Study On Effect Of Dorsal Raphe Nucleus - Anteriror Cingulate Cortex 5-HT Pathway In The Mechaniam Of Propofol-Induced Anesthesia

Objective:Recent years,Some studies have also found that many key nuclei in the sleep-wake pathway are involved in the loss and recovery of consciousness during general anesthesia by behavioral methods,functional area lesion,and microinjection.In the early stage,our team used optogenetics to specifically activate dosal Raphe Nucleus?DRN?5-hydroxytryptamine?5-HT?neurons in SERT-cre mice under propofol anesthesia.It can accelerate the recovery time of righting reflex in mice.This experimental result shows that the important arousal nucleus DRN participates in propofol-induced anesthesia.This experiment is mainly from the perspective of neural pathways,in order to further clarify the DRN-related pathways involved in propofol-induced anesthesia.This experiment uses optogenetics combined with c-fos staining to study the downward projection of 5-HT neurons in DRN.From the perspective of neural pathways,the mechanism of loss of consciousness caused by general anesthesia was studied.Methods:1.Twelve healthy male adult SERT-cre mice of SPF grade were randomly divided into the ChR2 experimental group?rAAV-Ef1?-DIO-hChR2-mCherry-WPRE-pA,n=6?and the control group?rAAV-Ef1?-DIO-mCherry-WPRE-pA,n=6?.This optogenetic virus has an antegrade tracking function,after 4 weeks of stable expression of the virus,the tail vein was given a single propofol?20 mg/kg?and light stimulation was given at the same time.After the recovery of righting reflex?RORR?,the light was stopped,and 90 minutes after the light stimulation was stopped,the brain was perfused and the whole brain section was used to track the downward projection of the 5-HT energy neurons in the DRN and perform c-Fos staining.2.Thirty-six healthy adult male C57 mice of SPF grade were selected and randomly divided into anterior cingulate cortex damage group?amanitaline,n=6?and its control group?saline,n=6?,Ethmoid thalamic nucleus damage group?amanita ammonia?Acid,n=6)and its control group?physiological saline,n=6?,Ventral posteromedial thalamic nucleus damaged group?amanitaline,n=6?and its control group?0.9%physiological saline,n=6?,The ACC,Eth,and VPM of the projected brain area of DRN were damaged.A single propofol?20 mg/kg?was administered to the tail vein 15 days later.The RORR time of the damaged group and the control group during the recovery period of propofol anesthesia and the cortical EEG Variety.3.Twelve healthy male adult SERT-cre mice of SPF level were randomly divided into the experimental group of optogenetic activation?rAAV-Ef1?-DIO-hChR2-mCherry-WPRE-pA,n=6?and mcherry control group?rAAV-Ef1?-DIO-mCherry-WPRE-pA,n=6?,establish an optogenetic experimental model,inject virus in DRN and embed fiber in ACC?anterior cingulate,ACC?,wait 4 weeks to transfer the virus to stable expression.Give light in ACC After stimulation,the DRN^5-HT-ACC pathway was activated to record changes in the RORR time and cortical EEG of mice in the experimental group and the control group under and without light stimulation.Results:1.The results of tracing experiments showed that after injection of the optogenetic antegrade tracing virus in DRN,the whole brain was stained and found that 5-HT neurons of DRN could project to ACC,Eth?Ethmoid thalamic nucleus,Eth?,VPM?Ventral posteromedial thalamic nucleus,VPM?,VO?ventral orbital cortex,VO?.And activating 5-HT neurons in DRN can increase the expression of c-Fos in the ACC of the descending brain region of DRN.2.The experimental results of the damage part showed that compared with the saline control group,the RORR time of the ACC damage group was prolonged,and the results of EEG analysis showed that the?wave increased and the?wave decreased;there were no significant changes in the RORR time and EEG changes of the Eth and VPM damage groups.No statistical difference.3.Twenty-four healthy male adult SERT-cre mice of SPF level were randomly divided into the experimental group of optogenetic activation?rAAV-Ef1?-DIO-hChR2-mCherry-WPRE-pA,n=6?and mcherry control group?rAAV-Ef1?-DIO-mCherry-WPRE-pA,n=6?,light stimulation group?rAAV-Ef1?-DIO-hChR2-mCherry-WPRE-pA,n=6?and non-light stimulation group?rAAV-Ef1?-DIO-hChR2-mCherry-WPRE-pA,n=6?,set up DRN optogenetic experimental model and embed fiber in ACC,and then transfer to stable expression of virus after 4 weeks.Give light stimulation at ACC,activate DRN^5-HT-ACC pathway,recording changes of RORR time and cortical EEG of the experimental and control mice?Conclusion:1.5-HT neurons in the DRN can project ACC,Eth,OV,VPM.Activating 5-HT neurons in the DRN can increase the expression of c-Fos in ACC2.ACC participates in the process of propofol-induced-anesthesia in mice3.DRN^5-HT-ACC exerts pro-awakening effect in propofol-induced-anesthesia.