The Role And Mechanism Of Dorsal Raphe Nucleus To Ventral Tegmental Area Pathway In Methamphetamine Addiction

Methamphetamine(METH)is a common class of psychostimulant drugs that can lead to psychostimulant effects and addiction.Although previous studies have extensively investigated the neural pathway,neurotransmitters and relevant molecules underlying METH addiction,the mechanism of METH addiction is still elusive.Recent studies have found that,the dorsal raphe nucleus(DRN)to ventral tegmental area(VTA)neural pathway is involved in the regulation of rewarding effects,but the role of DRN in addictive behaviors caused by METH and its mechanisms remain unclear.Objective:To investigate the role of DRN-VTA pathway in METH addiction and its effect on the release of dopamine transmitters;and to analyze the proteomic changes in the DRN after METH treatment,to identify the protein molecules related to METH addiction.Methods:(1)Detection of neuron activation in the DRN and VTA induced by METH:A conditioned place preference(CPP)animal model was established using C57BL/6J mice.The activation of neurons in the DRN and VTA of model mice was observed by immunofluorescence staining for the immediate early gene c-Fos.Ca2+ is an important indicator of neuron activation.Therefore,this work prepared an adenovirus vector containing the Ca2+ indicator GCaMP6s and injected it into the DRN to detect changes in neuronal Ca2+levels in the METH-induced model mice.In addition,the protein expression of CaMKII and its downstream molecule p-CREB was detected.(2)Detection of activation from DRN to VTA projection neurons:First,under the guidance of a stereotaxic instrument,retrograde fluorescent tracers were injected into the VTA for 7 days,and then METH was administered.The expression of c-Fos in labeled neurons within the DRN was detected in the model mice.Then,mice were injected with virus for anterograde transneuronal labeling based on the Cre-loxp system.scAAV2/1-hSyn-Cre was injected into the DRN,while AAV2/9-CMV-DIO-EGFP-WPRE was injected into the VTA.The colabeling of c-Fos and virus-labeled neurons in model mice was examined three weeks later.(3)Effect of chemogenetic inhibition of DRN neurons on METH-induced CPP in mice:In the present study,we injected two AA Vs:AAV9-hSyn-hM4D(Gi)-mCherry or AAV9-hSynmCherry into the DRN.hM4Di bound to clozapine N oxide(CNO)to activate the downstream Gi pathway,which caused neuronal hyperpolarization,thus inhibiting neurons in the DRN.After three weeks,the CPP model was established and the two groups were injected with CNO at 30 min before the CPP test to detect changes in CPP score.(4)Specific inhibition of DRN-VTA via the Cre-loxp virus:AAV2/retro-hSyn-EGFP-P2ACre-WPRE was injected into the VTA and Cre-dependent adenovirus virus AAV2/9-Ef1a-DIOhM4Di-mCherry-WPRE or AAV2/9-Efla-DIO-mCherry-WPRE was injected into the DRN.After three weeks,the CPP model was established and the two groups were injected with CNO at 30 min before the CPP test to detect changes in CPP score.(5)Detection of changes in DA neurotransmitter levels by carbon fiber electrodes:At an oxidative voltage of 780 mV,the carbon fiber electrode can oxidize the DA to generate current and the oxidation current accurately reflects the DA level.After specific inhibition of the DRN to VTA pathway,the changes in DA levels in the NAc was detected.(6)LC/MS-MS-based analysis of differential protein expression in DRN:DRN proteins from the METH group and control group were extracted and proteomic analysis was performed using liquid chromatography-Mass spectrometry(LC/MS-MS).To understand the biological characterization of differentially expressed proteins obtained from LC/MS-MS data,the cellular components,molecular functions,and biological processes of the proteins were analyzed by GO analysis.Biological function analysis of these differentially expressed proteins was performed using the KEGG database.Results:(1)METH induces c-Fos expression in DRN neurons:C-Fos expression significantly increased in the DRN of the METH group,which was mainly distributed in the midline ventral region.As revealed by the Ca2+level detection,the fluorescence intensity of Ca2+signal in DRN neurons was significantly higher after METH treatment than that of the control group.METH also increased the expression of CaMKII and p-CREB.(2)METH activates neurons projected by the DRN-VTA pathway:In retrograde labeling,c-Fos+retro cells neurons accounted for 46.72±5.79%among all retro cells significantly higher in METH group than control group.In addition,in anterograde-labeling,c-Fos+anterograde cells in the METH group accounted for 45.45±3.21%of all anterograde cells,which was significantly higher than control group.The results showed that METH activates neurons projected by the DRN-VTA pathway.(3)Inhibition of DRN significantly reduces METH-induced CPP preference:After chemogenetic inhibition of neurons in the DRN,the CPP score of the AAV-hM4di group was-110.8±30.86s,while the CPP score of the AAV-mCherry group was 29.01±28.80s.Inhibition of DRN reduced the preference of mice for the METH companion chamber.(4)Specific inhibition of the DRN-VTA pathway via the Cre-loxp virus system significantly reduced CPP preference:According to our results,the CPP score of the AAVhM4di group was-121.7±31.3s,which was lower than that of 55.94±4.42s in the AAVmCherry group.In other words,inhibition of DRN neurons projecting to VTA reduced the METH-induced CPP.(5)Specific inhibition of the DRN-VTA pathway via the Cre-loxp virus system reduced the DA level in the NAc:Electrochemical results demonstrated that after inhibiting the DRNVTA pathway,the concentration of DA in NAc in the AAV-mCherry group was significantly higher than that in the NS group(20.11±2.62 μM VS 1.60 ±0.67 μM),and was higher than that in the AAV-hM4Di group at 11.24±1.23μM.It was shown that inhibition of the DRN neurons that project to the VTA can reduce DA levels in the NAc.(6)Differentially expressed proteins in the DRN detected by LC/MS-MS:Altogether 583 proteins were obtained.By adopting the threshold of P<0.05 and the fold change(FC)≥1.5,and 23 upregulated proteins such as Cadm1,Syt1,Stxla,and 22 downregulated proteins such as Atp1b2,Nfasc and Sv2c were obtained.Bioinformatics analysis indicated that the differentially expressed proteins were closely related to neurotransmitter transport,catecholamine secretion and synaptic plasticity.CONCLUSION:This study demonstrated that METH abnormally activates DRN-VTA neurons,and that inhibiting this pathway significantly reduces the CPP preference behavior of METH model mice,which may be related to the reduction in DA release in the NAc.Additionally,METH induces the expression of proteins closely related to neurotransmitter transport and secretion and synaptic plasticity in the DRN,providing potential targets for the further study on the role of DRN in mediating METH addiction.