Synaptic Mechanism Of Basal Forebrain Neural Circuits During General Anesthesia

BackgroundIn modern medicine,the application of general anesthetics accelerates the rapid development of anesthesiology and surgery,and relives patients from pain.However,the mechanism of reversible loss of consciousness induced by general anesthetics in the central nervous system is still unclear.Research on the mechanism of general anesthesia has been carried out at the level of protein,molecule and neural network.At present,people prefer to follow the neural network theory to explain the loss of consciousness caused by the application of general anesthetics.It is reported that information is transmitted between different nuclei in the central nervous system through neural circuits.For example,thalamus,amygdala and basal forebrain regulate the activity of cortex through different neurotransmitter systems and participate in the process of general anesthesia.These large nucleus in the brain contain different sub-nucleus,and many types of neurons in the nucleus.The sub-nucleus and the different neurons in the nucleus also form microcircuits through synaptic connections.In recent years,it has been found that the complex microcircuit within the nucleus and the long projection between the nucleus play an important role in neural activities.Therefore,research on the mechanism of general anesthesia from multi-system and multi-angle will be more helpful for us to understand the whole process of loss of consciousness caused by general anesthesia.Basal forebrain(BF)is a relay station between the reticular ascending system and the cerebral cortex,which is directly involved in the activation of the cortex and maintain the sleep homeostasis.BF contains several sub-nucleus,including not only cholinergic neuron clusters,but also projection neurons containing a variety of neurotransmitters,which transmit glutamate,GABA,Ach and other neurotransmitters to the downstream nucleus.Meanwhile,BF neurons are also regulated by inhibitory transmitters such as adenosine and serotonin.In our previous study,we found that microinjection of orexin into the BF area activated the neurons in BF and promoting arousal from general anesthesia,indicating that the BF neural network plays an important role in the process of consciousness conversion during anesthesia.However,it is still not clear how various neurotransmitter systems in BF regulate each other and which transmitter regulates the activity of thalamus to mediate the transition between anesthesia and arousal.Traditionally,with the application of general anesthetics,the release of excitatory neurotransmitters such as Glutamate and Ach is reduced,while the release of inhibitory neurotransmitters such as GABA is increased,which inhibits cortical excitement and leads to a loss of consciousness.In our previous study,we found that general anesthesia has a "dual" role in regulating central neurotransmitters in BF region.At the same time,our previous in vivo experiments found that the calcium signals recorded from three types of BF neurons were all weakened after isoflurane(ISO)anesthesia,indicating that the activity of all three types of BF neurons was inhibited.These findings cannot be explained by traditional viewpoints.The reason may be closely related to the mutual regulation of different transmitter systems in BF nucleus.In order to resolve the problem,this research mainly use genetically modified mice,combined the technology of promoter,optogenetic method and in vitro electrophysiology recording,exploring the mechanism of neurotransmitter system regulate electrical activity and synaptic transmission in BF,and the mechanism of microcircuit in BF and the projections from BF to CMT during the anesthesia and arousal.Aims1.In vitro electrophysiology recording to verify the electrophysiological characteristics of glutamate,GABA and Cholinergic neurons of BF,as well as the change of firing rate in three types of BF neurons and subtypes of GABA neurons after ISO anesthesia.2.Promoter technology combined with optogenetic method was used to specifically regulate a certain type of BF neurons.In vitro electrophysiological recording of postsynaptic neurons which had synaptic connections with presynaptic neurons expressed ChR2.To observe the reciprocal regulation between BF neurons and the changes of relationship between different neurons after ISO administration.3.The optogenetic technique was used to observe the projection from cholinergic neurons,glutamate neurons,GABA neurons in BF to CMT(central medial thalamus)neurons in vitro,and to explore the electrophysiological mechanism of the BF-CMT circuit during ISO anesthesia.Methods1.Vglut2-td Tomato,Vgat-td Tomato and Chat-td Tomato mice aged from 4 to 8 weeks were selected to prepare isolated brain slices with BF nuclei,fluorescently labeled neurons were illuminated by green light under a fluorescence microscope,and the highlighted neurons in the field of vision were recorded by whole-cell recording.A step current stimulation was given under current clamp to record the electrophysiological characteristics of neurons.The firing activity of neurons was recorded under the current clamp.After the cell firing was stable,recorded 5 minutes for baseline,Isoflurane was applied for 10 minutes,and washed out for 25 minutes.The change of firing rate before and after ISO perfusion were observed.2.Vglut2-td Tomato mice aged 4-5 weeks were selected and microinjected with r AAV-DIO-ChR2-e GFP with Chat-promoter in BF nuclei.Two or three weeks after virus expression,brain slices were taken for in vitro electrophysiological experiments.The fluorescent Vglut2 neurons were clamped,and the ChR2 expressed cholinergic neurons were activated by local blue light(3ms,1-3m W)in BF,and the optogenetically evoked excitatory/inhibitory postsynaptic currents(o EPSC/o IPSC)induced by blue light on Vglut2 neurons were recorded.After the induction of stable postsynaptic current,the o EPSC/o IPSC amplitude changes were observed in 5 min of baseline,10 min of ISO and 25 min wash out.In the same way,the relationship between cholinergic neurons and GABA neurons,GABA neurons and Vglut2 neurons before and after ISO were observed.3.Vglut2-Cre mice aged 4-5 weeks were selected and injected into BF region with AAV-DIO-ChR2-m Cherry virus,which was expressed for 2-3 weeks for in vitro electrophysiological experiment.CMT neurons were recorded in whole-cell recording,and the axonal endings of Vglut2 neurons in CMT were activated by blue light.Once the photoinduced postsynaptic current on CMT neurons was observed,and ISO was applied to observe the changes of postsynaptic current.In the same way,we observed whether there was GABA projection and cholinergic projection from BF to CMT,and verified the functional change of projection after ISO.Results1.The firing activity of all three types of BF neurons changed significantly after ISO application,and the firing activity of each type of neurons increased and decreased.The firing rate of 31% cholinergic neurons increased after ISO administration(183±16% of baseline,P<0.01,n=8),the firing rate of 69% cholinergic neurons decreased after ISO administration(9.7±1% of baseline,P<0.0001,n=18);The firing rate of 27% Vglut2 neurons increased after ISO administration(156±4% of baseline,P<0.01,n=10),73% Vglut2 neurons discharge frequency decreased(17±2% of baseline,P<0.0001,n=27);41% of BF GABA neurons showed an increase in firing frequency after ISO(223.9±3% of baseline,P<0.0001,n=14),the firing frequency of 59% of neurons decreased(18.4±2% of baseline,P<0.001,n=20).We further observed the firing rate of PV and SOM neurons after ISO administration,and found that the firing activity of 26% PV neurons after ISO increased(264±7% of baseline,P<0.001,n=6),the discharge activity of 74% PV decreased(43.8±4% of baseline,P<0.01,n=17).While 69% of SOM neurons showed increased firing activity after ISO(290±9% of baseline,P<0.01,n=11),31% decreased discharge activity after ISO administration(20±4% of baseline,P<0.01,n=5).2.We verified relationships among the three types of BF neurons and the change of relationship after ISO.o IPSC could be record on Vglut2 neurons when photogenetic activation of cholinergic neurons in the local area of BF.After ISO was applied,the amplitude of o IPSC increased(58.84±21.15,P<0.05,n=5),suggesting that the inhibitory effect from cholinergic neurons to Vglut2 neurons increased after ISO.After recording GABA neurons and activating cholinergic neurons,o EPSC could be recorded.o EPSC increased after ISO(47.84±15.22,P<0.05,n=5),indicating that the excitability effect from cholinergic neurons to GABA neurons increased after ISO.Vglut2 neurons were recorded,GABA neurons were activated,and o IPSC was recorded,which was significantly increased after ISO(54.32±14.59,P<0.01,n=6),indicating that the inhibitory effect of GABA neurons on Vglut2 neurons was increased after ISO.Blue light activated Ca MKII neurons in BF,and o EPSC was recorded on cholinergic neurons.Current amplitude increased after ISO administration(46.13±18.46,P<0.01,n=5).Cholinergic neurons were recorded,and blue light activated BF GABA neurons,but o EPSC or o IPSC were not appeared(n=15).3.Vglut2,Cholinergic and GABA projection from BF to CMT were verified in vitro experiments.We found that after blue light activated the axonal endings of Vglut2 and cholinergic neurons in CMT,no photoinduced postsynaptic current(o EPSC or o IPSC)was recorded in CMT neurons.o IPSC can be recorded on CMT neurons after blue light activates the axon ends of GABA neurons,and the o IPSC can be blocked by GABA receptor blockers.At the same time,o IPSC can be blocked by adding TTX to the external fluid and then adding 4-AP can restore o IPSC.The amplitude of o IPSC in CMT neurons was decreased in 9 cases(49.74±2.274,P<0.001,n=9),increased in 2 cases(22.57±8.277,P<0.05,n=2),and totally decreased in the amplitude(32.91±2.955,P<0.001,n=11).Conclusion1.The electrical activity of glutamate neurons(73%),cholinergic neurons(69%),GABA neurons(59%)and PV neurons(74%)in BF was significantly inhibited under ISO anesthesia;The firing activity of SOM neurons(69%)increased after ISO anesthesia.2.The cholinergic neurons in BF nucleus directly and indirectly inhibit glutamate neurons,which is enhanced under ISO anesthesia,resulting in a stronger inhibitory effect on glutamate neurons after ISO anesthesia.3.Electrophysiology experiment further verified that there was no functional projection from glutamate neurons and cholinergic neurons in BF to CMT neurons,but there was a monosynaptic projection from GABA neurons in BF to CMT,and the projection was significantly inhibited after the ISO anesthesia.