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.     

Genetic deletion of MT1 and MT2 melatonin receptors differentially abrogates the development and expression of methamphetamine‐induced locomotor sensitization during the day and the night in C3H/HeN mice

Abstract: 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 MT₁ and/or MT₂ 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), MT₁KO and MT₂KO 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 MT₁/MT₂KO 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, MT₁KO and MT₂KO mice was statistically different from VEH‐treated controls. However, WT and MT₂KO, but not MT₁KO 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 MT₁ melatonin receptor activation by endogenous melatonin. We suggest that MT₁ and MT₂ melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.     

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 receptor structures shed new light on melatonin research

The tryptophan derivative melatonin is an evolutionary old molecule that is involved in a pleiotropy of physiological functions. In humans, age‐related decline of circulating melatonin levels and/or dysregulation of its circadian synthesis pattern have been associated with several disorders and disease states. Several molecular targets have been proposed for melatonin since its discovery, in 1959. Among them, melatonin MT₁ and MT₂ receptors are the best characterized melatonin targets, mediating melatonin effects in a variety of tissues. They belong to the superfamily of G protein‐coupled receptors. Two back‐to‐back articles published in the “Nature” Journal earlier this year present the first crystal structures of the human MT₁ and MT₂ in its inactive states. Here, we will briefly outline the discovery path of melatonin receptors until their structural elucidation and discuss how these new findings will guide future research toward a better understanding of their function and rational drug design.     

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.     

Melatonin‐mediated inhibition of Purkinje neuron P‐type Ca2+ channels in vitro induces neuronal hyperexcitability through the phosphatidylinositol 3‐kinase‐dependent protein kinase C delta pathway

Although melatonin receptors are widely expressed in the mammalian central nervous system and peripheral tissues, there are limited data regarding the functions of melatonin in cerebellar Purkinje cells. Here, we identified a novel functional role of melatonin in modulating P‐type Ca²⁺ channels and action‐potential firing in rat Purkinje neurons. Melatonin at 0.1 μm reversibly decreased peak currents (I_Ba) by 32.9%. This effect was melatonin receptor 1 (MT_R1) dependent and was associated with a hyperpolarizing shift in the voltage dependence of inactivation. Pertussis toxin pretreatment, intracellular application of QEHA peptide, and a selective antibody raised against the G_β subunit prevented the inhibitory effects of melatonin. Pretreatment with phosphatidylinositol 3‐kinase (PI3K) inhibitors abolished the melatonin‐induced decrease in I_Ba. Surprisingly, melatonin responses were not regulated by Akt, a common downstream target of PI3K. Melatonin treatment significantly increased protein kinase C (PKC) activity 2.1‐fold. Antagonists of PKC, but not of protein kinase A, abolished the melatonin‐induced decrease in I_Ba. Melatonin application increased the membrane abundance of PKC_δ, and PKC_δ inhibition (either pharmacologically or genetically) abolished the melatonin‐induced I_Ba response. Functionally, melatonin increased spontaneous action‐potential firing by 53.0%; knockdown of MT_R1 and blockade of P‐type channels abolished this effect. Thus, our results suggest that melatonin inhibits P‐type channels through MT_R1 activation, which is coupled sequentially to the βγ subunits of G_i/o‐protein and to downstream PI3K‐dependent PKC_δ signaling. This likely contributes to its physiological functions, including spontaneous firing of cerebellar Purkinje neurons.     

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.     

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.     

Expression of the orphan GPR50 protein in rodent and human dorsomedial hypothalamus,tanycytes and median eminence

Abstract: The melatonin receptor family is composed of three members, MT₁ and MT₂ receptors that bind melatonin with high affinity and the orphan GPR50 that does not bind melatonin but shares significant sequence homology with the two other subtypes. In the absence of any known ligand for this orphan receptor, little is still known about its function. We recently reported the development of the first anti‐GPR50 antibodies that reliably recognized the recombinant human GPR50. We here used these antibodies to study the expression of GPR50 in mouse, rat and human hypothalamus, a region reported to express GPR50 mRNA. GPR50 immunoreactivity (ir) was observed in dorsomedial hypothalamic (DMH) cells co‐stained with the neuronal marker HuC/D. GPR50‐ir was also observed in cells of the ependymal layer of the third ventricle that co‐stained with vimentin. More specifically, its localization in the lower region of the third ventricle and along the long basal processes contacting portal blood vessels in the median eminence (ME) suggested expression of GPR50 in tanycytes. Consistent staining patterns were observed in all three species with two different antibodies. Taken together, our study validates two GPR50‐specific antibodies for the use in rodent and human tissue. Evolutionary conserved expression of GPR50 in DMH neurons and tanycytes, together with previously reported expression of the receptor in the pituitary, support the potentially important role of GPR50 in key hypothalamic functions, including regulation of the hypothalamo‐pituitary axes.     

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.     

Melatonin‐mediated inhibition of Cav3.2 T‐type Ca2+ channels induces sensory neuronal hypoexcitability through the novel protein kinase C‐eta isoform

Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T‐type Ca²⁺ channels (T‐type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T‐type channels in small TG neurons via the melatonin receptor 2 (MT₂ receptor) and a pertussis toxin‐sensitive G‐protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT₂ receptor coprecipitated with Gα_o. Both shRNA‐mediated knockdown of Gα_o and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin‐induced T‐type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC‐eta (PKC_η) while antagonism of PKC_η or shRNA targeting PKC_η prevented the melatonin‐mediated effects. In a heterologous expression system, activation of MT₂ receptor strongly inhibited Cav3.2 T‐type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKC_η dependent and was accompanied by a negative shift in the steady‐state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant‐induced inflammatory pain. These actions were inhibited by T‐type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T‐type channel activity through the MT₂ receptor coupled to novel G_βγ‐mediated PKC_η signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.     

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.     

Separation of presynaptic Cav2 and Cav1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca2+ pump PMCA

Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca²⁺ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)–triggered presynaptic Ca²⁺ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Ca_v2 homolog, Dmca1A or cacophony, and the Ca_v1 homolog, Dmca1D) but with spatial and functional separation. Ca_v2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Ca_v1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca²⁺ influx but has a small impact on release. Instead, L-type calcium currents through Ca_v1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca²⁺ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Ca_v1-mediated Ca²⁺ influx. We show that the plasma membrane Ca²⁺ ATPase (PMCA) resides in between active zones and isolates Ca_v2-triggered release from Ca_v1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Ca_v2 may also contribute to. As L-type Ca_v1 channels also localize next to PQ-type Ca_v2 channels within axon terminals of some central mammalian synapses, we propose that Ca_v2, Ca_v1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.

Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca²⁺-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca²⁺ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca²⁺ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood.SV release probability depends on the sensitivity of the vesicular Ca²⁺ sensor and the positioning of VGCCs inside active zones (AZs) (1). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered (2–4). Irrespective of such modulation, efficient Ca²⁺-triggered SV release through presynaptic VGCCs (mainly Ca_v2.1 and Ca_v2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca²⁺ buffers (5, 6). It is thus conceivable that Ca²⁺ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity.Apart from the need for fast activating and inactivating Ca_v2 channels for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of Ca_v1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression (7). In hippocampal CA3 mossy fiber boutons (8–10) or in synapses of the lateral amygdala (11), Ca_V2.3 and Ca_V1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (9, 11).Differential functions of Ca_v2 and Ca_v1 channels in neurotransmitter release versus other Ca²⁺-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant (4, 10). Moreover, compared with Ca_v2.1 and Ca_v2.2, Ca_v1 channels display higher conductances (12), suggesting that additional mechanisms are required to allocate Ca_V1-related Ca²⁺ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca²⁺ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca²⁺-triggered SV release (13–15). We hypothesize that activity-dependent regulation of SV recycling employs Ca_v1-dependent Ca²⁺ entry and that active mechanisms exist to regulate the relative contributions of Ca_v2 and Ca_v1 channels to SV release versus recycling. We address these hypotheses at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (16–18).     

Melatonin inhibits serotonin transporter activity in intestinal epithelial cells

Abstract: Gastrointestinal serotonin (5‐HT) and melatonin are two closely related neuromodulators which are synthesised in the enterochromaffin cells of the intestinal epithelium and which have been shown to be involved in the physiopathology of the gastrointestinal tract. The effects of 5‐HT depend on 5‐HT availability which is, in part, modulated by the serotonin transporter (SERT). This transporter provides an efficient 5‐HT uptake after release and is expressed in the membrane of the enterocytes. Although the origin and effects of 5‐HT and melatonin are similar, the interrelationship between them in the gastrointestinal tract is unknown. The main aim of this study was to determine whether melatonin affects SERT activity and expression, and, if so, to elucidate the mechanisms involved. Caco‐2 cell line was used to carry out the study as these cells have been shown to endogenously express SERT. The results showed that melatonin inhibits SERT activity by affecting both V_max and kt kinetic constants although SERT synthesis or intracellular trafficking did not appear to be affected. The melatonin effect seemed to be independent of melatonin receptors MT₁ and MT₂ and protein kinase C and cAMP intracellular pathways. Our results suggest that the inhibition of SERT might be due to a catalytic effect of melatonin on the allosteric citalopram‐sensitive site in SERT. This study shows, for the first time, that melatonin modulates SERT activity, thus demonstrating the feedback system between melatonin and the serotoninergic system in the gastrointestinal tract.     

Melatonin improves the reprogramming efficiency of murine‐induced pluripotent stem cells using a secondary inducible system

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all‐iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10^?6, 10^?7, 10^?8, 10^?9, or 10^?10 m ) or with vitamin C (50 μg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10^?8 (0.81 ± 0.04%), and 10^?9 m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (P < 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT₂ was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT₁ and MT₂ receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis‐related genes p53 and p21 was lower in the group treated with 10^?9 m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53‐mediated apoptotic pathway.     

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.