Cerebellar Purkinje Cells Promote Wakefulness From Anesthesia Through Synchronizing The Activity Of Neurons In The Cerebral M1

Background The activity of the cerebellum undergoes dynamic changes during different phases of the sleep-wake cycle.Damage to the cerebellum not only leads to motor-related functional impairments,but also causes abnormalities in the sleep-wake state of patients.Currently,the neural regulatory mechanisms underlying the involvement of the cerebellum in arousal are not clear.Studying the role of the cerebellum in arousal through anesthesia-awakening as a probe may shed light on delayed emergence,agitation and delirium after anesthesia,as well as consciousness disorders,such as coma.Objective The neuronal activity and neurotransmitter release of the cerebellar vermis of the V lobule（5Cb）and the primary motor cortex（M1）,as well as their synchronous interaction with EEG,are clearly defined throughout the process of consciousness states induced by anesthssia.The study seeks to explore the neural mechanisms responsible for promoting arousal,with an emphasis on the functional role of Purkinje cells in the 5Cb.Method 1.The anterograde transsynaptic tracing method with herpes simplex virus（HSV）was used to map the neural circuits between the 5Cb and the M1.Also,functional magnetic resonance imaging（fMRI）was utilized to detect the functional connectivity between both brain regions.To further elucidate the molecular regulatory mechanisms underlying the functional connectivity,the optogenetics and fiber photometry were used.2.By combining fiber photometry with electroencephalography/electromyography（EEG/EMG）recordings,we monitored the dynamic changes in Ca²⁺ activity of Purkinje cells（PCs）in the 5Cb and CaMKIIα⁺ neurons in the M1 region,as well as the neurotransmitter release of glutamate（Glu）,gamma-aminobutyric acid（GABA）,and norepinephrine（NE）in both regions during the transitions from wakefulness to loss of consciousness（LOC）,burst suppression（BS）,and early recovery of consciousness（ROC）induced by 3% sevoflurane.3.Excitatory optogenetic virus AAV-DIO-Chrimson R or inhibitory virus AAV-DIOe Np HR was injected into the 5Cb of Pcp2/L7-Cre mice to study the time of loss of consciousness（LOC）and recovery of consciousness（ROC）during exposure and withdrawal of 3% sevoflurane.Optogenetic stimulation was administered during BS to detect the changes of burst suppression rate（BSR）.4.For a clinical case study,patient undergoing cerebellopontine angle（CPA）tumor resection was enrolled.The values of bispectral index（BIS）and the spontaneous electromyographic responses of the facial muscles were synchronously recorded in the process of surgery.Results 1.The cerebellar 5Cb and the cerebral M1 exhibited both structural and functional connectivity.Tracing the neuroanatomical pathways revealed that the 5Cb has structural connections to the M1 and secondary motor cortex（M2）through the thalamic posterior nucleus group（PO）,parafascicular nucleus（PF）,and ventral posterior medial nucleus（VPM）.Strong functional connectivity signals were revealed by fMRI between the 5Cb and PO and PF in the thalamus,as well as the M1 and the forelimb area of the primary somatosensory cortex（S1）in the anesthetized brain,with the 5Cb as region of interest.In addition,optogeneticspecific activation of PCs in the 5Cb led to rapid release of Glu and a rapid increase in Ca²⁺ activity in CaMKIIα⁺ neurons of the M1.2.The neuron activity and neurotransmitter release in the 5Cb and the M1 were synchronously coordinated during the anesthesia-awaking process.The consciousness transitions induced by sevoflurane can be divided into the states of wakefulness,LOC,BS,pre-recovery of consciousness（Pre-ROC）and ROC.（1）There was a synchronous decrease in Glu/GABA release between the 5Cb and the M1 during the transition from wakefulness to LOC and then to BS.From BS to the Pre-ROC stage,there was a synchronous increase in Glu/GABA release.At the same time,the activity of the PCs in the 5Cb and CaMKIIα⁺ neurons in the M1 shows consistency with the synchronous release of Glu/GABA.（2）During BS,the Ca²⁺ activity of CaMKIIα⁺ neurons and Glu release in the M1 show a high correlation with EEG gamma oscillations,while the PCs activity and Glu/GABA/NE release in the 5Cb were absent.（3）During Pre-ROC,the Ca²⁺ activity of PCs and the Glu/GABA/NE release in the 5Cb,not the M1,showed a preferential response and are highly correlated with EEG gamma oscillations.3.Optogenetic activation of the PCs in the 5Cb led to a switch from unconscious state to an awakened state.During sevoflurane-induced deep anesthesia with EEG burst suppression,optogenetic activation of the PCs in the 5Cb for 2 minutes led to an immediate significant reduction in BSR,and this effect persisted for 13 minutes.Upon sevoflurane withdrawal,optogenetic activation of the PCs in the 5Cb shortened the time to recovery of consciousness by 44%.Conversely,optogenetic inhibition prolonged the time to recovery of consciousness by 55% after sevoflurane withdrawal in mice.4.In a clinical case of acoustic neuroma resection in the CPA,the first stimulation near the cerebellum led to an increase in BIS values from 40 to 74.The second stimulation resulted in a sudden increase in BIS values from 40 to 92,accompanied by intense muscle electrical activity.Conclusion The cerebellar 5Cb participated in the regulation of anesthesia-arousal by synchronizing the activity of neurons in the cerebral M1.