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 MT_2, 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.     

The antidepressant-like effect of the melatonin receptor ligand luzindole in mice during forced swimming requires expression of MT2 but not MT1 melatonin receptors

We previously reported an antidepressant-like effect in C3H/HeN mice during the forced swimming test (FST) following treatment with the MT1/MT2 melatonin receptor ligand, luzindole. This study investigated the role melatonin receptors (MT1 and/or MT2) may play in the effect of luzindole in the FST using C3H/HeN mice with a genetic deletion of either MT1 (MT1KO) or MT2 (MT2KO) melatonin receptors. In the light phase (ZT 9-11), luzindole (30 mg/kg, i.p.) significantly decreased immobility during swimming in both wild type (WT) (135.6 +/- 25.3 s, n = 7) and MT(1)KO (132.6 +/- 13.3 s, n = 8) as compared with vehicle-treated mice (WT: 207.1 +/- 6.0 s, n = 7; MT1KO: 209.5 +/- 6.2 s, n = 8) (P < 0.001). In the dark phase (ZT 20-22), luzindole also decreased time of immobility in both WT (89.5 +/- 13.9 s, n = 8) and MT1KO (66.5 +/- 6.4 s, n = 8) mice as compared with the vehicle treated (WT: 193.8 +/- 3.5, n = 6; MT1KO: 176.6 +/- 6.2 s, n = 8) (P < 0.001). Genetic disruption of the MT1 gene did not alter the diurnal rhythm of serum melatonin in MT1KO mice (ZT 9-11: 1.3 +/- 0.6 pg/mL, n = 7; ZT 20-22: 10.3 +/- 1.1 pg/mL, n = 8) as compared with WT (ZT 9-11: 1.4 +/- 0.7 pg/mL; ZT 20-22: 10.6 pg/mL). Swimming did not alter the serum melatonin diurnal rhythm in WT and MT1KO mice. Decreases in immobility of WT and MT1KO mice by luzindole treatment were not affected by gender or age (3 months versus 8 months). In contrast, luzindole did not decrease immobility during the FST in MT2KO mice. We conclude that the antidepressant-like effect of luzindole may be mediated through blockade of MT2 rather than MT1 melatonin receptors.     

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.     

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Effect of MT1 melatonin receptor deletion on melatonin-mediated phase shift of circadian rhythms in the C57BL/6 mouse

In the mouse suprachiasmatic nucleus (SCN), melatonin activates MT1 and MT2 G-protein coupled receptors, which are involved primarily in inhibition of neuronal firing and phase shift of circadian rhythms. This study investigated the ability of melatonin to phase shift circadian rhythms in wild type (WT) and MT1 melatonin receptor knockout (KO) C57BL/6 mice. In WT mice, melatonin (90 microg/mouse, s.c.) administered at circadian time 10 (CT10; CT12 onset of activity) significantly phase advanced the onset of the circadian activity rhythm (0.60 +/- 0.09 hr, n = 41) when compared with vehicle treated controls (-0.02 +/- 0.07 hr, n = 28) (P < 0.001). In contrast, C57 MT1KO mice treated with melatonin did not phase shift circadian activity rhythms (-0.10 +/- 0.12 hr, n = 42) when compared with vehicle treated mice (-0.12 +/- 0.07 hr, n = 43). Similarly, in the C57 MT1KO mouse melatonin did not accelerate re-entrainment to a new dark onset after an abrupt advance of the dark cycle. In contrast, melatonin (3 and 10 pm) significantly phase advanced circadian rhythm of neuronal firing in SCN brain slices independent of genotype with an identical maximal shift at 10 pm (C57 WT: 3.61 +/- 0.38 hr, n = 3; C57 MT(1)KO: 3.45 +/- 0.11 hr, n = 4). Taken together, these results suggest that melatonin-mediated phase advances of circadian rhythms of neuronal firing in the SCN in vitro may involve activation of the MT2 receptor while in vivo activation of the MT1 and possibly the MT2 receptor may be necessary for the expression of melatonin-mediated phase shifts of overt circadian activity rhythms.     

Molecular and cellular pharmacological properties of 5‐methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT): a nonspecific MT3 ligand

Abstract: 5‐Methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT) has been initially described as a ligand at non MT₁, non MT₂ melatonin binding site (MT3) selective versus MT₁ and MT₂, two membrane melatonin receptors. MCA‐NAT activity has been reported by others in different models, in vivo, particularly in the intra‐ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA‐NAT is described. MCA‐NAT has micromolar range affinities at the melatonin receptors MT₁ and MT₂, while in functional studies, MCA‐NAT proved to be a powerful MT₁/MT₂ partial agonist in the sub‐micromolar range. These data strongly suggest that MCA‐NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA‐NAT is unable to elicit any type of receptor‐like functional responses from Chinese hamster ovary cells over‐expressing quinone reductase 2, the MT3.     

Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT₁ and MT₂ receptors, belonging to the G protein‐coupled receptor (GPCR) super‐family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT₁ and MT₂ receptors, which comprises 378 individual proteins. Among the proteins interacting with MT₁, but not with MT₂, we identified several presynaptic proteins, suggesting a potential role of MT₁ in neurotransmission. Presynaptic localization of MT₁ receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT₁ physically interacts with the voltage‐gated calcium channel Ca_v2.2 and inhibits Ca_v2.2‐promoted Ca²⁺ entry in an agonist‐independent manner. In conclusion, we show that MT₁ is part of the presynaptic protein network and negatively regulates Ca_v2.2 activity, providing a first hint for potential synaptic functions of MT₁.     

Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain

The involvement of melatonin in mammalian brain pathophysiology has received growing interest, but information about the anatomical distribution of its two G‐protein‐coupled receptors, MT₁ and MT₂, remains elusive. In this study, using specific antibodies, we examined the precise distribution of both melatonin receptors immunoreactivity across the adult rat brain using light, confocal, and electron microscopy. Our results demonstrate a selective MT₁ and MT₂ localization on neuronal cell bodies and dendrites in numerous regions of the rat telencephalon, diencephalon, and mesencephalon. Confocal and ultrastructural examination confirmed the somatodendritic nature of MT₁ and MT₂ receptors, both being localized on neuronal membranes. Overall, striking differences were observed in the anatomical distribution pattern of MT₁ and MT₂ proteins, and the labeling often appeared complementary in regions displaying both receptors. Somadendrites labeled for MT₁ were observed for instance in the retrosplenial cortex, the dentate gyrus of the hippocampus, the islands of Calleja, the medial habenula, the suprachiasmatic nucleus, the superior colliculus, the substantia nigra pars compacta, the dorsal raphe nucleus, and the pars tuberalis of the pituitary gland. Somadendrites endowed with MT₂ receptors were mostly observed in the CA3 field of the hippocampus, the reticular thalamic nucleus, the supraoptic nucleus, the inferior colliculus, the substantia nigra pars reticulata, and the ventrolateral periaqueductal gray. Together, these data provide the first detailed neurocytological mapping of melatonin receptors in the adult rat brain, an essential prerequisite for a better understanding of melatonin distinct receptor function and neurophysiology.     

Melatonin MT1 and MT2 receptor ERK signaling is differentially dependent on Gi/o and Gq/11 proteins

G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and β-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT₁ and MT₂ receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules. We show that ERK1/2 activation by both receptors is exclusively G protein-dependent, without any participation of β-arrestin1/2 in HEK293 cells. ERK1/2 activation by MT₁ is only mediated though G_i/o proteins, while MT₂ is dependent on the cooperative activation of G_i/o and G_q/11 proteins. In the absence of G_q/11 proteins, however, MT₂-induced ERK1/2 activation switches to a β-arrestin1/2-dependent mode. The signaling cascade downstream of G proteins is the same for both receptors and involves activation of the PI3K/PKCζ/c-Raf/MEK/ERK cascade. The differential G protein dependency of MT₁- and MT₂-mediated ERK activation was confirmed at the level of EGR1 and FOS gene expression, two ERK1/2 target genes. G_i/o/G_q/11 cooperativity was also observed in Neuroscreen-1 cells expressing endogenous MT₂, whereas in the mouse retina, where MT₂ is engaged into MT₁/MT₂ heterodimers, ERK1/2 signaling is exclusively G_i/o-dependent. Collectively, our data reveal differential signaling modes of MT₁ and MT₂ in terms of ERK1/2 activation, with an unexpected G_i/o/G_q/11 cooperativity exclusively for MT₂. The plasticity of ERK activation by MT₂ is highlighted by the switch to a β-arrestin1/2-dependent mode in the absence of G_q/11 proteins and by the switch to a G_i/o mode when engaged into MT₁/MT₂ heterodimers, revealing a new mechanism underlying tissue-specific responses to melatonin.     

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 enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing

Melatonin, a circadian rhythm–promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)‐MSCs pretreated with melatonin enhanced wound closure, granulation, and re‐epithelialization at mouse skin wound sites, where relatively more UCB‐MSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB‐MSCs. Melatonin (1 μm ) significantly increased the motility of UCB‐MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT₂). We found that Gαq coupled with MT₂ and that the binding of Gαq to MT₂ uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently downregulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization‐related proteins such as profilin‐1, cofilin‐1, and F‐actin in UCB‐MSCs. Finally, a lack of MT₂ expression in UCB‐MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT₂ triggers FAK/paxillin phosphorylation to stimulate reorganization of the actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB‐MSCs motility.     

Association of disease‐predisposition polymorphisms of the melatonin receptors and sunshine duration in the global human populations

Abstract: Melatonin is predominantly involved in signaling circadian and seasonal rhythms, and its synthesis is regulated by the environmental light/dark cycle. The selection pressure by geographically different environmental light/dark cycles, which is predominantly determined by sunshine duration, on the global distribution of genetic polymorphisms in the melatonin pathway is not well understood. Recent genetic association studies identified various disease‐predisposition polymorphisms in this pathway. We investigated the correlations between the prevalence of these clinically important single nucleotide polymorphisms (SNPs) and sunshine duration among worldwide human populations from twelve regions in the CEPH‐HGDP database rs4753426, a recently reported predisposition SNP for type 2 diabetes in the promoter of the MT₂ melatonin receptor gene (MTNR1B), which was not included in the CEPH‐HGDP genotyping array, was additionally genotyped. This SNP showed a marginally significant correlation in 760 CEPH‐HGDP DNA samples (r = ?0.5346, P = 0.0733), and it showed the most prominent association among the candidate melatonin pathway SNPs examined. To control for population structure, which may lead to a false positive correlation, we genotyped this SNP in a replication set of 1792 subjects from China. The correlation was confirmed among Chinese populations (r = ?0.8694, P = 0.0002), and was also statistically significant after correction of other climatic and geographical covariants in multiple regression analysis (β = ?0.907, P = 1.94 × 10^?5). Taken together, it suggests that the human melatonin signaling pathway, particularly MT₂ melatonin receptor may have undergone a selective pressure in response to global variation in sunshine duration.     

Melatonin regulates the activities of ovary and delays the fertility decline in female animals via MT1/AMPK pathway

Female fertility irreversibly declines with aging, and this is primarily associated with the decreased quality and quantity of oocytes. To evaluate whether a long‐term of melatonin treatment would improve the fertility of aged mice, different concentrations of melatonin (10^?3, 10^?5, 10^?7 mol/L) were supplemented into drinking water. Melatonin treatments improved the litter sizes of mice at the age of 24 weeks. Mice treated with 10^?5 mol/L melatonin had the largest litter size among other concentrations. At this optimal concentration, melatonin not only significantly increased the total number of oocytes but also their quality, having more oocytes with normal morphology that could generate more blastocyst after in vitro fertilization in melatonin (10^?5 mol/L)‐treated group than that in the controls. When these blastocysts were transferred to recipients, the litter size was also significantly larger in melatonin treated mice than that in controls. The increases in TAOC and SOD level and decreases in MDA were detected in ovaries and uterus from melatonin‐treated mice compared to the controls. Melatonin reduced ROS level and maintained mitochondrial membrane potential in the oocytes cultured in vitro. Mechanistically studies revealed that the beneficial effects of melatonin on oocytes were mediated by MT1 receptor and AMPK pathway. Thereafter, MT1 knocking out (MT1‐KO) were generated and shown significantly reduced number of oocytes and litter size. The expression of SIRT1, C‐myc, and CHOP were downregulated in the ovary of MT1‐KO mice, but SIRT1 and p‐NF‐kB protein level were elevated in response to disturbed redox balance. The results have convincingly proven that melatonin administration delays ovary aging and improves fertility in mice via MT1/AMPK pathway.     

Functional MT1 and MT2 melatonin receptors in mammals

Melatonin, dubbed the hormone of darkness, is known to regulate a wide variety of physiological processes in mammals. This review describes well-defined functional responses mediated through activation of high-affinity MT₁ and MT₂ proteinteoupled receptors viewed as potential targets for drug discovery. MT₁ melatonin receptors modulate neuronal firing, arterial vasoconstriction, cell proliferation in cancer cells, and reproductive and metabolic functions. Ativation of MT₂ melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. The melatonin-mediated responses elicited by activation of MT₁ and MT₂ native melatonin receptors are dependent on circadian time, duration and mode of exposure to endogenous or exogenous melatonin, and functional receptor sensitivity. Together, these studies underscore the importance of carefully linking each melatonin receptor type to specific functional responses in target tissues to facilitate the design and development of novel therapeutic agent.     

Melatonin recovers sleep phase delayed by MK-801 through the melatonin MT2 receptor- Ca2+-CaMKII-CREB pathway in the ventrolateral preoptic nucleus

Melatonin (MLT) is widely used to treat sleep disorders although the underlying mechanism is still elusive. In mice, using wheel-running detection, we found that exogenous MLT could completely recover the period length prolonged by N-methyl-D-aspartate receptor (NMDAR) impairment due to the injection of the NMDAR antagonist MK-801, a preclinical model of psychosis. The analysis of the possible underlying mechanisms indicated that MLT could regulate the homeostatic state in the ventrolateral preoptic nucleus (VLPO) instead of the circadian process in the suprachiasmatic nucleus (SCN). In addition, our data showed that MK-801 decreased Ca²⁺-related CaMKII expression and CREB phosphorylation levels in the VLPO, and MLT could rescue these intracellular impairments but not NMDAR expression levels. Accordingly, Gcamp6 AAV virus was injected in-vivo to further monitor intracellular Ca²⁺ levels in the VLPO, and MLT demonstrated a unique ability to increase Ca²⁺ fluorescence compared with MK-801-injected mice. Additionally, using the selective melatonin MT₂ receptor antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT), we discovered that the pharmacological effects of MLT upon NMDAR impairments were mediated by melatonin MT₂ receptors. Using electroencephalography/electromyography (EEG/EMG) recordings, we observed that the latency to the first nonrapid eye movement (NREM) sleep episode was delayed by MK-801, and MLT was able to recover this delay. In conclusion, exogenous MLT by acting upon melatonin MT₂ receptors rescues sleep phase delayed by NMDAR impairment via increasing intracellular Ca²⁺ signaling in the VLPO, suggesting a regulatory role of the neurohormone on the homeostatic system.     

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 MT 1, but not MT 2 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 MT 1 receptors to inhibit orexin neurons and promote sleep.     

Melatonin promotes regeneration of injured motor axons via MT1 receptors

Melatonin is an ancient multi‐tasking molecule produced by the pineal gland and by several extrapineal tissues. A variety of activities has been ascribed to this hormone in different physiological and pathological contexts, but little is known about its role in peripheral neuroregeneration. Here, we have exploited two different types of injury to test the capability of melatonin to stimulate regeneration of motor axons: (a) the acute and reversible presynaptic degeneration induced by the spider neurotoxin α‐Latrotoxin and (b) the compression/transection of the sciatic nerve. We found that in both cases melatonin administration accelerates the process of nerve repair. This pro‐regenerative action is MT₁‐mediated, and at least in part due to a sustained activation of the ERK1/2 pathway. These findings reveal a receptor‐mediated, pro‐regenerative action of melatonin in vivo that holds important clinical implications, as it posits melatonin as a safe candidate molecule for the treatment of a number of peripheral neurodegenerative conditions.