The Role And Mechanism Of Orexinergic Projections To VTA In Isoflurane Anesthesia-Arousal Regulation

Background:General anesthetics have been widely used in clinic for nearly two centuries,and its main pharmacological effect is to induce the loss of consciousness,but the mechanism of which is still unclear.Since the discovery of general anesthesia drugs,the study of the mechanism of general anesthesia has gone through lipid theory,protein theory,and the current network regulation theory.The theory of network regulation can be divided into two sub-thoeries based on the initial loop of regulation: Bottom-up mechanism and Top-down mechanism.The researchers who agree with the Bottom-up mechanism believe that general anesthesics act on different targets.They firstly affect the excitatory or inhibitory nuclei in the deep brain,inhibit the excitatory nuclei or activate the inhibitory nuclei,which further project the inhibitory signals to the cerebral cortex through the circuit,cut off the information connection of the cortex-cortex and the cortex-thalamus,resulting in the loss of consciousness.The awakening from general anesthesia originates from the restoration of the activity in the deep brain nuclei of the ascending activation system and then finally the cortex is activated.The orexinergic cell in the perifornical lateral hypothalamic area(Pe FLH)is an important part of the ascending activation system.It has been shown that the plasma concentration of orexin during wake after general anesthesia is higher than that before induction.The up-regulation of orexin neuron activity can significantly shorten the emergence time after general anesthesia,suggesting that orexin may be a key substance with active arousal characteristics.Orexin projects almost all over the brain,indicating that its arousal-promoting effect may be mediated by a variety of neural systems.The dopamine system located in the midbrain is involved in important physiological and pathological processes,such as sleep rhythm,learning and memory,reward feedback,drug addiction and so on.The dopamine neurons in the ventral tegmental area(VTA)project to the nucleus accumbens and prefrontal cortex,which are mainly involved in the biorhythm of sleep-awakening and drug addiction.Dopamine neurons of VTA were recently reported to be involved in the process of general anesthesia-arousal.Studies have shown that orexin can increase cortical dopamine release by regulating VTA,and orexin-VTA pathway plays an important role in learning and memory and drug addiction.In previous studies,it has been confirmed that microinjection of orexin into VTA can shorten the emergence time of rats after isoflurane anesthesia.However,due to the limitation of pharmacological methods,it is not clear what kind of VTA neurons are regulated by orexin.Therefore,the main purpose of this study was to investigate the role and mechanism of orexin neurons projecting to VTA in isoflurane anesthesia-arousal regulation by optogenetic and chemicogenetic techniques.Methods:1.Optogenetic and virus tracer techniques were used to observe the existence of orexin projection terminals in VTA,and the effects of orexinergic terminals on the emergence time and EEG signature during anesthesia maintenance in rats.2.The type of orexinergic target neurons in VTA was studied by using specific viruses,transgenetic animals and tracing technique.3.Chemicogenetic method was used to inhibit various neurons of VTA,and to observe whether photogenetically excited orexinergic endings in VTA could promote emergence.4.The expression of orexin-1 and orexin-2 receptors in VTA was observed by immunofluorescence staining,and the proportion of expression in GABA,dopamine and glutamate neurons was quantitatively analyzed.In vitro electrophysiological technique was used to observe and verify the effects of orexin-A/B ligands on the excitability of VTA neurons perfused with isoflurane.Results:1.The blue light at 473 nm stimulating orexinergic terminals in VTA could reduce the burst suppression rate(BSR)during 1.4% isoflurane anesthesia in rats from 40.5 ± 2.71%to 22.4 ± 11.79%,and shorten the emergence time from 915 ± 89 s to 685 ± 68 s.When yellow light at 594 nm was used to inhibit the orexinergic terminals in VTA,the emergence time was prolonged from 941 ± 92 s to 1279 ± 250 s.At the same time,under light anesthesia induced by 0.8% isoflurane,photogenetically excited orexinergic terminals of VTA could reduce the proportion of delta wave in EEG from 44.15 ± 11.85% to 29.07 ±3.93%,increase the proportion of α wave from 7.73 ± 2.51% to 10.57 ± 1.02%,and increase the proportion of β wave from 12.65 ± 5.46% to 19.26 ± 3.67%.The proportion of γ wave increased from 13.45 ± 3.23% to 18.57 ± 3.26%,accompanied by the dynamic awakening behavior of rats.Photogenetic inhibition of VTA orexinergic terminals reduced the proportion of θ wave from 18.73 ± 3.94% to 12.15 ± 5.08%,and the proportion of βwave from 12.7 ± 5.46% to 6.99 ± 3.67%.2.Among the VTA neurons,dopamine neurons accounted for 73.67 ± 12.85%,glutamate neurons accounted for 29 ± 7.96%,and Orexin neurons accounted for 15.89 ±6.42%.3.Using the method of chemicogenetics,intraperitoneal injection of CNO significantly inhibited the c-fos expression of TH neurons transfected with h M4 Di virus in VTA region: h M4Di+CNO 1.94 ± 2.37% vs.m Cherry+CNO 51.87 ± 16.86% journal p <0.001.Under the condition of intraperitoneal injection of CNO to inhibit TH neurons in the VTA area,the photogenetically excited orexinergic terminals in the VTA area could not shorten the awakening time of 1.4% isoflurane anesthesia in rats: h M4Di+CNO 1170± 91 s vs.h M4Di+saline 875 ± 73 s,m Cherry+saline 983 ± 168 s,m Cherry+CNO 901 ± 63 s p < 0.05,and also failed to reduce BSR:h M4Di+CNO:before 25.75 ± 15.52% vs.laser27.11 ± 16.89% vs.after 31.12 ± 16.56% vs.after in 1.4% isoflurane anesthesia in rats.4.In the VTA,the dopamine neurons expressing orexin-1 receptor accounted for73.38% ± 5.04% of the orexin-1 receptor positive neurons,and the dopamine neurons expressing orexin-2 receptor accounted for 74.41% ± 6.24% of the orexin-2 receptor positive neurons.GABA neurons expressing orexin-1 and orexin-2 receptors accounted for 6.82 ±0.56% and 8.1 ±1.91% of the total number of orexin-1 and orexin-2 receptor positive neurons,respectively.Glutamate neurons expressing orexin-1 and orexin-2receptors accounted for 8.96 ±1.87% and 7.3 ±1.58% of the total number of orexin-1 and orexin-2 receptor positive neurons,respectively.Perfusion with 0.6% and 1.4% isoflurane on isolated rat brain slices,additional perfusion with orexin-A(50n M)for 10 minutes increased the standardized action potential frequencies of dopamine neurons by 387 ±78%and 393 ± 69%,respectively.Conclusion:During the transition from isoflurane anesthesia to arousal,orexin neurons in the Pe FLH region play a central role.After excitation,orexin neurons can activate VTA nuclei through orexinergic terminals and then induce arousal.In the VTA nucleus,the arousal of orexin is mainly mediated by the dopamine neurons,and the orexinergic terminals are regulated by the orexin receptors on the dopamine neurons.