Cellular And Circuit Mechanisms Underlying Cerebellar Involvement In The Regulation Of Sleep-wakefulness Cycle

It has long been recognized that the cerebellum is an important subcortical motor regulation center,which participates in regulating muscle tension,maintaining balance and coordinating voluntary movement.Recently,the cerebellum has also been demonstrated to be involved in a variety of non-motor functions such as cognition,spatial navigation,rewards,social interaction,and fear memory.Intriguingly,clinical studies have implicated that patients with damage in the cerebellum show not only motor-related dysfunctions,but also sleep disorder.In addition,animal experiments have shown that impairment of the cerebellum leads to reduced arousal time and increased sleep time in cats.Neuroanatomical studies illustrated that the cerebellum innervates multiple brain regions involved in promoting and/or maintaining wakefulness.Taken together,the previous findings imply that the cerebellum may be involved in the regulation of sleep-wakefulness cycle.However,it remains unclear whether and how the cerebellum participates in this regulation.In this study,in vivo multi-channel recording was utilized to reveal the activity pattern of cerebellar neurons during the natural sleep-wakefulness transition in mice.Moreover,virus tracing methods were used to label the cerebellar nuclei neurons specifically innervating the ventral thalamus(referred to as DCN ^VL/VM neurons).Afterwards,the collateral circuit and morphological characteristics of these DCN ^VL/VM neurons were analyzed by the immunohistochemical techniques.Combined optogenetics with in vivo multi-channel recording,we further explored the potential mechanisms underlying the involvement of DCN ^VL/VM neurons in the regulation of sleep and wakefulness.Results:1.Neuronal activity in the cerebellum during sleep-wakefulness transition1)The averaged firing rates of Purkinje cells（PC）in the arousal state were higher than that in either NREM or REM sleep state.Moreover,the PCs exhibited significantly increased firing activities prior to the transition from sleep to wakefulness.In contrast,the PCs exhibited significantly reduced firing activities prior to the transition from wakefulness to NREM sleep.2)The averaged firing rates of non-PCs during the NREM sleep were lower than that in either arousal or REM sleep state.Likewise,the non-PCs exhibited significantly increased firing activities prior to the transition from NREM sleep to wakefulness.In contrast,the non-PCs exhibited significantly reduced firing activities prior to the transition from wakefulness to NREM sleep.3)During the transition,there were significant excitatory monosynaptic inputs from the non-PCs to the PCs.4)There were no significant differences in the averaged firing rates of deep cerebellar nuclei（DCN）neurons across the arousal,NREM,and REM sleep states.As the downstream of PCs,neurons in the DCN manifested decreased firing activities during the transition from NREM sleep to wakefulness.In contrast,the DCN neurons showed increased firing activities during the transition from wakefulness to NREM sleep.2.Circuit connections and working mechanisms of DCN ^VL/VML/VM neurons1)DCN ^VL/VML/VM neurons innervates both the ventral thalamus nucleus and the cerebellar cortex.2)Optogenetic inhibition of the DCN ^VL/VM neurons resulted in increased firing rates of PCs.In contrast,optogenetics activation of the DCN ^VL/VM neurons induced rapid inhibition of PCs.3)Optogenetic activation of the DCN ^VL/VM neurons causes more extensive neuronal activities in the DCN by means of their inhibitory synaptic connections with the PCs.4)Optogenetic activation of the DCN ^VL/VM neurons exerts heavy effects on the neuronal activities in the cingulate cortex.Conclusions:1.Both the Purkinje cells and DCN neurons show significant firing changes,which were prior to the sleep-wakefulness transitions in mice.2.The DCN ^VL/VML/VM neurons innervate both the ventral thalamus and the cerebellar cortex.In particular,the DCN ^VL/VML/VM neurons formed inhibitory synaptic connections with the PCs in the cerebellar cortex.3.Optogenetic activation of the DCN ^VL/VM neurons reduced the firing rates of PCs,which in turn indirectly caused more extensive firing changes in the DCN.This process might function as the amplification of cerebellar outputs to the ventral thalamus,which in turn activate the neurons widely distributed in the cerebral cortex.Taken together,the current study aimed to investigate the firing activities of various types of cerebellar neurons across the sleep-wakefulness cycles.We herein showed the temporal features of single unit activity in the mouse cerebellum,which are essential for us to get the hints about whether and how the cerebellum is actively involved in the regulation of sleep-wakefulness transition.