Role Of The Edinger-Westphal Nucleus In Sevoflurane General Anesthesia And Its Mechanisms

General anesthesia（GA）is featured as a revisable global brain and body state leading to unconsciousness,amnesia,analgesia and immobility along with physiological stability.It remains unclear how general anesthetics exert such effects.Investigations into the mechanisms of GA will advance our basic understandings and benefit its clinical applications.It is conventionally considered that general anesthetics work through exerting a wide range of inhibitory impacts on the nervous system.In recent years,a few studies have found that general anesthetics could work by exerting some excitatory effects in a few brain areas as well.However,it is unknown what receptors mediate these excitatory effects.Recent studies found that isoflurane could activate neurons in the central amygdala（Ce A）to suppress pain circuit to exert analgesic effects.Notably,isoflurane could activate the glutamatergic neurons in the supraoptic nucleus（SON）to promote sleep to cause sedation or loss of consciousness.However,compared to sevoflurane,isoflurane has several disadvantages including less favorable hemodynamic responses,more irritating to the airway,less favorable recovery profile,so that it has been prone to be gradually replaced by sevoflurane.Sevoflurane has become the most widely used inhalational anesthetics.However,the mechanisms underlying its anesthetic effects are still far from completely understood.Sevoflurane is predominately known to cause some inhibitory effects on some brain regions regarding its neural mechanisms.So far,no study has provided systemic evidence for the excitatory effects of sevoflurane anesthesia.Therefore,the present study aims to search for the neural targets that might be activated by sevoflurane and explore the possible mechanisms,so as to contribute to providing the precise targets for precise anesthesia,as well as providing evidence for improving or developing the anesthetics.In this study,adult mice were firstly subjected to sevoflurane-oxygen anesthesia,followed by detection of c-Fos expression throughout the whole brain to search for the neural targets,that was further confirmed using whole-cell patch-clamp recording on ex vivo level and multi-channel recording on in vivo level,respectively.Next,the cell types activated by sevoflurane were identified using immunofluorescence（IF）and fluorescence in situ hybridization（FISH）.Subsequently,the Fos-TRAP technology,combined with chemogenetic inhibition,was utilized to specifically inhibit and ablate the sevoflurane-activated neurons to investigate their roles in sevoflurane anesthesia.Afterwards,the target receptor was identified using FISH,which was then also confirmed by whole-cell patch-clamp recording in isolated brain slice.Finally,the antagonist or agonist was locally injected into the brain region to modulate the activity of the receptor to observe the influences both on sevoflurane anesthesia and sleep-wakefulness,so as to further investigate the mechanisms underlying sevoflurane anesthesia.The main results are as follows:1.Edinger-Westphal nucleus is one of the activated targets for sevoflurane.（1）To search for the activated neural targets of sevoflurane,adult mice were firstly subjected to sevoflurane-oxygen anesthesia to detect the c-Fos expression throughout the whole brain.Compared to control condition without continuous anesthesia,sevoflurane caused a more than fourfold increase in the number of c-Fos⁺neurons in the Edinger-Westphal nucleus（EW）,SON,Ce A,EW and the bed nucleus of the stria terminalis（BNST）,implying sevoflurane may activate the SON,Ce A,BNST,EW.（2）To confirm that sevoflurane indeed activated these neurons in the EW,whole-cell patch-clamp recording was next performed in ex vivo brain slice to detect the effects of sevoflurane on the EW.On one hand,the current changes were recorded in voltage clamp mode before and after application of sevoflurane at different concentration,and sevoflurane caused inward currents of these neurons in the EW in a concentration-dependent manner.On the other hand,the membrane potential changes were recorded in current clamp mode before and after application of sevoflurane,and sevoflurane increased the firing rate（P<0.05）and depolarized the membrane potential（P<0.001）.These results suggest that sevoflurane excites EW in a concentration-dependent manner.（3）To confirm that sevoflurane could excite the EW in vivo,multi-channel recording was then performed in the EW region to observe the firing changes before and after sevoflurane anesthesia.In total,77 cells from 16 mice were recorded,of which 43%showed an increase in their discharge frequency during sevoflurane anesthesia（P<0.001）,31%showed a decrease,and 26%remained unchanged,suggesting that sevoflurane excites the EW in vivo.（4）Given that EW is closely associated with sleep,we further detected the changes of discharge frequency of 19 cells among sevoflurane-activated cells,and found that the firing rates of all these sevoflurane-activated cells markedly increased during transitions from wakefulness to non-rapid eye movement（NREM）sleep.However,no difference was observed during transitions from NREM sleep to rapid eye movement（REM）sleep.On average,these sevoflurane-activated cells showed a higher level of discharge frequency during both NREM and REM sleep than that during wakefulness（P<0.001）,implying that sevoflurane may activate the EW to promote sleep to cause anesthesia.2.Sevoflurane-activated neurons in the EW are mainly peptidergic neurons that express CART,UCN1 and CCK.（1）To identify the cell types activated by sevoflurane,IF and FISH were used to detect the neurons possibly existing in and near the EW.There existed some glutaminergic neurons expressing vesicular glutamate transporter（Vglut2）,GABAergic neurons expressing glutamate decarboxylase（GAD）,dopaminergic neurons,cholinergic neurons and peptidergic neurons expressing calcitonin gene-related peptide alpha（CALCA）,substance P,cholecystokinin（CCK）,cocaine and amphetamine-regulated transcript protein（CART）and urocortin 1（UCN1）.CCK⁺、CART⁺and UCN1⁺neurons were highly enriched in the EW.No noradrenergic,histaminic or serotonergic neurons were found from anterior to posterior in the EW.No glutaminergic neurons expressing Vglut1 or Vglut3 were found,too.（2）Afterwards,co-localization between sevoflurane-activated neurons and these above neurons existing in and near the EW were detected using IF and FISH.98%of the sevoflurane-activated neurons were CART⁺and UCN1⁺,and 85%were CCK⁺.Only a very small proportion（5%）was Vglut2⁺.None of the sevoflurane-activated neurons was dopaminergic,cholinergic,GABAergic neuron or peptidergic neuron expressing CALCA or substance P.Further,FISH showed a complete overlap of CART⁺and UCN1⁺neurons（>98%）.3.Specific inhibition or ablation of the sevoflurane-activated neurons in the EW using Fos-TRAP technology remarkably changes the anesthetic effects of sevoflurane.（1）To investigate the role of the sevoflurane-activated neurons in sevoflurane anesthesia,the Fos-TRAP technology,combined with chemogenetic inhibition,was utilized to specifically inhibit the sevoflurane-activated neurons in the EW to observe the influences on sevoflurane anesthesia.Specific inhibition of the sevoflurane-activated neurons caused a slower and lower reduction in EEGθ/δratio during sevoflurane anesthesia,implying entering anesthesia more slowly and maintaining a shallower anesthesia.In addition,it caused a delay of entering deep anesthesia（P<0.05）and an earlier emergence（P<0.01）.（2）To further confirm these findings above,the Fos-TRAP technology,combined with diphtheria toxin A（DTA）,was used to specifically ablate the sevoflurane-activated neurons in EW in Fos^{2A-i Cre ERT2}mice to further observe the influences on sevoflurane anesthesia.Specific ablation of the sevoflurane-activated neurons also caused a slower and lower reduction in EEGθ/δratio during sevoflurane anesthesia,implying entering anesthesia more slowly and maintaining a shallower anesthesia.In addition,it also caused a delay of entering deep anesthesia（P<0.05）and an earlier emergence（P<0.001）.These findings suggest that sevoflurane may activate the EW to induce and maintain the state of anesthesia.Either specific inhibition or ablation of the sevoflurane-activated neurons in the EW can remarkably change the anesthetic effects of sevoflurane on induction,maintenance and emergence,entering anesthesia more slowly and maintaining a shallower anesthesia with a delay of entering deep anesthesia and an earlier emergence.4.Sevoflurane activated the EW through GHSR.（1）According to the Allen Mouse Brain Atlas and previous studies,growth hormone secretagogue receptor（GHSR）is highly enriched in EW.Thus,we next detected whether the sevoflurane-activated neurons co-localized with GHSR,and found that the overlap between GHSR and the sevoflurane-activated neurons reached above 94%,implying sevoflurane may activate the EW through GHSR.（2）Then the Autodock software,a structure-based computational simulation,was applied to predict the GHSR binding sites and the affinity for sevoflurane.Sevoflurane could dock into the allosteric pockets under the orthosteric sites with binding free energy of-5.4 kcal/mol.Its binding mode with orthosteric sites was also explored with a free energy of-4.9 kcal/mol,suggesting the allosteric pockets were more favorable for sevoflurane.In the sevoflurane/GHSR docking model,sevoflurane interacted with allosteric pockets by hydrophobic interactions with Thr276,Phe272,Ala271,Cys275,Ser315,Asn319,and Ile318.（3）The antagonist of GHSR[D-Lys³]-GHRP-6 was next applied to observe the influence on the excitatory effect of sevoflurane in isolated brain slice.On the sevoflurane-excited cells,the antagonist of GHSR[D-Lys³]-GHRP-6 abolished the excitatory effect of sevoflurane（P<0.001）,implying GHSR antagonist may block the excitatory effect of sevoflurane on the EW,and confirming sevoflurane activates the EW through GHSR on ex vivo level.（4）To investigate the role of GHSR in sevoflurane anesthesia on in vivo level,an intracranial cannulation was placed into the EW region to locally inject the antagonist,[D-Lys³]-GHRP-6,and the agonist,ghrelin,respectively,to observe the influences on sevoflurane anesthesia.GHSR antagonist caused a slower and lower reduction in EEGθ/δratio during sevoflurane anesthesia,implying entering anesthesia more slowly and maintaining a lighter anesthesia.In addition,it caused a delay of entering deep anesthesia（P<0.05）and an earlier emergence（P<0.05）.However,the agonist had the opposite effects,and caused a faster and higher reduction in EEGθ/δratio during sevoflurane anesthesia,implying entering anesthesia faster and maintaining a deeper anesthesia.In addition,it caused entering deep anesthesia faster（P<0.05）and a delayed emergence（P<0.05）.These findings suggest that GHSR blockade may attenuate the anesthetic effects of sevoflurane,prolong the induction time,maintain a shallower anesthesia and shorten the time to emergence.GHSR activation may strengthen the anesthetic effects of sevoflurane,shorten the induction time,maintain a deeper anesthesia and prolong the time to emergence.These findings also confirm that sevoflurane activates the EW through GHSR to induce and maintain the state of anesthesia in in vivo.Activating or blocking GHSR remarkably changes the anesthetic effects of sevoflurane on induction,maintenance and emergence.（5）Considering that CART⁺/UCN1⁺and CCK⁺neurons in the EW involve in sleep regulation proved by previous studies,plus that ghrelin,the endogenous ligand and agonist of GHSR,may promote sleep,as well as our observation that these sevoflurane-activated cells were active during sleep,we finally locally injected the antagonist,[D-Lys³]-GHRP-6,and the agonist,ghrelin into the EW,respectively,to modulate the activity of GHSR to observe the influences on sleep-wakefulness,so as to further investigate the mechanisms of sevoflurane anesthesia.Ghrelin or saline was locally injected at the beginning of the dark phase（18:00,ZT12）,when mice spend most of their time in wakefulness.Ghrelin caused a significant increase in NREM sleep（P<0.001）and a significant decrease in wakefulness（P<0.001）in 3 hours after injection,implying GHSR activation in EW may promote sleep,which may be the mechanism of sevoflurane anesthesia.To further confirm our hypothesis,GHSR antagonist[D-Lys³]-GHRP-6 or saline was locally injected into the EW at the beginning of the light phase（6:00,ZT0）,when mice spend most of their time asleep.GHSR antagonist caused a significant decrease in NREM sleep（P<0.001）and a significant increase in wakefulness（P<0.001）in 3 hours after injection,implying GHSR blockade in EW may promote wakefulness.These findings suggest that sevoflurane may activate GHSR in the EW to promote sleep to induce and maintain the state of anesthesia.Conclusions:Based upon these above,sevoflurane may activate CART⁺/UCN1⁺and CCK⁺peptidergic neurons in the EW via GHSR activation to promote sleep to induce and maintain the state of anesthesia.In the field of general anesthesia,these findings identify a novel activated target for sevoflurane and provide novel cellular and molecular mechanisms for both sevoflurane anesthesia and the sleep-promoting effect of ghrelin,also identify a novel receptor in the field of general anesthesia,further providing a molecular target mediating the excitatory effects.Search for the neural target and study on the mechanisms of sevoflurane will not only advance our basic understandings,but also provide novel accurate targets for improving or developing new anesthetics,especially for developing the reversal agent for inhalational anesthetics to fill the gap in clinical practice.