Hyperexcitable arousal circuits drive sleep instability during aging

INTRODUCTION: Sleep destabilization is strongly associated with aging and cognitive function decline. Despite sleep fragmentation being central to the most prevalent complaints of sleep problems in elderly populations, the mechanistic underpinnings of sleep instability remain elusive. Fragmented sleep during aging has been observed across species, indicating conserved underlying mechanisms across the phylogenetic tree. Therefore, understanding why the aging brain fails to consolidate sleep may shed light on translational applications for improving the sleep quality of aged individuals. RATIONALE: We hypothesized that the decline in sleep quality could be due to malfunction of the neural circuits associated with sleep/wake control. It has been established that hypocretin/orexin (Hcrt/OX) neuronal activity is tightly associated with wakefulness and initiates and maintains the wake state. In this study, we investigated whether the intrinsic excitability of Hcrt neurons is altered, leading to a destabilized control of sleep/wake states during aging. RESULTS: Aged mice exhibited sleep fragmentation and a significant loss of Hcrt neurons. Hcrt neurons manifested a more frequent firing pattern, driving wake bouts and disrupting sleep continuity in aged mice. Aged Hcrt neurons were capable of eliciting more prolonged wake bouts upon optogenetic stimulations. These results suggested that hyperexcitability of Hcrt neurons emerges with age. Patch clamp recording in genetically identified Hcrt neurons revealed distinct intrinsic properties between the young and aged groups. Aged Hcrt neurons were hyperexcitable with depolarized membrane potentials (RMPs) and a smaller difference between RMP and the firing threshold. Aged Hcrt neurons expressing ChR2-eYFP were more sensitive to optogenetic stimulations, with a smaller-amplitude attenuation upon repetitive light pulse stimulations. More spikelets were generated in aged Hcrt neurons upon current injections. Recording from non-Hcrt neurons postsynaptic to Hcrt neurons revealed that optogenetic stimulation of Hcrt neurons expressing ChR2-eYFP reliably evoked time-locked postsynaptic currents (PSCs) after optogenetic stimulation more often in the aged group. Aged Hcrt neurons were characterized with a functional impairment of repolarizing M-current mediated by KCNQ2/3 channels and an anatomical loss of KCNQ2, revealed with array tomography at ultrastructural resolution. Single-nucleus RNA-sequencing (snRNA-seq) revealed molecular adaptions, including up-regulated prepro-Hcrt mRNA expression and a smaller fraction of Kcnq family subtypes Kcnq1/2/3/5 in aged Hcrt neurons. CRISPR/SaCas9–mediated disruption of Kcnq2/3 genes selectively in Hcrt neurons was sufficient to recapitulate the aging-associated sleep fragmentation trait in young mice. Pharmacological augmentation of M-current repolarized the RMP, suppressed spontaneous firing activity in aged Hcrt neurons, and consolidated sleep stability in aged mice. Sleep fragmentation in a narcolepsy mouse model with genetic ablation of Hcrt neurons at young ages manifested a mechanism other than hyperexcitable arousal-promoting Hcrt neurons that drives sleep fragmentation during healthy aging. CONCLUSION: Our data indicate that emerging hyperexcitability of arousal-promoting Hcrt neurons is strongly associated with fragmented sleep in aged mice, which display a lowered sleep-to-wake transition threshold defined for Hcrt neuronal activity. We have demonstrated that the down-regulation of KCNQ2/3 channels compromising repolarization drives Hcrt neuronal hyperexcitability, which leads to sleep instability during aging. Pharmacological remedy of sleep continuity through targeting KCNQ2/3 channels in aged mice confers a potential translational therapy strategy for improving sleep quality in aged individuals.