The Mechanism Of PVT-BNST Neural Circuit In The Regulation Of Induction,Maintenance And Emergence Of Sevoflurane Anesthesia In Mice

Background Volatile anesthetics such as sevoflurane have been widely used in clinical practice,but how it causes loss of consciousness,anesthesia maintenance and consciousness recovery remains unclear.The understanding of its mechanism is not only beneficial to improve the quality and safety of general anesthesia,but also to further promote the understanding of consciousness formation.With the further discovery of the neural networks that regulate sleep-wakefulness states,modern scientists believe that general anesthetics can also regulate anesthesia-awakening by acting on targets with specific neurotransmitters of many different brain regions and the neural networks formed between different brain regions can jointly regulate the transition of anesthesia-awakening state.However,the key targets related to the synergistic action of multiple brain regions and multiple neurotransmitters that mediate the general anesthesia remain unkown.The 2018 Science reported that glutamatergic neurons in the paraventricular nucleus of the thalamus are the key areas for maintaining arousal,and photogenetic activation of glutamate neurons in the paraventricular nucleus of the thalamus in mice under isoflurane anesthesia can promote behavioral and EEG arousal.The paraventricular nucleus of thalamus are mainly glutamatergic neurons,which can receive afferent inputs from areas related to sleep-wakefulness or anesthesia-arousal regulation,such as parabrachial nucleus(PBN),periaqueductal gray(PAG),dorsal raphe nucleus(DR),lateral hypothalamus(LH),and the nerve fibers can also project to the limbic system and its related brain areas.However,whether the PVT glutamatergic neurons are related to sevoflurane induction,maintenance,recovery and anesthetic sensitivity is still unclear.Little has been reported about the specific brain regions and specific types of neurons that PVT glutamatergic neurons project to,and what kind of regulatory pathways they formed to mediate the transition from anesthesia to arousal remained unkown.The bed nucleus of stria terminalis(BNST),located in the limbic forebrain,regulates a variety of endocrine and autonomic nervous responses mainly by projecting to the relay nucleus of the autonomic nervous system,hypothalamus and central amygdala,and plays an important role in stress,fear and anxiety.Recent studies have shown that photogenetic or chemogenetics activation of GABAergic neurons in BNST can promote the rapid transition from non-REM sleep to wakefulness in mice.In addition,calretin(CR)positive neurons in PVT project directly to BNST,and the PVTCR+-BNST pathway mediates starvationpromoting arousal behavior.Recent studies have confirmed that there are overlapping neural mechanisms between general anesthesia and sleep-wake regulation.Therefore,we speculate that glutamatergic neurons in PVT also project to BNST and regulate sevoflurane anestheticawakening transition by activating GABAergic neurons in BNST.Based on the scientific questions above,we explored the role of PVT glutamatergic neurons in sevoflurane anesthesia induction,maintenance and recovery by using electrophysiology,photogenetics,chemicalgenetics and virus tracing techniques in transgenic mice,and explored the role of PVT-BNST neural circuit in sevoflurane anestheticawakening regulation.In order to improve the mechanism for the network regulation of sevoflurane and lay a scientific and theoretical basis for the further development of more safe,effective and high-quality general anesthetics,we further explore the role of PVT-BNST neural circut in pain and anxiety-like behavior.Methods and results1.The role of PVT glutamatergic neurons in the induction,maintenance and emergence of sevoflurane anesthesiaMethods: Firstly,c-Fos immunofluorescence staining was used to reveal the changes of neuronal activity in PVT under sevoflurane anesthesia.Secondly,we used chemogenetics to inhibit the PVT glutamatergic neurons and to observe its influence on the behavior and EEG characteristics of sevoflurane induction and recovery.To further test the effects of PVT glutamatergic neuron on the induction and recovery of sevoflurane anesthesia,we used optogenetics to stimulate the activities of PVT glutamatergic neurons,and we also explored its effect on the behavior and EEG burst suppression during the maintenance of sevoflurane.Results: 1.The results of immunofluorescence showed that,compared with the awake group,the expression of c-Fos in neurons of PVT was significantly decreased under sevoflurane anesthesia(p < 0.001).2.The behavioral results showed that the time to LORR of h M4D-CNO group(116.4 ± 36.02 s)was significantly shorter than that in control group of h M4D-saline(154.0 ± 23.22 s,p < 0.05)and m Cherry-CNO group(162.8 ± 22.36 s,p < 0.01)during sevoflurane induction period.During sevoflurane recovery period,the recovery time of righting reflex in h M4D-CNO group was significantly longer than that in control group of h M4D-saline group(266.5 ± 84.22 s vs.157.3 ± 40.76 s,p < 0.001)and m Cherry-CNO group(266.5 ± 84.22 s vs.168.9 ± 45.08 s,p < 0.001).And we found that both LORR and RORR doseresponse curve were left-shifted for the h M4D-CNO group compared with the h M4D-saline group and the m Cherry-CNO.3.During anesthesia induction period,group with delivery of CNO showed a significant increment in delta band compared with h M4D-saline group(p < 0.01),while the other bands showed no statistical difference.And during the recovery period of sevoflurane anesthesia,the power in delta band showed a significant increase in h M4D-CNO group compared with h M4D-saline group(p < 0.0001),and the power in theta band was decreased in the h M4D-CNO group compared with the control group(p < 0.01),while the other bands showed no statistical difference.4.We found that constantly optogenetic stimulation of PVT significantly increased the time to LORR in Ch R2-on group(187.6 ± 30.27 s)compared with the Ch R2-off group(138.4± 32.53 s,p < 0.05)and m Cherry-on group(143.5 ± 27.59 s,p < 0.05).For sevoflurane anesthesia emergence,the time to RORR was significantly reduced in Ch R2-on group(101.5 ± 26.59 s)compared with the Ch R2-off group(165.9 ± 52.20,p < 0.05)and m Cherry-on group(158.5 ± 32.55 s,p < 0.05)5.Photogenetic activation of PVT glutamatergic neurons was sufficient to induce behavior arousal during sevoflurane maintenance,significantly increase the duration and frequency of EEG bursts and decrease burst-suppression ratio(BSR)from 57 ± 0.18% to 14 ± 0.09%(p < 0.001)during sevoflurane-induced burst suppression condition.The average PSD analysis of EEG showed an increase in delta,alpha,beta,gamma band during photostimulation compared with pre-stimulation(p < 0.05).2.Structural identification of PVT-BNST neural circuitMethods: First,the Cre-dependent forward tracer virus AAV-EF1?-DIO-Ch R2-m Cherry was microinjected into the PVT region of vglut2-Cre mice to explore the downstream projection brain regions of PVT glutamatergic neurons in mice.The helper virus and modified rabies virus and two kinds of transgenic mice(vgat-Cre: labeled GABAergic neurons and vglut2-Cre: labeled glutamatergic neurons)were used for specific retrograde trans-monosynaptic tracing,combined with vglut2 fluorescence staining to observe whether PVT vglut2+ neurons directly project to BNST GABAergic neurons and glutamatergic neurons.Results: The BNST neurons could receive direct projections from PVT glutamatergic neurons,and both GABAergic neurons and glutamatergic neurons in BNST received direct inputs from PVT neurons.And most of the PVT neurons that projected onto BNST GABA and glutamatergic neurons were vglut2+.3.Role of PVT-BNST neural circuit in sevoflurane anesthesia induction,maintenance and arousalMethods: we specifically inhibited PVT-BNST neural circuit using chemical genetics,to observe its effects on sevoflurane induction and arousal behavior and EEG characteristics.The PVTvglut2+-BNST neural circuit were then stimulated by optogenetics to observe its effects on the induction and recovery of sevoflurane anesthesia,and we further explored its effects on the behavior and EEG burst suppression during sevoflurane maintenance.Results: 1.The time to LORR was reduced in the h M4D-CNO group(87.17 ± 24.00 s)compared with the h M4D-saline group(135.8 ± 35.96 s,p < 0.01)and the m Cherry-CNO group(123.7 ± 28.64 s,p < 0.05).And the time to RORR of sevoflurane anesthesia was prolonged in h M4D-CNO group(292.4 ± 108.4 s)compared with h M4D-saline group(174.2 ± 58.68 s,p < 0.01)and the m Cherry-CNO group(159.8 ± 72.54 s,p < 0.001).And the dose-response curve of LORR and RORR were all left-shifted in the suppression of PVT-BNST pathway group(h M4D-CNO group)compared with the controls(h M4 Dsaline group and the m Cherry-CNO).2.Inhibition of the PVT-BNST neural circuit significantly increased the power in delta band during induction(p < 0.0001)and recovery of sevoflurane(p < 0.01),and had no significant effect on the other frequency bands.3.Optical activation of PVTvglut2+ terminals in the BNST led to an extended time in LORR as compared to Ch R2-off group(153.6 ± 24.46 s vs.103.3 ± 36.41 s,p < 0.05)and m Cherry-on group(153.6 ± 24.46 s vs.109.3 ± 32.84 s,p < 0.05).And we also found that optical stimulation of PVTvglut2+-BNST projection reduced the time to RORR(106.9 ± 22.47 s)as compared to Ch R2-off group(168.6 ± 40.53 s,p < 0.05)and m Cherry-on group(159.5 ± 34.38 s,p < 0.05).4.Optical stimulation of PVTvglut2+ terminals in the BNST induced behavioral emergence during the maintenance of sevoflurane anesthesia,significantly increased the duration and frequency of EEG burst and significantly decreased BSR from 43 ± 0.10% to 3 ± 0.40%(p < 0.0001)during sevoflurane-induced burst suppression condition.EEG spectrum analysis showed that the optogenetic activation of PVTvglut2+-BNST neural circuit caused a significant increase in delta power compared with pre-stimulation(p < 0.05)and had no statistical difference in other frequency bands.4.Role of PVT-BNST neural circuits in pain and anxiety behaviorMethod: we selectively inhibited PVT-BNST neural circuit using chemical genetics,and optogenetics were used to excite PVTvglut2+-BNST neural circuit.Von Frey hairs were used to determine the mechanical threshold and hot plate test was used to determine thermal pain threshold.The open field tests(OF)and elevated plus maze(EPM)experiments were used to observe the influence of manipulation PVT-BNST neural circuits on anxiety behavior.Results: 1.Chemogenetic silencing of PVT-BNST projections could increase the time in and entries into the open arms during the EPM tests,and had no significant effects on the total distance and time spent in central area of the OF tests.2.Optical stimulation PVTvglut2+ terminals in the BNST decreased the time spent in centers during OF tests,reduced the time in and entries into the open arms during EMP tests.3.Neither chemogenetic silencing nor optical stimulation of PVT-BNST circuit affected mechanical threshold and heat pain latency in mice.Conclusion 1.PVT glutamatergic neurons are involved in the regulation of sevoflurane induction,maintenance,emergence and anesthesia sensitivity.2.PVT glutamatergic neurons had synaptic connections with the BNST GABAergic and glutamatergic neurons.3.The PVT-BNST neural circuit is involved in the regulation of sevoflurane induction,maintenance,recovery and anesthesia sensitivity.4.The PVT-BNST neural circuit may be involved in the regulation of anxiety-like behavior,but has no significant effect on pain regulation.