Nitric oxide (NO) has been implicated as an important neurotransmitter in stress responses and sleep regulatory processes. However, the role of NO in the relationship between stress and sleep remains unclear. The medial septum (MS) and vertical diagonal band (VDB), regions of the basal forebrain involved in sleep regulation, contain nitric oxide synthase (NOS) producing neurons. Additionally, NOS neurons in the dorsal raphe nucleus (DRN) encode information about stress duration. The role of nitrergic neurons in these regions in subserving sex-specific responses to stress and sleep loss has yet to be elucidated. In this study, NADPH-d, an index of NOS activity, was used to examine the effects of acute restraint stress and sleep loss on NOS activity in the MS, VDB, and DRN. We show that NOS activity in response to restraint stress, total sleep deprivation (TSD), and partial sleep restriction (PSR) differs based on sex and region. Initial analysis showed no effect of restraint stress or TSD on NOS activity in the basal forebrain. However, investigation of each sex separately revealed that restraint stress and TSD significantly decrease NOS activity in the MS of females, but not males. Interestingly, the difference in NOS activity between restraint stress and TSD in females was not significant. Furthermore, PSR was not sufficient to affect NOS activity in males or females. These data suggest that restraint stress and sleep loss regulate NOS activation in a sex-dependent manner, and that the NOS stress response in females may be mediated by sleep loss.

Background

Sleep disturbances are a prevalent and complex comorbidity in neurodevelopmental disorders (NDDs). Dup15q syndrome (duplications of 15q11.2-13.1) is a genetic disorder highly penetrant for NDDs such as autism and intellectual disability and it is frequently accompanied by significant disruptions in sleep patterns. The 15q critical region harbors genes crucial for brain development, notably UBE3A and a cluster of gamma-aminobutyric acid type A receptor (GABA_AR) genes. We previously described an electrophysiological biomarker of the syndrome, marked by heightened beta oscillations (12-30 Hz) in individuals with Dup15q syndrome, akin to electroencephalogram (EEG) alterations induced by allosteric modulation of GABA_ARs. Those with Dup15q syndrome exhibited increased beta oscillations during the awake resting state and during sleep, and they showed profoundly abnormal NREM sleep. This study aims to assess the translational validity of these EEG signatures and to delve into their neurobiological underpinnings by quantifying sleep physiology in chromosome-engineered mice with maternal (matDp/ + mice) or paternal (patDp/ + mice) inheritance of the full 15q11.2-13.1-equivalent duplication, and mice with duplication of just the UBE3A gene (Ube3a overexpression mice; Ube3a OE mice) and comparing the sleep metrics with their respective wildtype (WT) littermate controls.

Methods

We collected 48-h EEG/EMG recordings from 35 (23 male, 12 female) 12-24-week-old matDp/ + , patDp/ + , Ube3a OE mice, and their WT littermate controls. We quantified baseline sleep, sleep fragmentation, spectral power dynamics during sleep states, and recovery following sleep deprivation. Within each group, distinctions between Dup15q mutant mice and WT littermate controls were evaluated using analysis of variance (ANOVA) and student's t-test. The impact of genotype and time was discerned through repeated measures ANOVA, and significance was established at p < 0.05.

Results

Our study revealed that across brain states, matDp/ + mice mirrored the elevated beta oscillation phenotype observed in clinical EEGs from individuals with Dup15q syndrome. Time to sleep onset after light onset was significantly reduced in matDp/ + and Ube3a OE mice. However, NREM sleep between Dup15q mutant and WT littermate mice remained unaltered, suggesting a divergence from the clinical presentation in humans. Additionally, while increased beta oscillations persisted in matDp/ + mice after 6-h of sleep deprivation, recovery NREM sleep remained unaltered in all groups, thus suggesting that these mice exhibit resilience in the fundamental processes governing sleep-wake regulation.

Conclusions

Quantification of mechanistic and translatable EEG biomarkers is essential for advancing our understanding of NDDs and their underlying pathophysiology. Our study of sleep physiology in the Dup15q mice underscores that the beta EEG biomarker has strong translational validity, thus opening the door for pre-clinical studies of putative drug targets, using the biomarker as a translational measure of drug-target engagement. The unaltered NREM sleep may be due to inherent differences in neurobiology between mice and humans. These nuanced distinctions highlight the complexity of sleep disruptions in Dup15q syndrome and emphasize the need for a comprehensive understanding that encompasses both shared and distinct features between murine models and clinical populations.