Effect And Mechanism Of Cerebral Cortex In Regulating Isoflurane Anesthesia

【Background】 General anesthesia refers to a state of reversible loss of consciousness,amnesia,analgesia,and immobility induced by anesthetics.General anesthesia is widely used in clinical practice and has very important clinical significance.However,the core mechanism of its role has not yet been clarified.At present,the "Bottom-Up" and "Top-Down" theories are the main mechanisms of "general anesthetics-induced loss of consciousness".Compared with the "Bottom-Up" theory,the current research on the "Top-Down" theory has not received enough attention from researchers.The “Top-Down” theory believes that anesthetics can induce the loss of consciousness by regulating the cortex-cortex and cortex-thalamus circuits related to the integration of neural information.Existing exploration of the "TopDown" theory is mainly focused on the use of EEG,PET,and f MRI to conduct clinical research on changes in cortical functional connectivity caused by anesthetics.Using optogenetic manipulation,in vivo fluorescence imaging,and other technologies in the basic research to deeply analyze the molecular and circuits mechanism of the "Top-Down" theory will contribute to revealing the mechanism of general anesthetics and even the mystery of consciousness.The cerebral cortex has a very complex network structure and function,and the structure contains different areas.According to the different functions performed,the cerebral cortex can be divided into the high-order functional cortex and primary functional cortex,such as the medial prefrontal cortex and the primary visual cortex.The functional projection from the high-order functional cortex to the primary functional cortex constitutes the cortical feedback connectivity.The inhibition of feedback connectivity in the cortex is also an important aspect of the "Top-Down" theory of general anesthetic-induced loss of consciousness.In addition,the elaborate cortical network is mainly composed of excitatory pyramidal cells(PCs)and three molecularly distinct inhibitory interneurons expressing parvalbumin(PV),somatostatin(SOM),or vasoactive intestinal peptide(VIP).They form a complex cortical neural network through synaptic interconnections releasing gammaaminobutyric acid(GABA)and glutamate.Notably,newly developed genetically encoded fluorescent sensors of i GABASn FR and i Glu Sn FR have allowed tracking of the GABA and glutamate dynamics of the brain with a high spatiotemporal resolution,which would contribute to revealing the cortical excitatory-inhibitory network dynamics at the mesoscale.This project uses in vivo two-photon imaging,optical fiber recording,optogenetics,and other technologies to study the dynamics of various neurons under isoflurane anesthesia in the primary visual cortex and the medial prefrontal cortex.In particular,the regulatory effect of pyramidal cells in the medial prefrontal cortex under isoflurane anesthesia was further investigated.Part One In vivo two-photon imaging of the dynamics of the primary visual cortical network under isoflurane anesthesia【Objectives】 The purpose of this experiment is to observe the dynamics of GABA and glutamate transmitter input and calcium activity of pyramidal cells,PV neurons,SOM neurons,and VIP neurons in the primary visual cortex network under isoflurane anesthesia.To label the pyramidal cells,PV neurons,SOM neurons,and VIP neurons in the primary visual cortex,injecting AAV-DIO-i GABASn FR,AAV-DIO-i Glu Sn FR,and AAV-DIOGCa MP6 m viruses into the primary visual cortex of the C57BL/6J,PV-ires-Cre,SOM-iresCre,and VIP-ires-Cre transgenic mice(2-4 months old),respectively;The number of mice in each group is not less than 3;Embed the glass cranial window and EEG electrodes at the same time during virus injection;After the virus is successfully expressed,the mouse is placed in the head-fixation apparatus and connected to the self-made isoflurane inhalation device.Using in vivo two-photon microscopy imaging technology with high spatiotemporal resolution,record the GABA and glutamate transmitters input onto four types of neurons in the 2/3 layer of the primary visual cortex under 1.2% isoflurane anesthesia,as well as the calcium activity of themselves;At the same time,recording EEG to assess the consciousness level of mice;Immunofluorescence staining was used to observe the expression of AAV-DIO-i GABASn FR,AAV-DIO-i Glu Sn FR and AAV-DIO-GCa MP6 m viruses within the four types of cortical neurons.【Methods】【Results】(1)AAV-DIO-i GABASn FR,AAV-DIO-i Glu Sn FR,and AAV-DIO-GCa MP6 m viruses could be successfully expressed in the pyramidal cells,PV neurons,SOM neurons,and VIP neurons.(2)The EEG signal showed that within ten minutes after the administration of isoflurane,the standardized γ wave power gradually decreased,and the burst suppression ratio gradually increased,and the mice could stably enter a state of loss of consciousness.(3)Compared with the awake state,the GABA input onto the pyramidal cells(ΔF/F: 15.37 ± 15.3% vs.-4.22 ± 3.98%,n = 26/3),PV neurons(ΔF/F: 11.47 ± 18.59% vs.-5.98 ± 11.16%,n = 32/5),SOM neurons(ΔF/F: 22.49 ± 10.01% vs.-0.07 ± 5.65%,n = 27/4)and VIP neurons(ΔF/F: 10.4 ± 13.53% vs.-6.17 ± 6.37%,n = 82/7)were general decayed within the 2/3 layer of the primary visual cortex upon isoflurane administration,and there was no statistical difference in the ?F/F(Ana-awake)and slope values of the four types of neurons.(4)In the 2/3 layer of the primary visual cortex,the glutamate input onto pyramidal cells(ΔF/F: 6.09 ± 4.85% vs.-1.75 ± 2.82%,n = 24/3),PV neurons(ΔF/F: 13.99 ± 19.06% vs.-6.61 ± 8.44%,n = 49/6),SOM neurons(ΔF/F: 9.71 ± 19.3% vs.-11.93 ± 12.22%,n = 28/4)and VIP neurons(ΔF/F: 15.79 ± 18.32% vs.-5.77 ± 7.86%,n = 84/7)were decreased;Under isoflurane anesthesia,the decrease of glutamate input onto pyramidal cells(?F/F(Ana-awake)and slope value)was significantly lower than that onto the inhibitory interneurons.(5)3-6 minutes after isoflurane anesthesia,the calcium events and total calcium activity of VIP neurons(n = 103/5)were higher than those of PV neurons(n = 49/5)and SOM neurons(n = 52/7);Under isoflurane anesthesia,the average value of the correlation coefficient r of VIP neurons was higher than that of the awake state.(6)Under isoflurane anesthesia,the inhibitory level of PV neurons(?F/F(Ana-awake)and slope value)was higher than that of SOM neurons,and the average correlation coefficient r of PV neurons and SOM neurons was higher than that of the awake state.At the same time,the average value of the correlation coefficient r of PV neurons was higher than that of the other two types of interneurons.【Conclusions】 The GABA input onto the four types of neurons in the primary visual cortex network was generally decayed without cell-type specificity during the isoflurane-induced loss of consciousness;Unlike a dramatic decrease in glutamate input onto inhibitory interneurons,the glutamate input onto pyramidal cells under isoflurane anesthesia was preserved;Under isoflurane anesthesia,the calcium activity of inhibitory interneurons showed cell-type heterogeneity,VIP neuronal calcium activity showed a delayed inhibition effect,and PV neuronal calcium activity were the deepest inhibited and the highest synchronization.The results of this part suggested that the excitatory-inhibitory network of the primary visual cortex is destroyed during isoflurane-induced loss of consciousness,and a functional cortical inhibitory network may play an important role in the the maintenance of consciousness.Part Two The role of pyramidal cells within the medial prefrontal cortex(m PFC)in the regulation of isoflurane anesthesia【Objectives】 This experiment aims to observe the dynamics of calcium activity of various neurons within the medial prefrontal cortex(m PFC)under isoflurane anesthesia;To clarify the regulatory effect of pyramidal cells within the medial prefrontal cortex(m PFC)on isoflurane anesthesia.【Methods】(1)First,to label the pyramidal cells,PV neurons,SOM neurons,and VIP neurons in the medial prefrontal cortex(m PFC),the AAV-DIO-GCa MP6 m virus was injected into the medial prefrontal cortex(m PFC)of 7-8 week old C57BL/6J mice,PV-ires-Cre mice,SOM-ires-Cre mice,and VIP-ires-Cre mice,respectively;At the same time,EEG electrodes and optical fiber ceramic ferrules were embedded in these mice;after the virus was successfully expressed,the EEG signals under isoflurane anesthesia and the calcium activity of various neurons in the medial prefrontal cortex were simultaneously monitored in real-time.(2)The virus mixture of AAV-DIO-GCa MP6 m and AAV-CAMKⅡ-cre was injected into the primary visual cortex(V1)and primary somatosensory cortex(S1BF)of C57BL/6J mice aged 7-8 weeks,and the pyramidal cells in these two areas were marked;After the virus was successfully expressed,the EEG signal under isoflurane anesthesia and the calcium activity of the pyramidal cells in the primary sensory cortex(S1BF)are simultaneously monitored in real-time;Analyze the difference in calcium activity of the pyramidal cells in the medial prefrontal cortex(m PFC),primary visual cortex(V1),and primary somatosensory cortex(S1BF)during the burst wave phase and the suppression wave phase of EEG,namely ΔF/F(Burst-Suppression).(3)The virus mixture of AAV-DIO-Ch R2 and AAV-CAMKⅡ-cre was injected into the medial prefrontal cortex(m PFC)and primary visual cortex(V1)of C57BL/6J mice aged 7-8 weeks;EEG electrodes and optical fiber ceramic ferrules were embedded at the same time;After the virus was successfully expressed,perform the EEG monitoring experiment under isoflurane anesthesia;At the 30 th and 35 th minutes of isoflurane anesthesia,twice blue light stimulations were given to observe the effect on the burst suppression wave of EEG.【Results】(1)The calcium activity of pyramidal cells in the medial prefrontal cortex(m PFC)after isoflurane administration was higher than that of the other three types of inhibitory interneurons(PC:-12.02 ± 3.55%,n = 6,PV:-24.35 ± 7.08%,n = 7,SOM:-37.56 ± 3.5%,n = 6,VIP:-24.43 ± 5.16%,n = 5).(2)The calcium signal intensity of the pyramidal cells in the medial prefrontal cortex(m PFC)during the burst wave phase is higher than the calcium signal intensity during the suppression wave phase(-16.07 ± 6.8% vs.-19.55 ± 6.8%,n = 6).(3)The ΔF/F(Burst-Suppression)value of calcium activity of pyramidal cells in the medial prefrontal cortex(m PFC)was significantly higher than the ΔF/F(Burst-Suppression)value of calcium activity of pyramidal cells in the primary visual cortex(V1)and primary somatosensory cortex(S1BF)(m PFC: 3.48 ± 0.45%,n = 6,V1: 1.84 ± 0.65%,n = 5,S1BF: 1.33 ± 0.74%,n = 5).(4)Upon blue light activated the pyramidal cells in the medial prefrontal cortex(m PFC),the burst suppression ratio(BSR)of EEG was significantly reduced(BF-sti-1: 76.8 ± 8.52%,sti-1: 39.08 ± 15.54%,AF-sti-1: 59.74 ± 16.73%,n = 8;BF-sti-2: 72.49 ± 14.77%,sti-2: 54.89 ± 10.95%,AF-sti-2: 75.11 ± 9.82%,n = 8);blue light activated the pyramidal cells in the primary visual cortex(V1)had no effect on the burst suppression ratio of the EEG.【Conclusions】 Isoflurane anesthesia had the lowest inhibitory effect on the calcium activity of pyramidal cells in the medial prefrontal cortex(m PFC),and didn’t completely inhibit the calcium activity of pyramidal cells;Under isoflurane anesthesia,the calcium activity of pyramidal cells in the medial prefrontal cortex(m PFC)showed a burst-suppression oscillation pattern,and the time course was consistent with the burst suppression wave of EEG signals;The burst-suppression oscillation intensity of calcium activity of the pyramidal cells in the primary sensory cortex was weaker than that of the pyramidal cells in the medial prefrontal cortex(m PFC);Not all cortical pyramidal cells could regulate the formation of burst suppression waves of EEG signals.Activating the activity of pyramidal cells in the medial prefrontal cortex(m PFC)can effectively reverse burst suppression waves of EEG signals.The results of this part confirmed that the pyramidal cells within the medial prefrontal cortex(m PFC)are the important switch for the formation of burst suppression waves of EEG signals,and are involved in regulating the depth of isoflurane anesthesia.