Study On The Mechanism Of Lateral Hypothalamus GABA Neurons Regulating Sevoflurane General Anesthesia To Promote Arousal

BackgroundSevoflurane has been widely applied in clinical anesthesia practice owing to the extensive clinical data supporting its benefits such as fast elimination and minor effects on respiratory system function.However,little is known about how sevoflurane anesthetic reversibly caused loss of consciousness and recovery of consciousness.Previous studies indicated that some key nodes and brain areas in the subcortex are known to mediate the transition from general anesthesia to wakefulness,such as basal forebrain(BF),ventral tegmental area(VTA),lateral hypothalamus(LH),locus coeruleus(LC),dorsal raphe nucleus(DRN).Among the various neuronal populations involving conscious modulation,the LH plays a critical role in integrating neuronal responses to a variety of peripheral signals to regulate survival.Studies have confirmed that LH regulates wakeful behaviors such as sleep-wake cycles,anesthesia-arousal transition,food intake,attack,stress,etc.Numerous neurotransmitters and neuropeptides,including γ-aminobutyric acid(GABA),Orexin,MCH,and Glu,are found in the LH.As the LH mixes various neuronal types with diverse molecular signatures and participates as one of the central core brain areas in regulating multiple fundamental physiological behaviors,admittedly,this directly leads to its complex roles in conscious modulation.The LH GABA system is involved in a broad range of neurophysiological modulations,including the regulation of sleep and wakefulness.However,it is unknown whether the LH GABA neurons influence consciousness transition during general anesthesia.Here,in combination with the EEG features and behaviors in the induction,maintenance and awakening stages during sevoflurane general anesthesia,we reported that LH GABA neurons and a specific neurocircuit projecting to the lateral periaqueductal gray(LPAG)selectively regulated the process of general anesthesia and produced obvious arousal effect.MethodsIn this study,immunofluorescence staining and fiber photometry recording have been used to observe the alterations in neural activity,especially the activity of LH GABA neurons during the period from induction to wakefulness of sevoflurane anesthesia.Using chemogenetic and optogenetic methods combined with EEG recording and general anesthetic behaviors to investigate the role of this neurocircuit in sevoflurane anesthesia induction,maintenance and emergence.Anterograde tracing has been used to observe the downstream of LH GABA neurons.The possible upstream input nuclei of the LH and LPAG were explored by virus reverse tracing technology.Results(1)Sevoflurane anesthesia inhibited the activity of GABA neurons in the LH,activation of GABA neurons in the LH promoted arousal,LH GABA neurons have projection connections to many nuclei in the whole brain。Immunofluorescence staining results showed that c-Fos expression in the LH was reduced after sevoflurane anesthesia.GABA,Orexin,and Glu neuronal activities showed different degrees of decrease,except for the increased activity of MCH neurons.Fiber photometry recordings further indicated that sevoflurane anesthesia inhibited the calcium activity of GABA neurons in the LH.Under sevoflurane anesthesia,LH GABA neuron activity was decreased sharply under sevoflurane anesthesia and sustained at lower levels for prolonged durations.Optogenetic and chemogenetic activation of GABA neurons in the LH decreased the burst-suppression ratio(BSR);optogenetic and chemogenetic activation of GABA neurons in the LH prolonged the loss of righting reflex(LORR)and shortened the recovery of righting reflex(RORR)in mice.Therefore,we confirmed that activation of LH GABA neurons could promote the transition from sevoflurane general anesthesia to arousal in mice.Anterograde tracing results showed that LH GABA neurons projected to multiple brain areas involved in the modulation of arousal,which including the lateral habenular nucleus,the VTA,the LPAG,and the DRN.(2)LH GABA neurons projected to LPAG,activation of LH GABAergic terminals in the LPAG promoted awakening.Optogenetic and chemogenetic activation of the LH GABAergic terminals in the LPAG reduced the BSR;optogenetic and chemogenetic activation of the LH GABAergic terminals in the LPAG prolonged LORR and shortened RORR in mice.These results confirmed that activation of LH GABAergic terminals in the LPAG could promote the transition from sevoflurane general anesthesia to arousal in mice.Reverse tracing results suggested that BLA,LPO,MPA,DRN,VTA,DMH,and other locations may have a third-level loop projecting to the LH GABA-LPAG.ConclusionThis study adopted a variety of neuron regulation technologies,the data revealed the crucial role for LH GABA neurons and terminal project to the LPAG in the proper formation of arousal from sevoflurane anesthesia,and that GABA neurons are important neurons within the LH for control of anesthesia-arousal transition.