The Role And Function Of Thalamic Reticular Subnuclei In Propofol Concerned Mechanism Of General Anesthesia

Background and Aims: Approved by the US FDA,general anesthetic Propofol was officially listed in 1989,which was a milestone event.Since Propofol has the advantage of rapid onset,less accumulation and quick recovery,it is widely used clinically.At present,tens of millions of cases of general anesthesia are carried out under Propofol every year over the world.Propofol is already an indispensable general anesthetic in clinic.However,like other general anesthetics,the mechanism of Propofol is still unclear.Revealing the mechanism of general anesthesia is the dream of Neuroscience and Anesthesiology.Donald Kennedy,editor-inchief of Science,ranked the general anesthesia mechanism as one of the 125 th century challenges of academics in the next 100 years in 2005(Kennedy D et al.,2005),breaking the fortress will be a milestone in the field of neuroscience.The reversal of consciousness caused by general anesthesia is a special nonphysiological state of the brain,which is closely related to the brain’s advanced functions such as learning,memory,sleeping and awareness.(Sarasso S et al.,2015).In recent years,more and more researching results supported that the mechanism of general anesthesia drugs was not as the previous view to take the brain as a whole non-specific broad inhibition,but the dissecting position and functional characteristics of different neural circuits constitute a complex network of synergies involved in narcotic drugs(Wu TL et al.,2016;Akeju O et al.,2016).That is to say,narcotic drugs affect specific brain areas,blocking the function of different brain areas and breaking the balancen of positive and negative feedback from information to the high-level central nervous system.Ying’s study suggested that the Thalamic Reticular Nucleus(TRN)played an important role in the general anesthesia of propofol(Ying SW et al.,2005).However,whether Propofol is and how it acts on the Thalamic Reticular Nucleus and produces anesthetic effects remains unclear.Thalamic Reticular Nucleus is extremely special in the regulation of brain information.TRN is composed of GABA-inhibitory neurons that regulates the thalamic-cortical loops.Since 2011,there have been several literature reported that different functional subunits exist within TRN by Optogenetic and Electrophysiological techniques,that is,the GABAergic neurons are projecting into different regions of the thalamus and cortex and play different physiological roles.Due to the special anatomical location and physiological function of the TRN,which is closely related to the advanced function of the brain like sleeping,awakening,consciousness,etc.(Lewis LD et al.,2015),we are encouraged to explore its role in the nonphysiological anesthesia state.Our study focuses on exploring the specific molecular,cellular,network and behavioral mechanisms the internal subunits of TRN in Propofol concerned anesthesia,providing new reference for the network theory of Propofol anesthetic mechanism and related nucleus localization.Research methods:(1)Chemical drug Ibotanic was used to damage different parts of TRN respectively and to detect the rats’ response to and sensitivity of Propofol anesthesia after destruction of the TRN subnucleus.(2)The functional states of GABAergic neurons of a TRN and p TRN were investigated in three stages: awake,Propofol anesthesia and resuscitation by using immediate expression gene C-Fos.(3)The presence and distribution of GABAa R-β3(β3 subunit of GABAa receptor)in TRN were stained by Immunoblotting and Immunofluorescence.(4)GABAa receptors in a TRN were blocked by injecting its antagonist Bicuculline(BIC).When the rats were stable,their response to Propofol was detected.(5)Virus containing small interfering RNA(si RNA)to GABAa R-β3 were designed and injected to a TRN.The effect of interference was verified by Immunoblotting.The rats’ response to and sensitivity of Propofol were detected after that.Research results:(1)After the damage of a TRN neurons,rats’ sensitivity to Propofol significantly and continuously improved.(1)After a TRN neurons were damaged,the rat received Propofol intravenously and its sensitivity to Propofol improved significantly and continuously but no such effect when receiving Dexmedetomidine intravenously.There was no significant change of the recovery time.(2)After p TRN neurons were damaged,the rats received Propofol and Dextromethorphan intravenously and did not change significantly in the time of LORR and recovery time.(2)GABAergic neurons of a TRN were active in awake and relatively inhibited during Propofol anesthesia.(1)Fluorescence co-labeling of C-Fos and GAD-67 showed functional state of a TRN and p TRN GABAergic neurons during awakeness,propofol anesthesia,recovery.During the state of awakeness,GABAergic neurons of a TRN were more active than p TRN and were relatively inhibited after Propofol anesthesia.(2)GABAergic neurons of p TRN were significantly activated after Propofol anesthesia and were significantly inhibited after recovery.(3)Neurons of a TRN highly expressed GABAa R-β3.(1)The specificity of anti-GABAa R-β3 antibody was confirmed by immunoblotting.The expression of GABAa R-β3 in a TRN,p TRN,thalamus,hippocampus and cortex was detected by the same antibody.The results showed that there was a rich expression of GABAa R-β3 in the a TRN region and significantly higher than that in the thalamus region.(2)Immunofluorescence showed that GABAa R-β3 was expressed in a TRN and p TRN.GABAa R-β3 was more abundant and dense in a TRN than in p TRN at the same field of view.(4)Antagonizing GABAa R or knocking down GABAa R-β3 of a TRN decreased rats’ sensitivity to Propofol.(1)Bilateral injection of GABAa receptor antagonist BIC to a TRN blocking the GABAa R,the animals immediately showed a very active condition.(2)Compared with the control group,Propofol-related LORR time of BIC injection group was significantly prolonged,which means the rats’ sensitivity to Propofol decreased.(3)The expression of Grbrb3 was down-regulated by si RNA targeting GABAa R-β3,and the time of LORR in the virus-treated group was significantly longer than that in the control group,which means the sensitivity to Propofol also decreased.Conclusion: After the neurons of a TRN were damaged,the time of loss of righting reflex(LORR)was reduced after intravenous injection of Propofol,which means the rats’ sensitivity to Propofol was significantly and sustainably improved.Chemical damage to other parts of TRN showed no such difference,suggesting that a TRN is likely to be specifically involved in the process of Propofol anesthesia in the central nervous system.It was found that the GABAergic neurons of TRN subnuclei showed opposite functional state in the three stages,which indicated that TRN subnuclei also played different roles during Propofol anesthesia.Neurons of a TRN had more abundant and dense GABAa R-β3 expression than p TRN and also supported the specificity of a TRN as a target of Propofol at morphological point of view.Rats became immediately hyperactive and its activity increased significantly after BIC blocked the GABAa R of a TRN,where the neuronal excitability increased and the release of neurotransmitters in the axon terminals increased,suggesting that the downstream of GABAergic neurons of a TRN may also be inhibitory neurons.It was found that the time of LORR was significantly prolonged in BIC group compared with control group.The rats’ sensitivity to Propofol was decreased.It is presumed that the effect of Propofol is reduced due to the blocking of its target GABAa R by the antagonist.We also found that the rats’ sensitivity to Propofol decreased after the expression of GABAa R-β3 was down-regulated by si RNA.The results of this study suggest that GABAa R-β3 on neurons at a TRN is closely related to the anesthetic effect of Propofol and is an important molecular site that mediates a TRN in Propofol anesthesia.In summary,we hypothesized that Propofol is most likely to inhibit GABAergic neurons by binding to GABAa R-β3 located in a TRN,thereby allowing GABAergic neurons in downstream p TRN to be disinhibited and thus releasing multiple inhibitory signals into the thalamus and cortex,resulting in anesthetic-related sedation(see hypothesis).