| Methamphetamine(MA)abuse is increasing rapidly in China and even in the world.Compared traditional drugs such ad heroin,cocaine,MA is more addictive and has more serious damage to the central nervous system.However,the research on MA addiction is lagging behind,and there is no approved pharmacological intervention for treating MA addiction at home and abroad.Drug addiction,core characterized by compulsive drug use,is a continuous process from recreational drug use to regular drug use and to compulsive drug use.Current studies suggest that the behavioral basis of this continuous process is a gradual transition from goal-directed to habit learning,while the nucleus involved in behavioral control gruadually migrates from ventral striatum(vStr),with the nucleus accumbens(NAc)at the core,to the dorsal striatum(dStr),especially the dorsolateral striatum(dlStr).Nevertheless,the underlying cellular and molecular mechanism remains unclear.Therefore,reseach on the cellular and molecular mechanisms underlying the transition from regular drug use to compulsive drug use is the key to elucidate the mechanism of MA addiction.MicroRNA(miRNA)are a class of noncoding short-chain RNAs that regulate gene expression at post-transcription.Many studies have revealed linkages between the abnormal miRNAs levels and cocaine,opioids and alcohol addiction,but few reports have focused on methamphetamine exposure,especially the research related to compulsive MA use is almost blank.Through the miRNA microarray screening and verification,our previous studies have found that miR-30a was significantly down-regulated while miR-134 was significantly up-regulated in dStr of rat with compulsive MA use.However,the molecular mechanisms underlying the role of miR-30a or miR-134 in compulsive MA use remain unclear.ObjectiveThe aim of this project was to study whether miR-30a and miR-134 regulated compulsive MA use and its molecular mechanism,providing experimental basis for elucidating the neurobiological mechanism of MA addiction.Methods and results1 The effect of dIStr miR-30a on MA addiction1.1 The correlation between dlStr miR-30a and compulsive MA use1.1.1 The establishment of MA self-administration models characterized with regular or compulsive drug use in ratsEstablishing an experimental animal model with "compulsive characteristics" is of great significance for revealing the neurological mechanism of drug addiction.With Reference to the literature,we established the MA self-administration models characterized with regular or compulsive drug use in rats.The results showed that the rats with extended access to MA(6 h/d,LgA group)displayed excessive and progressive increase in drug intake,whereas those with limited access to MA(2 h/d,ShA group)showed moderate and controlled drug consumption.As a result,the total MA consumption in LgA group was significantly higher than that in ShA group.Meanwhile,LgA self-administering rats cumulatively consumed more MAin the first 2 h of each session than ShA rats.The above results suggested that ShA rats displayed moderate and controlled regular drug use,while LgA rats showed excessive and uncontrolled compulsive drug use.This animal model was used in our subsequent studies in this dissertation.1.1.2 The miR-30a levels in subregions of striatum in MA self-administrated and passive treatment ratsIn MA self-administered rats,using RT-qPCR,compared with the saline control group(Con group),we found that miR-30a in NAc was significantly down-regulated in ShA group,while miR-30a in dlStr and dmStr was significantly down-regulated in LgA group,with greater down-regulation in dlStr(30%)than that in dmStr(15%).In the MA passive administration model,the short-term(7 days)exposure to low dose(1 mg/kg)MA resulted in decreased miR-30a level in NAc,while the long-term(28 days)exposure to high dose(5 mg/kg)MA resulted in down-regulated miR-30a in dlStr.The above results suggested that accompanying with the transition from regular to compulsive MA use,the down-regulation of miR-30a transferred from NAc to dlStr,which mediated by extended excessive drug exposure.1.1.3 The effect of up-regulated miRNA-30a in dlStr on compulsive MA use in rats The lentivirus carrying pre-miR-30(LV-miR-30a)was microinjected into dlStr to up-regulate miR-30a levels in rats.Compared with the negative control lentivirus(LV-Con),LV-miR-30a selectively increased miR-30a in dlStr.Using the immunofluorescence staining,we found that LV-miR-30a was mainly localized in neurons.Compared with microinjecting LV-Con bilaterally,LV-miR-30a up-regulated dlStr miR-30a,and decrease the incremental drug use curve and total drug intake in rats with extended access to MA,suggesting that dlStr miR-30a negatively regulated compulsive MA use in rats.1.2 The neurobiological mechanism of miR-30a modulating compulsive MA use in rats.1.2.1 Differential expressions of miR-30a targets induced by compulsive MA useConsidering the bioinformaties analysis that the function of predicted targets of miR-30a mainly clustered in gene silence and regulation of synaptic plasticity,Tnrc6a and BDNF,as important molecules in mediating gene silence and regulating synaptic plasticity respectively,were chosen for the following study.(BDNF and Tnrc6a were verified as targets of miR-30a by our and other published work.)In dlstr of MA self-administered rats,the expression of BDNF and Tnrc6a robustly increase in LgA group,but not in ShA group,compared with Con group.However,there was no significant difference in BDNF and Tnrc6a levels in dmStr among the three groups.Therefore,the expression of BDNF and Tnrcoa in dlStr was specifically up-regulated by compulsive MA use,consistent with the negative regulation of miR-30a on its targets,suggesting that BDNF and Tnrc6a may be related to the mechanism underlying miR-30a participating in compulsive MA use.1.2.2 The effect of Tnrc6a,a target of miR-30a,on compulsive MA use in ratsAt present,the research on Tnrcoa is mainly related to tumors,but there has been no report about neuropsychiatric disorders including drug addiction.ShRNA targeting Tnrc6a(sh-Tnrc6a)was used to down-regulate Tnrc6a in dlStr.It was confirmed that shRNA could effectively down-regulate the expression of Tnrc6a in vitro and in vivo.Lentivirus-mediated sh-Tnrc6a(LV-sh-Tnrc6a)and control sequences(LV-Con)were microinjected into bilateral dlStr of rats,respectively.Subsequently,rats were trained with MA self-administration.Compared with LV-Con,LV-sh-Tnre6a significantly reduced the expression of Tnrcoa in dlStr,but did not alter the incremental drug use and total drug intake in rats with extended access to MA,suggesting that dlStr miR-30a took part in compulsive MA use not via its target Tnrc6a.1.2.3 The effect of BDNF,a target of miR-30a,on compulsive MA use in rats It has been reported that BDNF is related to cocaine,opioid and alcohol addiction,but there has been no study about the role of dlStr BDNF in MA addiction.The same strategy with Tnrc6a was used here to down-regulate BDNF expression in diStr.Compared with LV-Con,LV-sh-BDNF significantly down-regulated the expression of BDNF in dlStr and also decreased MA-induced incremental drug use and total drug intake in rats with extended access to MA,which simulated the effect of up-regulated miR-30a in dlStr,suggesting that miR-30a might via its target BDNF,participate in regulating compulsive MA use.1.2.4 The effect of BDNF downstream pathway on compulsive MA use in ratsBDNF exerts biological effects by activating tyrosine kinase receptor B(TrkB),and stimulating extracellular signal-regulated kinase(ERK)and phosphoinositide-3 kinase(PI3K)pathways.Which pathway is involved in BDNF participating in compulsive MA use?The differential expression of important molecules in downstream pathway of BDNF were determined in the MA self-administration model.Compared with those in Con group,the levels of p-TrkB/TrkB and p-PI3K/PI3K in LgA group significantly increased,while the levels of p-ERK/ERK in ShA group significantly elevated in dlStr of rats.In addition,bilaterally microinjection of the PI3K pathway inhibitor LY294002 into dlStr significantly reduced self-administration in rats with extended access to MA,while the ERK pathway inhibitor U0126 did not affect the drug use behavior of rats.Therefore,our results suggested that dlStr BDNF,a target of miRNA-30a,positively regulated the compulsive MA use possibly through activating downstream PI3K pathway by BDNF-TrkB.Given that previous research on compulsive alcohol use suggested that up-regulated BDNF in dlStr reduced compulsive alcohol intake in rats by activating TrkB-ERK pathway,which was contrary to our results,it is suggested that BDNF might have complex regulatory effects on addiction behaviors induced by different types of addictive substances,and can mediate the opposite behavioral effects even in the same brain region(such as dlStr)through activating different downstream pathways.1,3 The correlation between miR-30a and BDNF as well as cue-induced relapse in MA-addicted ratsWe have found that the miR-3Oa and its target BDNF in dlStr are involved in the regulation of compulsive MA use.Then,is it also related to relapse after withdrawal?In this part,we established MA-induced compulsive drug use model in rats.After withdrawal for one month,cue-induced re-establishment of drug-seeking was conducted.The results showed that some rats exhibited high tendency to relapse,with active pokes at 51.88± 5.86,while others displayed low tendency to relapse,with active pokes at 25.83± 2.41.The levels of miR-30a and BDNF in dlStr were determined after relapse.Compared with those in Con group(saline self-administered rats),the miR-30a in dlStr of rats with low tendency to relapse increased significantly,and the BDNF mRNA did not alter;while the miR-30a in rats with high tendency to relapse remained unchanged but the BDNF mRNA increased significantly.In addition,compared with the low tendency to relapse group,the high tendency to relapse group showed lower miR-30a level and higher expression of BDNF.Linear regression analysis revealed that the miR-30a level was negatively correlated with the active pokes(r2 = 0.39,P<0.05),and the expression of BDNF was positively correlated with the active pokes(r2 = 0.62,P<0.01).These results suggested that the miR-30a and its target BDNF in dlStr may be related to cue-induced relapse after MA withdrawal and may be used to predict relapse tendency.2.The effect of dlStr miR-134 on MA addictionIn this part,we focused on the regulation of miR-134,another important molecule involved in synaptic transmission and plasticity,on compulsive MA use.2.1 The levels of miR-134 and its target LIMK1 in subregions of striatum in MA-induced self-administrated or passive adiministered ratsSimilar to previous parts about miR-30a,MA-induced short-term self-administration(ShA group)and long-term self-administration(LgA group)models were established.In dlStr,compared with those in Con group and ShA group,miR-134 in LgA group significantly increased and its target LIMK1 significantly decreased in both mRNA and protein levels,while there was no difference between ShA and Con groups in the levels of miR-134 and LIMKl.In dmStr,there were no significant differences in the levels of miR-134 and LIMK1 among the three groups.In NAc,compared with Con group,the level of miR-134 in LgA group increased slightly,while the level of LIMK1 did not change significantly.In the passive MA exposure model,rats in Con,low-dose MA(1 mg/kg,sc)and high-dose MA(5 mg/kg,sc)groups were administered with saline or MA for 7 days or 28 days continuously,respectively.After the continuous administration,no differential levels of miR-134 and LIMK1 mRNA were found in dlStr,dmStr or NAc among the three groups.These results suggested that compulsive MA use induced increase in miR-134 and decrease in its target LIMK1 in dlStr of rats,which might depend on the S-R habit learning.2.2 The effect of up-regulated miRNA-134 in dlStr on compulsive MA use in ratsLentivirus-mediated miR-134 sponge was used to silence endogenous miR-134.Using the luciferase reporter,we found that the sponge could down-regulate functional miR-134 in vitro.Next,we used a lentiviral vector to express the miR-134 sponge(LV-miR-134-Sil)in dlStr.Using immunofluorescence staining,we found that LV-miR-134-Sil mainly located at neurons rather than astrocytes in dlStr of rats.LV-miR-134-Sil and negative control virus(LV-NC)were microinjected into bilateral dlStr of rats respectively,and then MA-induced self-administration training was carried out for extended or limited access.In rats with extended access to MA,compared with LV-NC,LV-mir-134-Sil significantly decreased the level of miR-134 in dlStr,accompaning by increase in LIMK1 level and decrease in MA self-administration and total drug intake.However,in rats with limited access to MA,the decrease in miR-134 level in dlStr did not change the regular drug use and drug intake.These results suggested that down-regulation of dlStr miR-134 may reduce the MA-induced compulsive drug use via decreasing the expression of LIMK1 in rats,supporting the hypothesis that S-R(Stimulus-Response)habitual learning controlled by dlStr plays an important role in compulsive drug use.Conclusions1.In the MA-induced self-administration model of rats,shift from casual and regular drug use to compulsive drug use can induce the down-regulation of miR-30a transited from NAc,which controls motivation,to dlStr,which drives stimulus-response habit learning.Moreover,dlStr miR-30a,perhaps via its target BDNF acting on TrkB receptors and its downstream PI3K pathway,negatively regulated the MA-induced compulsive drug use.In addition,we also found that the levels of miR-30a and its target BDNF in dlStr were correlated with cue-induced relapse tendency after withdrawal in rats,suggesting that miR-30a and BDNF may be potential biomarkers for early warning of MA addiction relapse,which deserves further study.2.The study of miR-134,another key molecule involved in synaptic transmission and plasticity,revealed that MA-induced compulsive drug use up-regulated miR-134 in dlStr specifically,which depended on S-R habit learning.Furthermore,miR-134 in dlStr might positively regulate MA-induced compulsive drug use through its target LIMK1To sum up,we revealed the regulatory effects of miR-30a and miR-134 in dlStr on MA-induced compulsive drug use and the possible mechanisms underlying,supporting the hypothesis that S-R habit formation mediated by dlStr is an important mechanism underlying the transition from regular drug use to compulsive drug use and addiction at molecular level,providing experimental evidence for accurately elucidating the mechanism of drug addiction. |
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