| BackgroundSince the birth of general anesthesia in 1846,the study of its pharmacodynamic mechanism has gone through the lipid theory,the protein theory and the current mainstream theory of neural network regulation,but it has not been fully clarified at present.Studies on the background of neural network regulation theory have confirmed that dopaminergic and gamma-aminobutyric acid(GABA)neurons in ventral tegmental area(VTA)are involved in the regulation of general anesthesia-arousal process.However,whether glutamatergic(Glu)neurons of VTA participate in the regulation of general anesthesia and its mechanism remains unclear.Therefore,the purpose of this study is to determine whether VTA glutamatergic neurons are involved in the regulation of general anesthesia-arousal process and the mechanism of neural circuits involved.In this study,neuron-specific calcium signal recording analysis and somatic electrophysiological techniques were used to observe the changes of VTA glutamatergic neurons during general anesthesia-arousal,and then optogenetics was used to regulate them and observe the effects on the general anesthesiaarousal process.Finally,neural circuits specific regulation technique was used to observe which downstream brain regions were involved in the regulation of VTA glutamatergic neurons on general anesthesia-arousal.Methods1.r AAV2/9-EF1α-DIO-GCa Mp6s-WPRE-h GH p A was stereotactically injected into the VTA of Vglut2-Cre mice,and fiber photometry was performed to measure the intensity of calcium signals throughout the isoflurane(ISO)anesthesia and arousal;r AAV2/9-Ef1α-DIO-h Ch R2-m Cherry-WPRE-h GH p A was injected into the VTA of Vglut2-Cre mice and opto-tetrode was implanted into the VTA simultaneously.Firing rates of glutamatergic neurons in the VTA were measured during ISO anesthesia and arousal by in vivo electrophysiology techniques.2.Excitatory optogenetic virus r AAV2/9-Ef1α-DIO-h Ch R2-m Cherry-WPRE-h GH p A or inhibitory optogenetic virus r AAV2/9-Ef1α-DIO-e Np HR3.0-m Cherry-WPRE-h GH p A were injected into the VTA of Vglut2-Cre mice,the optical fiber was implanted above the VTA.The effects of optogenetic regulation on electroencephalogram(EEG),the burst suppression ratio(BSR),power spectrum,and anesthesia behavior during the maintenance period were observed while activating glutamatergic neurons by 473 nm blue light or inhibiting by 594 nm yellow light.3.The virus was injected into the VTA of Vglut2-Cre mice with r AAV2/9-Ef1α-DIOm Cherry-WPRE-h GH p A.The efferent projections of glutamatergic neurons in the VTA were observed,and the downstream brain regions that may regulate general anesthesiaarousal were selected.4.r AAV2/9-Ef1α-DIO-h Ch R2-m Cherry-WPRE-h GH p A or r AAV2/9-Ef1α-DIOe Np HR3.0-m Cherry-WPRE-h GH p A was injected into the VTA of Vglut2-Cre mice,the optical fiber was implanted above the medial prefrontal cortex(m PFC),lateral hypothalamus(LH),nucleus accumbens(NAc)and lateral septum(LS)to observe the effect of activation or inhibition of VTA glutamatergic terminals in the four nuclei on the BSR of EEG during anesthesia maintenance.LS was selected for further investigation.To determine the synaptic connections between the glutamatergic neurons in VTA and GABAergic neurons in LS,retrograde virus tracing and immunofluorescence staining were used.The virus RV-Env ADG-m Cherry and its auxiliary virus were injected into LS of Vgat-Cre mice,and glutamatergic neurons in the VTA were stained.5.Using fiber photometry,the fiber was implanted in the LS.The intensity of calcium signals of VTA glutamatergic terminals in the LS was determined by optical fiber recording technology during isoflurane anesthesia.Then,the effects of excitation or inhibition of VTA Glu-LS circuit on EEG and anesthesia behavior during anesthesia maintenance were observed by optogenetic regulation.6.r AAV2/9-Ef1α-DIO-h Ch R2-EGFP-WPRE-h GH p A was injected into the VTA of Vglut2-Cre mice,r AAV2/9-Ef1α-Vgat-e Np HR3.0-m Cherry-WPRE-h GH p A was injected into the LS and the opto-tetrode was implanted above the LS.The changes in the firing of LS GABAergic neurons during isoflurane anesthesia and emergence when regulated by activation of VTA glutamatergic terminals in the LS were measured by using dual optogenetic regulation technique and in vivo electrophysiological technique.Results1.The population calcium signaling activity of glutamatergic neurons in the VTA decreased following the initiation of isoflurane anesthesia,and recovered following termination of isoflurane inhalation(0.052±0.006 vs.-0.074±0.018 vs.-0.040±0.012,n=7,F=23.86,P<0.001);The firing rates of glutamatergic neurons in the VTA decreased during isoflurane anesthesia(5.685±1.055 vs.1.448±0.301 vs.3.854±0.898,n=34,F=6.741,P=0.001).2.Optogenetic activation of glutamatergic neurons in the VTA significantly reduced the BSR during 1.4% isoflurane maintenance(62.22±3.645 vs.20.92±3.048 vs.46.73±6.454,n=6,F=34.13,P<0.001),reduced total power percentages of the δ wave(30.99±3.504 vs.17.57±3.108 vs.29.38±2.565,n=6,F=10.83,P=0.003)during 0.8% isoflurane maintenance and shortened emergence time(484.7±21.47 s vs.395.7±13.95s;n=6,P=0.006).In contrast,optogenetic inhibition of glutamatergic neurons in the VTA increased the BSR(58.00±3.941 vs.71.65±4.214 vs.57.07±4.298,n=6,F=12.05,P=0.013),increased total power percentage of the δ wave(29.76±1.829 vs.44.91±3.972 vs.33.16±2.236,n=6,F=10.14,P=0.004)during 0.8% isoflurane maintenance and prolonged the emergence time(535±34.22 s vs.653.2±22.74s;n=6,P=0.016).3.The glutamatergic neurons in the VTA mainly sent projection fibers to the ventral orbital cortex(VO),NAc,LS,lateral habenular nucleus(LHb),LH,and dorsal raphe nucleus(DRN).In addition,they also project to reuniens thalamic nucleus(Re),thalamic parafascicular nucleus(Pa F),pontine reticular nucleus(Pn O),and giant cell reticular nucleus(Gi).4.Optogenetic activation of VTA glutamatergic terminals in the LH significantly reduced the BSR during 1.4% isoflurane maintenance(65.60±7.885 vs.37.77±3.508 vs.60.62±5.232,n=6,F=26.51,P=0.002);Optogenetic activation of VTA glutamatergic terminals in the LS significantly reduced the BSR during 1.4% isoflurane maintenance(60.69±4.930 vs.8.537±2.134 vs.31.57±5.950,n=6,F=55.30,P<0.001);Optogenetic activation of VTA glutamatergic terminals in the NAc significantly reduced the BSR during 1.4% isoflurane maintenance(75.98±6.475 vs.44.72±10.80 vs.68.51±7.240,n=6,F=8.288,P=0.027);Optogenetic activation of VTA glutamatergic terminals in the m PFC had little effect on BSR.Retrograde tracing shows that glutamatergic neurons in the VTA monosynaptically innervate GABAergic neurons in the LS.5.Activity of VTA glutamatergic terminals in the LS gradually decreased during isoflurane anesthesia and recovered during emergence(0.034±0.006 vs.-0.031±0.011 vs.-0.009±0.016,n=8,F=7.023,P=0.004).Optogenetic activation of VTA Glu-LS circuit significantly reduced the BSR during 1.4% isoflurane maintenance(60.69±4.930 vs.8.537±2.134 vs.31.57±5.950,n=6,F=55.30,P<0.001),reduced the δ power(39.63±2.788 vs.22.76±2.810 vs.30.02±3.114,n=6,F=16.67,P=0.001)during 0.8% isoflurane and shortened emergence time(636.5±41.33 s vs.493.8±26.24s;n=6,P=0.015).Optogenetic inhibition increased the BSR during 1.4% isoflurane maintenance(60.50±3.496 vs.73.41±3.768 vs.60.13±3.412,n=6,F=8.657,P=0.008),increased the δ power(28.50±5.565 vs.44.73±3.192 vs.31.16±3.686,n=6,F=16.94,P=0.001)and prolonged the emergence time(605±40.83 s vs.736.5 ±40.20s;n=6,P=0.044).6.Optogenetic activation of VTA glutamatergic terminals in LS increased the firing rate of GABAergic neurons in the LS in isoflurane anesthesia maintenance(0.515±0.129 vs.2.444±0.762;n=23,P=0.008)and emergence(1.472±0.281 vs.2.646±0.537;n=23,P=0.002).ConclusionIn this study,calcium signaling,in vivo electrophysiology,optogenetics,virus tracing and other technologies confirmed that glutamatergic neurons in the VTA participate in the regulation of isoflurane general anesthesia-arousal process and up-regulate the activity of glutamatergic neurons in the VTA can promote arousal from anesthesia.Glutamatergic neurons in the VTA project to multiple regions throughout the brain,and excitation of VTA glutamatergic terminals in the LH,NAc,and LS promotes arousal from isoflurane anesthesia.Glutamatergic neurons in the VTA can promote arousal by excitation of GABAergic neurons in the LS. |
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