Dopaminergic Neuron Projections From The Ventral Tegmental Area To The Parabrachial Nucleus Modulate The Effects Of Propofol Anesthesia In Rats

Objective:Research suggests that brain regions that control sleep-wake cycles may play a vital role in the regulation of general anesthetic arousal.Different arousal areas form complex neural loops through neuronal projections and connections that regulate general anesthesia arousal.The role of the ventral tegmental area（VTA）and the parabrachial nucleus（PBN）in sleep-wake and general anesthesia has been demonstrated,but whether this unique pathway of dopaminergic neurons in the PBN and dopaminergic projections from the VTA to the PBN modulate general anesthesia has not been investigated.In this study,we investigated the modulatory role of dopaminergic neurons in the PBN and the dopaminergic pathway from the VTA to the PBN in propofol anesthesia by chemical destruction,optogenetics and chemogenetics combined with c-Fos immunofluorescence staining and in vivo EEG recording.Methods:（1）We demonstrated the role of dopaminergic neurons in the PBN in propofol anesthesia by lesioning PBN dopaminergic neurons with 6-OHDA.SD rats were randomly divided into the 6-OHDA group and the NC group.6-OHDA group:6-hydroxydopamine was injected bilaterally into the PBN;NC group:an equal amount of saline was injected bilaterally into the PBN.In the NC group,the same amount of saline was injected into the PBN bilaterally.Loss of righting reflex（LORR）,recovery of consciousness（RORR）time,and electroencephalography（EEG）changes were observed under propofol anesthesia.（2）To demonstrate the role of the VTA^DA-PBN pathway in propofol anesthesia using chemogenetic methods.SD rats were randomly divided into the h M3Dq group,the h M4di group,and the m Cherry group.The VTA^DA-PBN pathway was activated or inhibited,and the changes in LORR,RORR time and corresponding EEG were recorded in rats under propofol anesthesia.Immunofluorescence verified viral injection sites,and changes in aminergic neuronal activity in the rat VTA were observed by c-Fos staining.（3）In addition,the VTA^DA-PBN pathway was more precisely regulated by optogenetic methods to demonstrate the role of this pathway in propofol anesthesia.SD rats were randomly divided into Ch R2,Np HR and m Cherry groups.The LORR,RORR time and EEG changes of rats in each group were observed by activation or inhibition of this pathway.Immunofluorescence was used to verify the viral injection site and to observe the changes in aminergic neuronal activity in the PBN of rats by c-Fos immunofluorescence staining.Results:（1）The results of the damage experiment showed that compared with the NC group,the induction time of propofol anesthesia in the 6-OHDA group had no statistical difference（P>0.05）,and the recovery time was significantly prolonged（P<0.01）.The results of EEG analysis showed that there was no statistical difference in waveforms during LORR（P>0.05）.During RORR process,theδwave power percentage of the 6-OHDA group was higher than that of the NC group（P>0.001）,while theβwave power percentage of the 6-OHDA group was lower than that of the NC group（P>0.001）.（2）Chemogenetic activation of the VTA^DA-PBN pathway showed no significant difference in the induction time of propofol anesthesia（P>0.05）,and the recovery time was shortened（P<0.001）.During RORR process,the percentage ofαandβwave power was higher in the h M3Dq group than in the m Cherry group（P<0.001）.The percentage ofδandθpower was lower than that in the m Cherry group（P<0.001）.Chemogenetic inhibition of the VTA^DA-PBN pathway showed no statistical difference in LORR time（P>0.05）,RORR time was longer（P<0.05）,EEG showed that the h M4Di group had a higher percentage ofδwave power（P<0.001）and a lower percentage ofβwave power（P<0.001）compared with m Cherry group.（3）There was no significant difference in LORR time（P>0.05）and RORR time was shortened（P<0.001）.During the RORR process,EEG showed that the percentage ofβ-wave power in the Ch R2 group was higher than that in the m Cherry group（P<0.05）.The percentage ofδ-wave power in the m Cherry group was lower than that in the m Cherry group（P<0.01）.There was no significant difference in LORR time（P>0.05）and RORR time（P<0.001）.During the RORR process,EEG showed that the percentage ofδwave power was higher in the Np HR group than in the m Cherry group（P<0.01）.The percentage ofβ-wave power was lower（P<0.01）.Conclusion:The dopaminergic neurons in the PBN are involved in the regulation of the emergence stage of propofol anesthesia,and the lesion of dopaminergic neurons in the PBN can prolong the emergence time of propofol anesthesia in rats;furthermore,the dopaminergic pathway of the PBN may be involved in the regulation of the emergence stage of propofol anesthesia through VTA projections to the PBN,and activation of this pathway may play a pro-arousal role.