Non-rapid eye movement (NREM) sleep is not uniform but characterized by brief intrusions of wake-like brain activity, known as micro-arousals or brief awakenings. Although micro-arousals are an inherent feature of both human and animal sleep, knowledge about the neural correlates of micro-arousals across the cortical column is sparse. We here developed an algorithm for automatic detection of micro-arousals based on EMG activity and used it to explore how micro-arousals modulate laminar neural activity in the motor cortex, and how this relationship is affected by sleep pressure in mice. Our analysis showed that micro-arousals were associated with a general suppression of local field potential (LFP) power across the cortical column. Slow wave activity (SWA, 1-4 Hz) immediately after micro-arousals was tightly correlated with sleep pressure and even surpassed average SWA levels during NREM sleep in sleep deprived animals. In addition, analysis of single-channel firing showed that some channels exhibited increased activity immediately prior to micro-arousals, while others exhibited decreased activity. This study provides new insights into the neural mechanisms underlying micro-arousals and identifies a new link between micro-arousals and sleep homeostasis. ### Competing Interest Statement The authors have declared no competing interest.

Growing evidence suggests that brain activity during sleep, as well as sleep regulation, are tightly linked with synaptic function and network excitability at the local and global levels. We previously reported that a mutation in synaptobrevin 2 (Vamp2) in restless (rlss) mice results in a marked increase of wakefulness and suppression of sleep, in particular REM sleep (REMS), as well as increased consolidation of sleep and wakefulness. In this study, using finer-scale in vivo electrophysiology recordings, we report that spontaneous cortical activity in rlss mice during NREM sleep (NREMS) is characterized by an occurrence of abnormally prolonged periods of complete neuronal silence (OFF-periods), often lasting several seconds, similar to the burst suppression pattern typically seen under deep anesthesia. Increased incidence of prolonged network OFF-periods was not specific to NREMS but also present in REMS and wake in rlss mice. Slow-wave activity (SWA) was generally increased in rlss mice relative to controls, while higher frequencies, including theta-frequency activity, were decreased, further resulting in diminished differences between vigilance states. The relative increase in SWA after sleep deprivation was attenuated in rlss mice, suggesting either that rlss mice experience persistently elevated sleep pressure or, alternatively, that the intrusion of sleep-like patterns of activity into the wake state attenuates the accumulation of sleep drive. We propose that a deficit in global synaptic neurotransmitter release leads to "state inertia,"reflected in an abnormal propensity of brain networks to enter and remain in a persistent "default state"resembling coma or deep anesthesia.

Modulatory neurotransmitters exert powerful control over neurons and the brain vasculature. Gamma Entrainment Using Sensory Stimuli (GENUS) promotes amyloid clearance via increased perivascular cerebral spinal fluid (CSF) flux in mouse models of Alzheimer's Disease. Here we use whole-brain activity mapping to identify the cholinergic basal forebrain as a key region responding to GENUS. In line with this, GENUS promoted cortical acetylcholine release, vascular dilation, vasomotion and perivascular clearance. Inhibiting cholinergic signaling abolished the effects of GENUS, including the promotion of arterial pulsatility, periarterial CSF influx, and the reduction of cortical amyloid levels. Our findings establish cholinergic signaling as an essential component of the brain's ability to promote perivascular amyloid clearance via non-invasive sensory stimulation.

Here, we characterized the dynamics of sleep spindles, focusing on their damping, which we estimated using a metric called oscillatory-Quality (o-Quality), derived by fitting an autoregressive model to electrophysiological signals, recorded from the cortex in mice. The o-Quality of sleep spindles correlates weakly with their amplitude, shows marked laminar differences and regional topography across cortical regions, reflects the level of synchrony within and between cortical networks, is strongly modulated by sleep-wake history, reflects the degree of sensory disconnection, and correlates with the strength of coupling between spindles and slow waves. As most spindle events are highly localized and not detectable with conventional low-density recording approaches, o-Quality thus emerges as a valuable metric that allows us to infer the spread and dynamics of spindle activity across the brain and directly links their spatiotemporal dynamics with local and global regulation of brain states, sleep regulation, and function.

APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.

Patients with cancer undergoing chemotherapy frequently experience a neurological condition known as chemotherapy-related cognitive impairment, or “chemobrain,” which can persist for the remainder of their lives. Despite the growing prevalence of chemobrain, both its underlying mechanisms and treatment strategies remain poorly understood. Recent findings suggest that chemobrain shares several characteristics with neurodegenerative diseases, including chronic neuroinflammation, DNA damage, and synaptic loss. We investigated whether a noninvasive sensory stimulation treatment we term gamma entrainment using sensory stimuli (GENUS), which has been shown to alleviate aberrant immune and synaptic pathologies in mouse models of neurodegeneration, could also mitigate chemobrain phenotypes in mice administered a chemotherapeutic drug. When administered concurrently with the chemotherapeutic agent cisplatin, GENUS alleviated cisplatin-induced brain pathology, promoted oligodendrocyte survival, and improved cognitive function in a mouse model of chemobrain. These effects persisted for up to 105 days after GENUS treatment, suggesting the potential for long-lasting benefits. However, when administered to mice 90 days after chemotherapy, GENUS treatment only provided limited benefits, indicating that it was most effective when used to prevent the progression of chemobrain pathology. Furthermore, we demonstrated that the effects of GENUS in mice were not limited to cisplatin-induced chemobrain but also extended to methotrexate-induced chemobrain. Collectively, these findings suggest that GENUS may represent a versatile approach for treating chemobrain induced by different chemotherapy agents.

Electrophysiological recordings from freely behaving animals are a widespread and powerful mode of investigation in sleep research. These recordings generate large amounts of data that require sleep stage annotation (polysomnography), in which the data is parcellated according to three vigilance states: awake, rapid eye movement (REM) sleep, and non-REM (NREM) sleep. Manual and current computational annotation methods ignore intermediate states because the classification features become ambiguous, even though intermediate states contain important information regarding vigilance state dynamics. To address this problem, we have developed "Somnotate"-a probabilistic classifier based on a combination of linear discriminant analysis (LDA) with a hidden Markov model (HMM). First we demonstrate that Somnotate sets new standards in polysomnography, exhibiting annotation accuracies that exceed human experts on mouse electrophysiological data, remarkable robustness to errors in the training data, compatibility with different recording configurations, and an ability to maintain high accuracy during experimental interventions. However, the key feature of Somnotate is that it quantifies and reports the certainty of its annotations. We leverage this feature to reveal that many intermediate vigilance states cluster around state transitions, whereas others correspond to failed attempts to transition. This enables us to show for the first time that the success rates of different types of transition are differentially affected by experimental manipulations and can explain previously observed sleep patterns. Somnotate is open-source and has the potential to both facilitate the study of sleep stage transitions and offer new insights into the mechanisms underlying sleep-wake dynamics.

The glymphatic movement of fluid through the brain removes metabolic waste 1 – 4 . Noninvasive 40 Hz stimulation promotes 40 Hz neural activity in multiple brain regions and attenuates pathology in mouse models of Alzheimer’s disease 5 – 8 . Here we show that multisensory gamma stimulation promotes the influx of cerebrospinal fluid and the efflux of interstitial fluid in the cortex of the 5XFAD mouse model of Alzheimer’s disease. Influx of cerebrospinal fluid was associated with increased aquaporin-4 polarization along astrocytic endfeet and dilated meningeal lymphatic vessels. Inhibiting glymphatic clearance abolished the removal of amyloid by multisensory 40 Hz stimulation. Using chemogenetic manipulation and a genetically encoded sensor for neuropeptide signalling, we found that vasoactive intestinal peptide interneurons facilitate glymphatic clearance by regulating arterial pulsatility. Our findings establish novel mechanisms that recruit the glymphatic system to remove brain amyloid.

Sleep spindles are traditionally defined as 10-15Hz thalamo-cortical oscillations typical of NREM sleep. While substantial heterogeneity in the appearance or spatio-temporal dynamics of spindle events is well recognised, the physiological relevance of the underlying fundamental property - the oscillatory strength - has not been studied. Here we introduce a novel metric called oscillatory Quality ( o-Quality ), which is derived by fitting an auto-regressive model to short segments of electrophysiological signals, recorded from the cortex in mice, to identify and calculate the damping of spindle oscillations. We find that the o-Quality of spindles varies markedly across cortical layers and regions and reflects the level of synchrony within and between cortical networks. Furthermore, the o-Quality of spindles varies as a function of sleep-wake history, determines the strength of coupling between spindles and slow waves, and influences the responsiveness to sensory stimulation during sleep. Thus, the o-Quality emerges as a metric that, for the first time, directly links the spatio-temporal dynamics of sleep spindles with their functional role.

Findings by the Tsai lab and others 1–8 demonstrate that 40 Hz frequency sensory stimulation induces electrophysiological responses and attenuates pathology in mouse models of Alzheimer’s disease (AD). A recent study in Nature Neurosciene 9 concluded that the stimulation does not affect endogenous gamma oscillations or amyloid burden. We welcome research investigating 40 Hz sensory stimulation, and the article by Soula et al enhances our understanding of the brain’s electrophysiological response to 40Hz. However, we respectfully suggest that the data in Soula et al are consistent with a neuronal response to 40 Hz, which we further support with new data in humans. Moreover we contend the non-significant effects on amyloid are due to technical limitations of the study.

Abstract Background During non-rapid eye movement sleep (NREM), alternating periods of synchronised high (ON period) and low (OFF period) neuronal activity are associated with high amplitude delta band (0.5–4 Hz) oscillations in neocortical electrophysiological signals termed slow waves. As this oscillation is dependent crucially on hyperpolarisation of cortical cells, there is an interest in understanding how neuronal silencing during OFF periods leads to the generation of slow waves and whether this relationship changes between cortical layers. A formal, widely adopted definition of OFF periods is absent, complicating their detection. Here, we grouped segments of high frequency neural activity containing spikes, recorded as multiunit activity from the neocortex of freely behaving mice, on the basis of amplitude and asked whether the population of low amplitude (LA) segments displayed the expected characteristics of OFF periods. Results Average LA segment length was comparable to previous reports for OFF periods but varied considerably, from as short as 8 ms to > 1 s. LA segments were longer and occurred more frequently in NREM but shorter LA segments also occurred in half of rapid eye movement sleep (REM) epochs and occasionally during wakefulness. LA segments in all states were associated with a local field potential (LFP) slow wave that increased in amplitude with LA segment duration. We found that LA segments > 50 ms displayed a homeostatic rebound in incidence following sleep deprivation whereas short LA segments (< 50 ms) did not. The temporal organisation of LA segments was more coherent between channels located at a similar cortical depth. Conclusion We corroborate previous studies showing neural activity signals contain uniquely identifiable periods of low amplitude with distinct characteristics from the surrounding signal known as OFF periods and attribute the new characteristics of vigilance-state-dependent duration and duration-dependent homeostatic response to this phenomenon. This suggests that ON/OFF periods are currently underdefined and that their appearance is less binary than previously considered, instead representing a continuum.

Alzheimer's disease (AD) is the most common type of neurodegenerative disease and a health challenge with major social and economic consequences. In this review, we discuss the therapeutic potential of gamma stimulation in treating AD and delve into the possible mechanisms responsible for its positive effects. Recent studies reveal that it is feasible and safe to induce 40 Hz brain activity in AD patients through a range of 40 Hz multisensory and noninvasive electrical or magnetic stimulation methods. Although research into the clinical potential of these interventions is still in its nascent stages, these studies suggest that 40 Hz stimulation can yield beneficial effects on brain function, disease pathology, and cognitive function in individuals with AD. Specifically, we discuss studies involving 40 Hz light, auditory, and vibrotactile stimulation, as well as noninvasive techniques such as transcranial alternating current stimulation and transcranial magnetic stimulation. The precise mechanisms underpinning the beneficial effects of gamma stimulation in AD are not yet fully elucidated, but preclinical studies have provided relevant insights. We discuss preclinical evidence related to both neuronal and nonneuronal mechanisms that may be involved, touching upon the relevance of interneurons, neuropeptides, and specific synaptic mechanisms in translating gamma stimulation into widespread neuronal activity within the brain. We also explore the roles of microglia, astrocytes, and the vasculature in mediating the beneficial effects of gamma stimulation on brain function. Lastly, we examine upcoming clinical trials and contemplate the potential future applications of gamma stimulation in the management of neurodegenerative disorders.

The slow oscillation (SO) is a central neuronal dynamic during sleep and is generated by alternating periods of high and low neuronal activity (ON and OFF states). Mounting evidence causally links the SO to sleep’s functions, and it has recently become possible to manipulate the SO non-invasively and phase-specifically. These developments represent promising clinical avenues, but they also highlight the importance of improving our understanding of how ON/OFF states affect incoming stimuli and what role they play in neuronal plasticity. Most studies using closed-loop stimulation rely on the electroencephalogram (EEG) and local field potential (LFP) signals, which reflect neuronal ON and OFF states only indirectly. Here we develop an online detection algorithm based on spiking activity recorded from laminar arrays in mouse motor cortex. We find that online detection of ON and OFF states reflects specific phases of spontaneous LFP SO. Our neuronal-spiking-based closed-loop procedure offers a novel opportunity for testing the functional role of SO in sleep-related restorative processes and neural plasticity.

Cortical and subcortical circuitry are thought to play distinct roles in the generation of sleep oscillations and global state control, respectively. Here we silenced a subset of neocortical layer 5 pyramidal and archicortical dentate gyrus granule cells in male mice by ablating SNAP25. This markedly increased wakefulness and reduced rebound of electroencephalographic slow-wave activity after sleep deprivation, suggesting a role for the cortex in both vigilance state control and sleep homeostasis.

Sleep and wakefulness are not simple, homogenous all-or-none states but represent a spectrum of substates, distinguished by behavior, levels of arousal, and brain activity at the local and global levels. Until now, the role of the hypothalamic circuitry in sleep-wake control was studied primarily with respect to its contribution to rapid state transitions. In contrast, whether the hypothalamus modulates within-state dynamics (state "quality") and the functional significance thereof remains unexplored. Here, we show that photoactivation of inhibitory neurons in the lateral preoptic area (LPO) of the hypothalamus of adult male and female laboratory mice does not merely trigger awakening from sleep, but the resulting awake state is also characterized by an activated electroencephalogram (EEG) pattern, suggesting increased levels of arousal. This was associated with a faster build-up of sleep pressure, as reflected in higher EEG slow-wave activity (SWA) during subsequent sleep. In contrast, photoinhibition of inhibitory LPO neurons did not result in changes in vigilance states but was associated with persistently increased EEG SWA during spontaneous sleep. These findings suggest a role of the LPO in regulating arousal levels, which we propose as a key variable shaping the daily architecture of sleep-wake states.

Central serotonin (5-HT) orchestrates myriad cognitive processes and lies at the core of many stress-related psychiatric illnesses. However, the basic relationship between its brain-wide axonal projections and functional dynamics is not known. Here we combine optogenetics and fMRI to produce a brain-wide 5-HT evoked functional map. We find that DRN photostimulation leads to an increase in the hemodynamic response in the DRN itself, while projection areas predominately exhibit a reduction of cerebral blood volume mirrored by suppression of cortical delta oscillations. We find that the regional distribution of post-synaptically expressed 5-HT receptors better correlates with DRN 5-HT functional connectivity than anatomical projections. Our work suggests that neuroarchitecture is not the primary determinant of function for the DRN 5-HT. With respect to two 5-HT elevating stimuli, we find that acute stress leads to circuit-wide blunting of the DRN output, while the SSRI fluoxetine noticeably enhances DRN functional connectivity. These data provide fundamental insight into the brain-wide functional dynamics of the 5-HT projection system.

Manual sleep stage annotation is a time-consuming but often essential step in the analysis of sleep data. To address this bottleneck several algorithms have been proposed that automate this process, reporting performance levels that are on par with manual annotation according to measures of inter-rater agreement. Here we first demonstrate that inter-rater agreement can provide a biased and imprecise measure of annotation quality. We therefore develop a principled framework for assessing performance against a consensus annotation derived from multiple experienced sleep researchers. We then construct a new sleep stage classifier that combines automated feature extraction using linear discriminant analysis with inference based on vigilance state-dependent contextual information using a hidden Markov model. This produces automated annotation accuracies that exceed expert performance on rodent electrophysiological data. Furthermore, our classifier is shown to be robust to errors in the training data, robust to experimental manipulations, and compatible with different recording configurations. Finally, we demonstrate that the classifier identifies both successful and failed attempts to transition between vigilance states, which may offer new insights into the occurrence of short awake periods between REM and NREM sleep. We call our classifier ‘Somnotate’ and make an implementation available to the neuroscience community.