The Role And Mechanism Of VTA-mPFC Pathway In Sevoflurane Anesthesia-Emergence Regulation

Background:General anesthesia has been carried out for more than 160 years,and there are many arguments about the interpretation of general anesthesia mechanism.In recent years,neuroscientists believe that general anesthesia drugs acting on different brain regions and nuclei regulate the level of neurotransmitters through specific targets,and form neural network interaction so that the body can make a transition between anesthesia and arousal.In 2016,PNAS reported:Under isoflurane anesthesia,dopaminergic neurons in the ventral tegmental area(VTA)of the middle brain were activated by photogenetic technique to make the mice immediately awake.VTA dopaminergic neurons receive projections from other nuclei nerve fibers such as the ventrolateral preoptic nucleus(VLPO)and dorsal raphe nucleus(DR).It is also capable of projecting nerve fibers into other nuclei such as the prefrontal cortex(PFC)and the nucleus accumbens(NAc).So how are VTA dopaminergic neurons regulated by other nuclei?And by which pathways it mediates the transition between anesthesia and arousal,is unclear.Scientific problems based on the above,this research will use the free-moving animal model,micro injection of brain kerne,nerve electrophysiology and chemical genetics technology,research in the role of VTA dopaminergic neuron which in the neural circuits process of transformation sevoflurane anesthesia induction to awakening,discusses the key role of VTA-mPFC pathways dopaminergic neurons which regulate anesthetic-awakening,preliminary explore its action mechanism.Part 1Objective:To investigate the effect and mechanism of VTA dopaminergic neurons in sevoflurane anesthesia and awareness regulation by using systematic drug delivery,DREADD technique and other methods.Methods:experimental animal,male SD rat,weight 250?280g.1.The effect of dopaminergic neurons on sevoflurane anesthesia-emergence was confirmed by systematic administration.18 SD rats were randomly divided into 3 groups(n=6),CON group,SCH group and APB group.Behavioral experiments were conducted after the experimental environment was adjusted for 5d.CON group was intraperitoneally injected with saline;SCH group was intraperitoneally injected with D1 receptor antagonist SCH23390(2mg/kg);APB group was intraperitoneally injected with Chloro-APB(3mg/kg).Then the effects of drugs were observed on sevoflurane anesthesia induction and emergence time in rats.The observation index of anesthesia induction was the time of loss righting reflex(LORR)and the observation index of anesthesia emergence was the time of recovery of righting reflex(RORR).2.The effect of VTA on sevoflurane anesthesia-awareness was confirmed by pharmacology method.60 SD rats were randomly divided into 5 groups(n=12),CON group,MK-801 group,SCH group,Bicu(Bicuculline)group and DMSO group.Before the experiment,microinjection catheters were implanted.After recovery for 5d,6 rats in each group were randomly selected to inject corresponding drugs.CON group was injected with Saline,SCH group was injected with D1 receptor antagonist SCH23390(5mg/ml),Bicu group was injected with GABAA receptor antagonist bicuculline(2.5mg/ml),and MK-801 group was injected with NMDA receptor antagonist MK-801(0.6mg/ml).DMSO group was the control group of Bicu group(DMSO was the organic solvent of Bicuculline).The effects of drugs were observed on anesthesia induction time of sevoflurane after injection for 15 minutes.The other 6 rats in each group were injected 15 minutes after the start of anesthesia to observe the effects of drugs on the emergence time of anesthesia.3.The influence of VTA dopaminergic neurons on sevoflurane anesthesia-awareness was confirmed by DREADD technique12 rats were randomly divided into 2 groups(n=6),hM3Dq group and CON group.The hM3Dq group VTA was injected with containing hM3Dq recombinant adenovirus(rAAV-DIO-hM3Dq-mCherry),and the CON group was injected with "blank virus"(rAAV-DIO-mCherry)for control.After 28 days of recovery and intraperitoneal injection of CNO,dopaminergic neurons were activated,and the effects of dopaminergic neurons on anesthesia induction and arousal time in rats were observed.Results:1.After intraperitoneal injection of D1 receptor antagonist SCH23390,compared with CON group the induction time of sevoflurane anesthesia was significantly shortened(P<0.01),and the emergence time was significantly prolonged(P<0.01).After intraperitoneal injection of Chloro-APB,compared with CON group the induction time of sevoflurane anesthesia was significantly prolonged(P<0.01),and the emergence time was significantly shortened(P<0.05).2.In the MK-801 group,the induction time of anesthesia compared with CON group was significantly shortened(P<0.01),and the emergence time was significantly prolonged(P<0.01).after the administration of NMD A receptor antagonist MK-801 in VTA.After VTA administration of D1 receptor antagonist SCH23390 in SCH group,compared with CON group the induction time of anesthesia was significantly shortened(P<0.01),and the emergence time was significantly prolonged(P<0.01).No significant changes were found in the induction time and emergence time of sevoflurane anesthesia between DMSO group and CON group after VTA administration of DMSO(P>0.05).In Bicu group,VTA administration of bicuculline compared with DMSO group significantly prolonged the induction time of anesthesia(P<0.05),and shortened the duration of emergence(P<0.05).3.After intraperitoneal injection of CNO to activate dopaminergic neurons,compared with CON group,the induction time of hM3Dq group was prolonged(P<0.05),and the emergence time was significantly shortened(P<0.01).Part 2Objective:To investigate the effect and mechanism of VTA-mPFC pathway in sevoflurane anesthesia-awareness regulation by using microinjection and DREADD technique.Methods:experimental animal,male SD rat,weight 250?280g.1.The effect of dopaminergic neurons in mPFC on sevoflurane anesthesia-awareness was observed by pharmacology method.36 rats were randomly divided into 3 groups(n=12):CON group,SCH group and APB group.Before the experiment,microinjection catheters were implanted.After recovery for 5d,6 rats in each group were randomly selected to receive microinjection of 0.5ul drug in mPFC.The SCH group was injected with the D1 receptor antagonist SCH23390(5mg/ml),and the APB group was injected with the D1 receptor agonist Chloro-APB(6mg/ml)for 15min before anesthesia.To observe the effect of drugs on anesthesia induction time,the other 6 rats in each group were injected at 15 minutes after the start of anesthesia.2.The effect of VTA-mPFC pathway on sevoflurane anesthesia-awareness was observed by pharmacology method.48 rats were randomly divided into 4 groups(n=12):CON group,VTA-Bicu(Bicuculline)group,mPFC-SCH group,Bicu/SCH group.Before the experiment,the micro injection catheters were implanted.After recovery for 5d,6 rats in each group were randomly selected to inject corresponding drugs.The drug concentrations were Bicuculline(2.5mg/ml)and SCH23390(5mg/ml)respectively.In the CON group,0.3ul DMSO was injected in VTA,and 0.5ul saline was injected in mPFC.VTA-Bicu group received GABAA receptor antagonist bicuculline 0.3ul microinjection in VTA and 0.5ul normal saline injection in mPFC.mPFC-SCH group received 0.3ul DMSO microinjection in VTA and D1 receptor antagonist SCH23390 0.5ul injection in mPFC.Rats in Bicu/SCH group were received VTA microinjected with 0.3ul bicuculline and mPFC microinjected with 0.5ul SCH23390.15 minutes after injection,the effects of various drugs on sevoflurane anesthesia induction time were observed.The other 6 rats in each group were injected with drugs 15 minutes after the start of anesthesia and observed the effect of drugs on anesthesia emergence time.3.The influence of VTA-mPFC pathway dopaminergic neurons on sevoflurane anesthesia and emergence was observed by DREADD technique.12 rats were randomly divided into two groups(n=6),hM3Dq group and CON group.hM3Dq group:the recombinant adeno-associated virus(rAAV-DIO-hM3Dq-mCherry)contain hM3Dq was injected in VTA.CON group:VTA microinjection of "empty virus"(rAAV-DIO-mCherry)without hM3Dq as control.After 28 days of recovery,VTA-mPFC pathway dopaminergic neurons were activated by mPFC microinjection of CNO,the anesthesia induction and emergence time of two groups were observed.Results:1.The induction time of sevoflurane anesthesia after mPFC microinjection of D1 receptor antagonist SCH23390 compared with CON group was significantly shortened(P<0.01),and the emergence time was significantly prolonged(P<0.01).After mPFC microinjection of Chloro-APB,compared with CON group the induction time of sevoflurane anesthesia significantly prolonged(P<0.01)and emergence time significantly shortened(P<0.01).2.Compared with CON group,the induction time of sevoflurane anesthesia was prolonged and the emergence time was shortened in VTA-Bicu group after VTA microinjection of bicuculline(P<0.01).Compared with CON group,the induction time of sevoflurane anesthesia was shortened and the emergence time was prolonged in mPFC-SCH group after mPFC microinjection of SCH23390(P<0.01).However,compared with CON group,the induction time and emergence time in Bicu/SCH group was no significant difference(P>0.05).3.Compared with CON group,the induction time of sevoflurane anesthesia in hM3Dq group was significantly prolonged(P<0.01)and the emergence time was significantly shortened(P<0.01).Conclusion:This study demonstrated that the change of VTA dopaminergic neurons and their receptors can affect sevoflurane anesthesia-arousal,suggesting that VTA dopaminergic neurons plays an important role in the neural network of general anesthesia mechanism,and VTA-mPFC pathway dopamine neurons plays an important role in the regulation of anesthesia-arousal.