the circadian system under entrained conditions and for the determination of the chronotype is still poorly understood. Mice with deletions in the melatoninergic system (melatonin deficiency or the lack of melatonin receptors, respectively) do not display any obvious defects in either their spontaneous (circadian) or entrained (diurnal) rhythmic behavior. However, there are effects that can be detected by analyzing the periodicity of the locomotor behaviors in some detail. We found that melatonin-deficient mice (C57Bl), as well as melatonin-proficient C3H mice that lack the melatonin receptors (MT) 1 and 2 (C3H MT1,2 KO), reproduce their diurnal locomotor rhythms with significantly less accuracy than mice with an intact melatoninergic system. However, their respective chronotypes remained unaltered. These results show that one function of the endogenous melatoninergic system might be to stabilize internal rhythms under conditions of a steady entrainment, while it has no effects on the chronotype.

Melatonin receptor deficiency decreases and temporally shifts ecto-5'-nucleotidase mRNA levels in mouse prosencephalon.

Ecto-5'-nucleotidase (eN) is the major extracellular adenosine-producing ecto-enzyme in mouse brain. Via the production of adenosine, eN participates in many physiological and pathological processes, such as wakefulness, inflammation, nociception and neuroprotection. The mechanisms regulating the expression of eN are therefore of considerable neurobiological and clinical interest. Having previously described a modulatory effect of melatonin in the regulation of eN mRNA levels, we decided to analyze the melatonin receptor subtype involved in the regulation of eN mRNA levels by comparing eN mRNA patterns in melatonin-proficient transgenic mice lacking either the melatonin receptor subtype 1 (MT1 KO) or both melatonin receptor subtypes (MT1 and MT2; MT1/2 KO) with the corresponding melatonin-proficient wild-type (WT) controls. By means of radioactive in situ hybridization, eN mRNA levels were found to be diminished in both MT1 and MT1/2 KO mice compared with WT controls suggesting stimulatory impacts of melatonin receptors on eN mRNA levels. Whereas eN mRNA levels increased during the day and peaked at night in WT and MT1 KO mice, eN mRNA levels at night were reduced and the peak was shifted toward day-time in double MT1/2 KO mice. These data suggest that the MT2 receptor subtype may play a role in the temporal regulation of eN mRNA availability. Notably, day-time locomotor activity was significantly higher in MT1/2 KO compared with WT mice. Our results suggest melatoninergic signaling as an interface between the purinergic system and the circadian system.

Melatonin Signaling Controls the Daily Rhythm in Blood Glucose Levels Independent of Peripheral Clocks.

Melatonin is rhythmically secreted by both the pineal gland and retina in a circadian fashion, with its peak synthesis occurring during the night. Once synthesized, melatonin exerts its effects by binding to two specific G-protein coupled receptors-melatonin receptor type 1(MT1) and melatonin receptor type 2(MT2). Recent studies suggest the involvement of MT1 and MT2 in the regulation of glucose homeostasis; however the ability of melatonin signaling to impart timing cues on glucose metabolism remains poorly understood. Here we report that the removal of MT1 or MT2 in mice abolishes the daily rhythm in blood glucose levels. Interestingly, removal of melatonin receptors produced small effects on the rhythmic expression patterns of clock genes within skeletal muscle, liver, and adipose tissue. Taken together, our data suggest that the loss of the daily rhythm in blood glucose observed in MT1(-/-) and MT2(-/-) mice does not occur as a consequence of 'disrupted' clocks within insulin sensitive tissues. Finally our results highlight a diurnal contribution of melatonin receptor signaling in the daily regulation of blood glucose levels.

Abnormal response of the proliferation and differentiation of growth plate chondrocytes to melatonin in adolescent idiopathic scoliosis.

Abnormalities in the melatonin signaling pathway and the involvement of melatonin receptor MT2 have been reported in patients with adolescent idiopathic scoliosis (AIS). Whether these abnormalities were involved in the systemic abnormal skeletal growth in AIS during the peripubertal period remain unknown. In this cross-sectional case-control study, growth plate chondrocytes (GPCs) were cultured from twenty AIS and ten normal control subjects. Although the MT2 receptor was identified in GPCs from both AIS and controls, its mRNA expression was significantly lower in AIS patients than the controls. GPCs were cultured in the presence of either the vehicle or various concentrations of melatonin, with or without the selective MT2 melatonin receptor antagonist 4-P-PDOT (10 µM). Then the cell viability and the mRNA expression of collagen type X (COLX) and alkaline phosphatase (ALP) were assessed by MTT and qPCR, respectively. In the control GPCs, melatonin at the concentrations of 1, 100 nM and 10 µM significantly reduced the population of viable cells, and the mRNA level of COLX and ALP compared to the vehicle. Similar changes were not observed in the presence of 4-P-PDOT. Further, neither proliferation nor differentiation of GPCs from AIS patients was affected by the melatonin treatment. These findings support the presence of a functional abnormality of the melatonin signaling pathway in AIS GPCs, which might be associated with the abnormal endochondral ossification in AIS patients.

Genetic deletion of MT1/MT2 melatonin receptors enhances murine cognitive and motor performance.

Melatonin, an indoleamine hormone secreted into circulation at night primarily by the brain's pineal gland, has been shown to have a wide variety of actions on the development and physiology of neurons in the CNS. Acting via two G-protein-coupled membrane receptors (MT1 and MT2), melatonin modulates neurogenesis, synaptic functions, neuronal cytoskeleton and gene expression. In the present studies, we sought to characterize the behavior and neuronal biology of transgenic mice lacking both of these melatonin receptors as a way to understand the hormone's receptor versus non-receptor-mediated actions in CNS-dependent activities, such as learning and memory, anxiety, general motor performance and circadian rhythmicity. Assessment of these behaviors was complemented by molecular analyses of gene expression in the brain. Our results demonstrate mild behavioral hyperactivity and a lengthened circadian period of free-running motor activity in melatonin receptor-deficient mice as compared to receptor-intact control mice beginning at an early age. Significant improvement in cognitive performance was found using the Barnes Maze and the Y-Maze. No behavioral changes in anxiety levels were found. Electrophysiological measures in hippocampal slices revealed a clear enhancement of long-term potentiation in mice lacking melatonin receptors with no significant differences in paired-pulse facilitation. Quantitative analysis of brain protein expression levels of phosphoCREB and phosphoERK1/2 and key markers of synaptic activity (synapsin, glutamate receptor 1, spinophilin, and glutamic acid decarboxylase 1) revealed significant differences between the double-knockout and wild-type animals, consistent with the behavioral findings. Thus, genetic deletion of melatonin receptors produces mice with enhanced cognitive and motor performance, supporting the view that these receptors play an important role in neurobehavioral development.

Genetic deletion of the MT1 or MT2 melatonin receptors abrogates methamphetamine-induced reward in C3H/HeN mice.

The drug of abuse methamphetamine (METH) is known for its ability to enhance reward responses. The rewarding properties of psychostimulants have been shown to vary across time of day in mice. The goal of this study was to determine the role of the MT1 and MT2 melatonin receptors in METH-induced reward, as measured by the conditioned place preference (CPP) paradigm during the light and dark phases. C3H/HeN wild-type mice were trained for METH-induced CPP at either ZT 6-8 (ZT: Zeitgeber time; ZT 0=lights on), when endogenous melatonin levels are low, or ZT 19-21, when melatonin levels are high. These time points also correspond to the high and low points for expression of the circadian gene Period1, respectively. The locomotor response to METH (1.2mg/kg, ip) treatment was of similar magnitude at both times; however only C3H/HeN mice conditioned to METH at ZT 6-8 developed a place preference. C3H/HeN mice with a genetic deletion of either the MT1 (MT1KO) or MT2 (MT2KO) receptor tested at ZT 6-8 or ZT 19-21 did not develop a place preference for METH, though both showed a similar increase in locomotor activity following METH treatment when compared to wild-type mice. We conclude that in our mouse model METH-induced CPP is dependent on time of day and the presence of the MT1 or MT2 receptors, suggesting a role for melatonin in METH-induced reward.

Sleep-wake characterization of double MT1/MT2 receptor knockout mice and comparison with MT1 and MT2 receptor knockout mice.

The neurohormone melatonin activates two G-protein coupled receptors, MT1 and MT2. Melatonin is implicated in circadian rhythms and sleep regulation, but the role of its receptors remains to be defined. We have therefore characterized the spontaneous vigilance states in wild-type (WT) mice and in three different types of transgenic mice: mice with genetic inactivation of MT1 (MT1(-/-)), MT2 (MT2(-/-)) and both MT1/MT2 (MT1(-/-)/MT2(-/-)) receptors. Electroencephalographic (EEG) and electromyographic sleep-wake patterns were recorded across the 24-h light-dark cycle. MT1(-/-)mice displayed a decrease (-37.3%) of the 24-h rapid eye movement sleep (REMS) time whereas MT2(-/-)mice showed a decrease (-17.3%) of the 24-h non rapid eye movement sleep (NREMS) time and an increase in wakefulness time (14.8%). These differences were the result of changes occurring in particular during the light/inactive phase. Surprisingly, MT1(-/-)/MT2(-/-) mice showed only an increase (8.9%) of the time spent awake during the 24-h. These changes were correlated to a decrease of the REMS EEG theta power in MT1(-/-)mice, of the NREMS EEG delta power in MT2(-/-)mice, and an increase of the REMS and wakefulness EEG theta power in MT1(-/-)/MT2(-/-) mice. Our results show that the genetic inactivation of both MT1 and MT2 receptors produces an increase of wakefulness, likely as a result of reduced NREMS due to the lack of MT2 receptors, and reduced REMS induced by the lack of MT1 receptors. Therefore, each melatonin receptor subtype differently regulates the vigilance states: MT2 receptors mainly NREMS, whereas MT1 receptors REMS.

Evidence of the receptor-mediated influence of melatonin on pancreatic glucagon secretion via the Gαq protein-coupled and PI3K signaling pathways.

Melatonin has been shown to modulate glucose metabolism by influencing insulin secretion. Recent investigations have also indicated a regulatory function of melatonin on the pancreatic α-cells. The present in vitro and in vivo studies evaluated whether melatonin mediates its effects via melatonin receptors and which signaling cascade is involved. Incubation experiments using the glucagon-producing mouse pancreatic α-cell line αTC1 clone 9 (αTC1.9) as well as isolated pancreatic islets of rats and mice revealed that melatonin increases glucagon secretion. Preincubation of αTC1.9 cells with the melatonin receptor antagonists luzindole and 4P-PDOT abolished the glucagon-stimulatory effect of melatonin. In addition, glucagon secretion was lower in the pancreatic islets of melatonin receptor knockout mice than in the islets of the wild-type (WT) control animals. Investigations of melatonin receptor knockout mice revealed decreased plasma glucagon concentrations and elevated mRNA expression levels of the hepatic glucagon receptor when compared to WT mice. Furthermore, studies using pertussis toxin, as well as measurements of cAMP concentrations, ruled out the involvement of Gαi- and Gαs-coupled signaling cascades in mediating the glucagon increase induced by melatonin. In contrast, inhibition of phospholipase C in αTC1.9 cells prevented the melatonin-induced effect, indicating the physiological relevance of the Gαq-coupled pathway. Our data point to the involvement of the phosphatidylinositol 3-kinase signaling cascade in mediating melatonin effects in pancreatic α-cells. In conclusion, these findings provide evidence that the glucagon-stimulatory effect of melatonin in pancreatic α-cells is melatonin receptor mediated, thus supporting the concept of melatonin-modulated and diurnal glucagon release.

Genetic deletion of MT(1) and MT(2) melatonin receptors differentially abrogates the development and expression of methamphetamine-induced locomotor sensitization during the day and the night in C3H/HeN mice.

This study explored the role of the melatonin receptors in methamphetamine (METH)-induced locomotor sensitization during the light and dark phases in C3H/HeN mice with genetic deletion of the MT(1) and/or MT(2) melatonin receptors. Six daily treatments with METH (1.2 mg/kg, i.p.) in a novel environment during the light phase led to the development of locomotor sensitization in wild-type (WT), MT(1)KO and MT(2)KO mice. Following four full days of abstinence, METH challenge (1.2 mg/kg, i.p.) triggered the expression of locomotor sensitization in METH-pretreated but not in vehicle (VEH)-pretreated mice. In MT(1)/MT(2)KO mice, the development of sensitization during the light phase was significantly reduced and the expression of sensitization was completely abrogated upon METH challenge. During the dark phase the development of locomotor sensitization in METH-pretreated WT, MT(1)KO and MT(2)KO mice was statistically different from VEH-treated controls. However, WT and MT(2)KO, but not MT(1)KO mice receiving repeated VEH pretreatments during the dark phase expressed a sensitized response to METH challenge that is of an identical magnitude to that observed upon 6 days of METH pretreatment. We conclude that exposure to a novel environment during the dark phase, but not during the light phase, facilitated the expression of sensitization to a METH challenge in a manner dependent on MT(1) melatonin receptor activation by endogenous melatonin. We suggest that MT(1) and MT(2) melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.

The endogenous melatonin (MT) signal facilitates reentrainment of the circadian system to light-induced phase advances by acting upon MT2 receptors.

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors.

Loss of melatonin signalling and its impact on circadian rhythms in mouse organs regulating blood glucose.

The transmission of circadian rhythms is mediated by specific promoter sequences binding a particular circadian clock factor. The pineal hormone melatonin acts via G-protein-coupled receptors to synchronise these clock-generated circadian rhythms. The study was aimed to elucidate the possible role of melatonin as a zeitgeber for peripheral clocks in pancreas and liver. Reverse transcription polymerase chain reaction (RT-PCR) provided evidence of the simultaneous expression of the melatonin receptors MT(1) and MT(2) in mouse pancreas, liver and hypothalamus. Melatonin receptor knockout mice were analysed with respect to the clock gene- or clock-output transcripts PER1, DBP and RevErbalpha in pancreas and liver, and both the occurrence of phase shifts and amplitude changes were detected. Circadian PER1 protein expression was found to be retained in melatonin receptor double knockout mice with an increased amplitude as measured by semiquantitative Western blot analysis. Moreover, an impact of melatonin receptor deficiency on insulin transcripts, and altered regulation of insulin secretion and glucose homeostasis were monitored in the knockout animals. Insulin secretion from isolated islets of melatonin receptor MT(1), MT(2) or MT(1) and MT(2) double melatonin receptor-knockout animals was found to be increased relative to the wild type. These data support the idea that melatonin synchronises the functions of the major organs involved in blood glucose regulation and negatively acts on the insulin secretion.

Melatonin receptor agonist ramelteon activates the extracellular signal-regulated kinase 1/2 in mouse cerebellar granule cells.

The melatonin receptors MT1 and MT2 take part in the regulation of the activity (i.e. phosphorylation) of extracellular-signal-regulated kinase (ERK1/2), an enzyme involved in neuroplasticity. Primary cultures of mouse and rat cerebellar granule cells (CGC), which express both MT1 and MT2 receptors, have been widely used as an in vitro model to study neuronal ERK1/2. A novel MT1/MT2 agonist, ramelteon, has recently become clinically available. In this study, we characterized its action on neuronal ERK1/2. We used CGC cultures prepared from the cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout (KO) mice (MT1 KO CGC and MT2 KO CGC, respectively), and we employed a Western blot assay to evaluate ERK1/2 phosphorylation. Ramelteon increased ERK1/2 phosphorylation not only in MT1/MT2 CGC but also in CGC expressing only one of the two melatonin receptors. In the MT1 KO CGC, the stimulatory effect of ramelteon was blocked by an MT2 antagonist, 4P-PDOT, whereas in the MT2 KO CGC, this effect of ramelteon was blocked by luzindole. Pertussis toxin treatment did not prevent ramelteon from activating ERK1/2 but pretreatment with a tyrosine kinase (Trk) inhibitor, K252a, did, suggesting that an activation of Trk may mediate melatonin-receptor dependent ERK1/2 activation. In conclusion, we showed for the first time that a clinically used MT1/MT2 agonist, ramelteon, is capable of activating neuronal ERK1/2.

Melatonin signaling in mouse cerebellar granule cells with variable native MT1 and MT2 melatonin receptors.

Although G protein-coupled MT1 and MT2 melatonin receptors are expressed in neurons of the mammalian brain including in humans, relatively little is known about the influence of native MT1 and MT2 melatonin receptors on neuronal melatonin signaling. Whereas human cerebellar granule cells (CGC) express only MT1 receptors, mouse CGC express both MT1 and MT2. To study the effects of altered neuronal MT1/MT2 receptors, we used CGC cultures prepared from immature cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout mice (MT2 and MT1 CGC, respectively). Here we report that in MT1/MT2 cultures, physiological (low nanomolar) concentrations of melatonin decrease the activity (phosphorylation) of extracellular-signal-regulated kinase (ERK) whereas a micromolar concentration was ineffective. Both MT1 and MT2 deficiencies transformed the melatonin inhibition of ERK into melatonin-induced ERK activation. In MT1/MT2 CGC, 1 nM melatonin inhibited serine/threonine kinase Akt, whereas in MT1 and MT2 CGC, this concentration was ineffective. Under these conditions, both MT1 and MT2 deficiencies prevented melatonin from inhibiting forskolin-stimulated cAMP levels and cFos immunoreactivity. We demonstrated that selective removal of native neuronal MT1 and MT2 receptors has a profound effect on the intracellular actions of low/physiological concentrations of melatonin. Since the expression of MT1 and MT2 receptors is cell-type-specific and species-dependent, we postulate that the pattern of expression of neuronal melatonin receptor types in different brain areas and cells could determine the capabilities of endogenous melatonin in regulating neuronal functioning.

Stimulatory effects of a melatonin receptor agonist, ramelteon, on BDNF in mouse cerebellar granule cells.

Melatonin receptor activation has been linked to the regulation of neurotrophic factors, including the brain-derived neurotrophic factor (BDNF). To further characterize the effects of melatonin receptor stimulation on neuronal BDNF, we used a clinically available novel agonist for MT1 and MT2 melatonin receptors, ramelteon. Primary cultures of cerebellar granule cells (CGC) have been established as an in vitro model for studying neuronal BDNF. We took advantage of the availability of MT1- and MT2-deficient (knockout; KO) mice to employ primary CGC prepared from wild type (WT), MT1 KO, and MT2 KO mice. We investigated the effects of ramelteon on BDNF protein and mRNA content. Administered in a low nanomolar range, ramelteon increased BDNF protein content in all three types of mouse CGC. This ramelteon-triggered BDNF protein elevation was not preceded by a BDNF mRNA increase. However, it was prevented by treatment of cultures with a protein synthesis inhibitor cycloheximide. These results demonstrated that the MT1/MT2 melatonin receptor agonist ramelteon is capable of increasing BDNF protein in neurons expressing either of the two melatonin receptor types and that this action of ramelteon involves translational mechanisms. Further research is needed to explore the putative influence of ramelteon on BDNF-associated neuroplasticity.