| Methamphetamine(MA)is a psychostimulant that is used extensively world-wide.MA abuse has become a serious social problem.MA is characterized as psychological dependence and neurotoxicity.MA administration can cause the alteration of behavior and cognition of individuals immediately,and induce neuronal damage.The long term MA abuse can result in the deficits of attention,working memory and decision-making,and lead to psychosis and aggressive behavior in addicts,even cause abvious neurode gene rat ion.After MA exposure,the individuals usually demonstrate severe drug dependence and great risk of relapse,which is also account for the severe social harm of MA.Although we already have deep understanding on the harm of MA,no effective method has been found to treat MA addiction and toxicity to date.Therefore,elucidating the mechanisms underlying MA dependence and toxicity is critical for the clinical treatment of MA addicts.Drug dependence is a phenomenon of compulsive drug craving and using.regardless of all consequences.When the individuals are exposed in the environment having drug associated cues during withdrawal,they would experience with strong drug craving and result in relapse.After withdrawal,the individuals have permanent and increasing drug craving associated with the drug cues.which is termed incubation of drug craving.It is supposed that repeated drug stimulation would cause the neural circuits to be sensitized to the incentive effects of the drug,and it would demonstrate as locomotor sensitization in animals.Considered as one of the manifestations of drug craving,locomotor sensitization is implicated in drug relapse.Locomotor sensitization is a phenomenon of the enhancement of locomotor activity after exposure to psychostimulants.The model of locomotor sensitization composed of three stages:induction,incubation and expression.After repeated administration of psychostimulants(5-7 days),the individuals should followed with the 7-14 days of the incubation of sensitization,then exposed with the drug again on the last day and the locomotion is recorded.The locomotor activities of the animals would be intensified after the incubation.Literatures mainly focus on the induction and the expression of sensitization,and rarely pay attcntion to the incubation of sensitization,especially the mechanisms of the locomotor enhancement after the incubation.Given the incubation of sensitization is also the withdrawal period,and the drug withdrawal would lead to the incubation of drug craving,there should be some common neurobio logical mechanisms underlying these two kinds of incubations.Investigations indicate that drug withdrawal.especially the MA withdrawal would cause anxiety and depression,besides drug craving.The depressed and anxious syndromes are common during MA withdrawal.and these syndromes have significant correlation with the impulsive MA craving and relapse.Therefore.the treatment which could effectively counteract the complications of MA withdrawal may be capable of attenuating the drug dependence.A great amount of studies indicate that stress can enhance drug craving and re lapse,and chronic stress would lead to depression.Stress can stimulate HPA axis,and induce the release of glucocorticoid,which is similar to the impact of M A exposure.Given that HPA axis is crucial for depression,the stimulation of HPA axis through MA may be one of the intrinsic reasons that MA withdrawal would be liable to cause depression.Moreover,stress,anxiety and depression also can inhibit the hippocampal neurogenesis,and the increase of hippocampal neuro genes is can effectively counteract stress and attenuating depression.In addition,various kinds of antidepressant treatment could lead to the increase of hippocampal neurogenesis.Investigations demonstrate that the self-ad minstr at io n of MA could decrease the hippocampal neuro genes is and proliferation,and reduce the number of granular cells and the volume of the hippocampus.Besides,the effects of MA exposure exert on hippocampal neurogenesis also are implicated in the neurotoxicity of MA.MA shows obvious neurotoxicity.The neurotoxicity of MA could cause the lost of gray matter,the thickening of white matter and the proliferation of microglia.Long term of MA exposure would lead to neurodegeneration.MA could induce the damage of the dopaminergic and serotonergic terminals,the injury of nigrostriatal pathway and increase the risk of suffering Parkinson’s disease.The neurotoxicity of MA could be related to the acute accumulating of dopamine in the brain.The oxidation of dopamine results in the production of reactive oxygen species(ROS)and oxidative damage,which is considered as the main factor in MA induced neurotoxicity.The subsequence investigations indicate that MA could also induce neuronal damage without dopamine.Thus,there should be mechanisms of MA induced damage beyond dopamine.Studies suggest that mitochondrional apoptotic pathways also taking part.MA exposure could cause the decrease of mitochondrional membrane potential and the over-loading of Ca2+ Therefore,the antioxidants and mitochondrional protector may have the ability to counteract the MA induced neurotoxicity,and melatonin is one of them.Melatonin is a kind of darkness hormone mainly released by the pineal gland.Melatonin can induce sleep and is used successfully in treating sleep related diseases.Melatonin is a biomarker of depression and can attenuate the depressive behaviors in mice caused by chronic stress.Moreover,melatonin is proved to be an antioxidant and free radical scavenger.Melatonin can inhibit the releasing the dopamine in specific brain regions and modulating the dopaminergic neuronal pathways related to motor diseases.MA abuse would decrease the level of the dopaminergic transporters and leads to the aberration of locomotion.It is demonstrated that the acute treatment of melatonin could ameliorate the MA induced hyperpyrexia,but have no effects on locomotor sensitization.The acute treatment of melatonin in high dose can enhance locomotor sensitization,and the melatonin treatment just before the expression of sensitization can not attenuate the expression of locomotor sensitization.Besides,the melatonin treatment during the induction of sensitization can decrease the expression of sensitization.However,there is no article performed the chronic melatonin treatment during the incubation of sensitization,and the mechanisms underlying the intervention of melatonin are also barely mentioned.Thus,we used melatonin to treat the incubation sensitization and expecting it could finally inhibit the expression sensitization In addition we also investigated the effects of melatonin on MA induced neuronal damage and their molecular mechanisms in vitro.The epigenetic mechanisms of drug dependence are gradually concerned by the investigators at present.As a part of epigenetic,emerging evidence has implicated microRNA in addiction.As a kind of microRNA which is abundant in the brain,miR-132 is regulated by pCREB.Investigations indicate that miR-132 is crucial for the synaptic plasticity of the nervous system.miR-132 can regulate the expression of MeCP2 as a target,and MeCP2 is critical in maintaining the synaptic functions of neurons.Investigations indicate that MeCP2 implicates in the compulsive cocaine administration and amphetamine induced locomotor sensitization.Moreover,MeCP2 can regulate the expression of BDNF,which is definitely implicated in drug addiction.It is reported that BDNF can counteract the MA induced neural death,and the regulation of AKT is critical for the process.Also based on the primary cultured neurons,another investigation reveals that BDNF exposure significantly increases the expression of miR-132.Interestingly,miR-132 can enhance the growth of neurites.and as a kind of prosurvival factor,AKT is also implicated in the regulation of neurite growth.Moreover,miR-132 can reduce the expression of PTEN as a target,and PTEN can inhibit the phosphorylation of AKT.Therefore,miR-132 indirectly activate AKT pathway via reducing PTEN expression.AKT activates a cascade of down-stream molecues,including GSK3β and mTOR.Investigations indicate that amphetamine can dephosphorylate AKT/GSK3β pathway,and MA exposure reduces the phophorylation of mTOR.Moreover,melatonin can counteract the amyloid-βprotein induced neuronal damage via phosphorylating AKT/GSK3β pathway,and exert its effect on neuronal protection via activating mTOR.Based on the investigations mentioned above,we suppose that the expression of miR-132 and MeCP2 in the PFC and NAc should be modulated by MA,and implicated in the locomotor sensitization in mice.Moreover,the expression of miR-132 and MeCP2 may also play a role in the intervention of melatonin on the expression of sensitization.Therefore,the sensitization behavior in mice could be manipulated via alteration the expression of miR-132 and MeCP2 in the NAc.With regard to neurotoxicity,we treated the primary cultured neurons with MA,and also protected the neurons with melatonin.We studied the effects of the treatments and their underlying molecular mechanisms.We performed the transfection of primary cultured neurons with lenti-virus to over-express miR-132 and reduce the expression of MeCP2,anticipating them to counteract the MA induced neuronal damage.We investigated the relationship of MA induced neuronal damage and AKT/GSK3β/mTOR pathway as well.Part I The investigation of the mechanisms and the effects of melatonin intervention on methamphetamine induced locomotor sensitizationIn this part of experiment,we hypothesize that the intervention of melatonin on the incubation of sensitization would attenuate the withdrawal syndrome of MA,and counteract MA induced neuronal damage,because of which finally leads to the mitigation of the expression of sensitization.The protocol of the locomotor sensitization in mice and the intervention of melatonin is as follow:after three days of acclimation,the mice were injected with MA consecutively for seven days as the induction of sensitization and their locomotor distance were recorded:then followed with the 14 days of the incubation of sensitization.among which we treated the mice with low dose of melatonin;finally,on day 22,the expression of sensitization was recorded.We found that the melatonin treatment during the incubation of sensitization could attenuate the expression of sensitization.Based on the results mentioned above.we further investigated the mechanisms of melatonin on the expression of sensitization.Given melatonin has an obvious effect on depression,and depression as well as anxiety are the main withdrawal syndrome of MA,we suppose melatonin may counteract the expression of sensitization via attenuating the withdrawal syndrome during the incubation of sensitization.We used light/dark boxes,forced swimming test,novelty-suppressed feeding test,and sucrose preference test et al.to confirm that the mice have obvious anxiety and depression during the incubation of sensitization and melatonin could significantly attenuate the anxious and depressive behaviors in mice.Moreover,because the hippocampal neurogenesis plays an important role in the MA withdrawal,depression and anxiety.we detected the expression of Brdu as well as DCX in the hippocampus via immunofluorescence.Our results indicate that the incubation of sensitization decreases the number of hippocampal neurogenesis and the intervention of melatonin could increase the level of hippocampal neurogenesis.Thus,melatonin could attenuate the withdrawal syndrome during the incubation of sensitization and because of which counteract the expression of sensitization.In order to further elucidate the molecular mechanism of MA induced locomotor sensitization and the intervention of melatonin,we probed the expression of miR-132 and MeCP2 in the NAc as well as PFC via PCR,immunofluorescence,and western blot.Our results showed that the expression of miR-132 was decreased in the PFC and NAc after the expression of sensitization,and the intervention of melatonin could recover the expression of miR-132 in the brain regions mentioned above:correspondingly,the results of immunofluorescence and western blot showed that the MA induced expression of sensitization could substantially increase the expression of MeCP2 in the NAc and PFC.According to the reports,pGSK3β is implicated in both the initiation and expression of sensitization.We found that MA increased the expression of pGSK3P in the NAc correspondingly,and melatonin can partially decrease its expression(not significantly).Interestingly,the correlation analysis indicates that the expression of MeCP2 in the NAc has linear correlation with the locomotor distance of mice in both the MA control group and saline control group.Based on the discoveries mentioned above,we further investigated the effect of the regulation of the expression of miR-132 and MeCP2 in the NAc on the locomotor sensitization.We injected lentivirus into the NAc bidirectionally at the initiation of the incubation of sensitization,and by which we increased the expression of miR-132 and decreased the expression of MeCP2 respectively.Then,after the incubation of sensitization,we observed their effects on the final expression of sensitization.Our results demonstrate that the downregulation of MeCP2 expression in the NAc could effectively decrease the final expression of sensitization,and the upregulation of the expression of miR-132 in the NAc could partially decrease the expression of sensitization,but not significantly.Together,the results of this part demonstrate that the intervention of melatonin on the incubation of sensitization can significantly decrease the MA induced expression of sensitization.Such effects could be partially related to the impact of melatonin on MA withdrawal induced anxiety and depression.We also investigated the miR-132/MeCP2 pathway in the expression of sensitization and the intervention of melatonin,and found that MeCP2 play a key role in the expression of sensitization.Part II The investigation of the mechanisms and the effects of melatonin intervention on methamphetamine induced neurotoxicityIn this part,we investigated the mechanisms of MA induced neuronal damage and melatonin intervention.First,we obtained the cortical neurons from the new born mice and performed the primary culture.We treated the neurons with different concentrations of MA on the fifth day of culture,detected the cell viability with CCK-8,and determined the optimal density.After that,we used different concentration of melatonin to protect the neurons and identified the optimal concentration for protection.Based on the results,we designed the experiment as three groups:control,MA induced damage,MA induced damage plus melatonin protection.Morphologically.MA can significantly reduce the number and length of neurites of the primary cultured neurons and melatonin treatment could significantly recover the number and length of neurites.We also detected the apoptosis and cell death via Hoechst/PI staining,and found that MA exposure could greatly increase the rate of apoptosis and cell death,and melatonin could significantly counteract the MA induced cell damage.In order to elucidate the molecular mechanisms of MA induced damage and the protective effects of melatonin,we detected the expression of miR-132 via PCR and the expression of pCREB,MeCP2 and PTEN with western blot.We found that MA exposure could significantly reduce the expression of pCREB and miR-132,and melatonin could effectively increase their expression.However,as the downstream targets of miR-132,the expression of MeCP2 and PTEN increased,and melatonin treatment could decrease their expression.We performed the transfection of primary cultured neurons with lentivirus,and found that the over-express ion of miR-132 could significantly attenuate the MA induced neuronal damage.Furthermore,the downregulation of MeCP2 could also protect the neurons against the neurotoxicity of MA.Finally,we used the inhibitor to down regulate the expression of PTEN.and got the protective effects as well.Given the impact of miR-132/PTEN on the downstream AKT pathway,we further investigated the relation of AKT/GSK3β/mTOR and the intervention of melatonin on MA induced neuronal damage.We found that MA exposure could significantly decrease the phosphorylation of the AKT/GSK3(3/mTOR pathway,and melatonin could significantly increase the phosphorylation of AKT/GSK3β/mTOR pathway.Then,we used LY294002 and rapamycin to inhibit the activation of AKT and mTOR,respectively.Our results show that both of the inhibitors could decrease the protective effects of melatonin.which suggest that AKT/mTOR pathway is implicated in the protective effects of melatonin.For the sake of elucidating the effects of AKT/GSK3β pathway in the process,we select lithium to inhibit the activation of GSK3β(increase the phosphorylation of GSK3β).The results show that lithium can protects the neurons against MA induced neuronal damage,and significantly increase the phosphorylation of the AKT/GSK3β/mTOR pathway.Moreover,LY294002 and rapamycin could inhibit the protective effects of lithium,which suggest that lithium protects the neurons against MA induced neurotoxicity viaAKT/GSK3β/mTOR pathway as well.Therefore,our investigation suggest that the dephosphorylation and phosphorylation of AKT/GSK3β/mTOR pathway is crucial for regulating the process of MA induced neurotoxicity and intervention.Together,in this part,we confirm that melatonin can protect the neurons against MA induced neurotoxicity,and identify miR-132/MeCP2/PTEN pathway and its downstream AKT/GSK3β/mTOR pathway are implicated in the MA induced neurotoxicity and melatonin protection.ConclusionOur investigation based on the specific characters of MA,select melatonin to treat the MA induced addiction and neurotoxicity.We study and elucidate the mechanisms of melatonin on the MA induced sensitization and neuronal damage.We originally use melatonin to treat the incubation of sensitization,and effectively reduced the expression of sensitization.Given the phase of the incubation of sensitization is also the drug withdrawal period.and the definite effect of melatonin on the depression and anxiety,we found that melatonin could significantly attenuate the depression and anxiety emerged during the incubation of sensitization,and suggest which may be implicated in the effect of melatonin on the expression of sensitization.Moreover.in the aspect of molecular mechanism,we originally find that the MA induced expression of sensitization can cause the decrease of miR-132 and increase of MeCP2 expression in the PFC and NAc,respectively.The intervention of melatonin can obviously increase the expression of miR-132 and significantly decrease the expression of MeCP2 in the brain regions mentioned above.Given our results demonstrate linear correlation of MeCP2 expression in the NAc and the locomotor distance in mice,we downregulate the expression of MeCP2 in the NAc via lenti-virus,and significantly decrease the final expression of sensitization.The results indicate that the expression of MeCP2 in the NAc is critical for regulation of the expression of sensitization.In order to further elucidate the mechanisms underlying the MA induced neurotoxicity and the protection effects of melatonin,we designed experiments in vitro.Our investigation demonstrates that melatonin can effectively counteract the MA induced neuronal damage.Furthermore,we find that melatonin can counteract the MA induced neuronal damage via miR-132/MeCP2/PTEN pathway and down streaming AKT/GSK3β/mTOR pathway.Together,our investigations provide new clues for elucidating the mechanisms of the effects of melatonin intervention on MA induced locomotor sensitization and neuronal damage. |
