Role And Mechanism Of Melatonin And Its Receptor MT2 In Learning And Memory

AIM Melatonin,the neurohormone synthesised and secreted by the mammalian pineal gland,shows great relationship with the day-light cycle.The secreted melatonin exists in blood,travelling to different parts of body,and functions through MT1 and MT2,the two main receptors of melatonin in mammals.It has been known to maintain the day-light circadian and promote sleep phase.Other functions of melatonin such as neuroprotection and free radical removel have been found in recent years.It has also been found that melatonin could improve some symptoms in neurodegenerative diseases like Alzheimer's disease,Parkinson's disease,Huntington's disease,amyotrophic lateralizing sclerosis and broad developmental disorders like autism.Learning and memory is the advanced function of human brain,most parts of the brain are related to this function,and the most studed parts are hippocampus and its neighboring region,cortex,amygdala and thalamus.Synaptic plasticity,including structure plasticity and function plasticity,is the neurobiological basis of learning and memory in animals.Long-term potentiation(LTP)and long-term depression(LTD)are two main forms of long term plasticity in mammalian brain,also the ideal cellular model in learning and memory study.Other than the transmission between neurons,the modification of glia and neural biochemical substances like neurosteroids and neuropeptides could also affect synaptic plasticity.The last few years have seen rapid advances in our understanding of the molecular mechanisms that are involved in the generation of LTD and in the functions of LTD in health and disease.There are two major forms of LTD,i.e.N-methyl-d-aspartate receptor(NMDAR)dependent LTD and metabotropic glutamate receptor(m Glu R)dependent LTD,act through different signaling pathways,causing ?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor(AMPAR)trafficking from neuron membrane to cytoplasm,decreasing the postsynaptic excitability,finally inducing LTD.The representative cognitive disorder disease of excessive LTD is fragile X syndrome(FXS),the most common inherited mental retardation diseases.FXS model mice show abnormal synaptic plasticity,like morphological abnormalities in neuronal development and synaptic transmission disorders.Electrophysiological recordings show that LTP is decreased and LTD is enhanced in the hippocampus and anterior cingulated cortex(ACC),and behavioral study results show the reduced fear memory performance involved by prefrontal cortex in FXS model mice.The role and mechanism of melatonin in learning and memory are still not clear,the melatonin-induced inhibition of LTP in different regions of hippocampus has been reported,and this inhibition acts through the activation of receptor MT2,but no one ever reported the influence of melatonin on LTD.Our research aims to find the influence of melatonin on the neuronal excitatory synaptic transmission,illuminate the function and mechanism of melatonin on LTD,and then discuss its role in FXS.It will provide both new ideas and theoretical foundation for exploring not only potential drug targets but also clinical treatment strategies.METHODS1.Rat cortical neurons were cultured in vitro,then treated with melatonin receptor MT2 agonist.Western blot method was used to test the changes of phosphorylation sites of AMPAR subunits Glu A1 and Glu A2.2.After MT2 antagonist and agonist treatments on cultured rat cortical neurons,biotin label membrane proteins were used to test the levels of membrane Glu A1 and Glu A2.3.Whole-cell patch clamp technique was used to test the influence of melatonin on the amplitudes and firing frequency of s EPSC and m EPSC in cultured rat hippocampus neurons.Evoked EPSC was recorded after melatonin application on CA1 neurons from the acute rat hippocampus brain slices.4.In vivo recording was used to find the influence of melatonin(i.p).on hippocampus CA1 f EPSP of anaesthetic rat.5.Melatonin-induced changes of evoked EPSC were recorded after pretreatment of NMDAR or m Glu R antagonist on brain slices,NMDAR or m Glu R dependent LTD were induced and the mechanism of melatonin on LTD induction was explored.6.Melatonin-induced changes of f EPSP were recorded in hippocampus CA1 from the mice with MT2 or m Glu R antagonist injection.7.Melatonin receptors levels were detected in hippocampus and ACC regions from Fmr1 KO mice and wild-type mice,to test if there is any overexpression of melatonin receptors.8.Stereotaxic inject sh RNA were performed to knock down the overexpressed melatonin receptor.Trace fear conditioning was performed to explore the change of learning and memory after fourteen days of injection.RESULTS 1.MT2 agonist could reduce the phosphorylation level of AMPAR subunit Glu A1 Ser 831 and enhance the phosphorylation level of AMPAR subunit Glu A2 Ser 880.2.MT2 agonist could reduce the membrane AMPAR Glu A1 and Glu A2 level,promote the trafficking of AMPAR from membrane to cytoplasm,and this trafficking could be blocked by MT2 antagonist pretreatment.3.Melatonin could significantly reduce the amplitude and firing frequency in both s EPSC and m EPSC,indicating that it could depress the excitatory synaptic transmission.Application of melatonin o rat hippocampus slices could reduce the amplitude of evoked EPSC in CA1,inducing LTD.4.Melatonin injection(i.p.)in anaesthetic rat could reduce the slope of f EPSP in hippocampus CA1 region,inducing LTD.5.The LTD induced by melatonin was not affected by NMDAR antagonist but could be totally blocked by m Glu R antagonist.After the evoked EPSC getting steady during the melatonin application,NMDAR dependent LFS could further induce LTD,but m Glu R dependent pp LFS didn't change the former LTD.6.Injection of MT2 or m Glu R antagonist could block the LTD in hippocampus CA1 region induced by following i.p.injection of melatonin in anaesthetic rat.7.In the hippocampus,the levels of melatonin receptors didn't show any significant difference between two types of mice,but overexpressed MT2 could be found in Fmr1 KO mice compared with wild-type mice.8.Knocking down MT2 in ACC of Fmr1 KO mice could significantly improve trace fear memory of Fmr1 KO.CONCLUSIONS 1.Melatonin could induce AMPAR trafficking from membrane to cytoplasm and,induced LTD through activation of MT2.2.The mechanism of melatonin-induced LTD was as similar to Group I m Rlu R-LTD.3.MT2 was overexpressed in Fmr1 KO mice,suppression of this overexpression could partially rescue the behavioral symptom of FXS model mice.