It is assumed that synaptic strengthening and weakening balance throughout learning to avoid runaway potentiation and memory interference. However, energetic and informational considerations suggest ...that potentiation should occur primarily during wake, when animals learn, and depression should occur during sleep. We measured 6920 synapses in mouse motor and sensory cortices using three-dimensional electron microscopy. The axon-spine interface (ASI) decreased ~18% after sleep compared with wake. This decrease was proportional to ASI size, which is indicative of scaling. Scaling was selective, sparing synapses that were large and lacked recycling endosomes. Similar scaling occurred for spine head volume, suggesting a distinction between weaker, more plastic synapses (~80%) and stronger, more stable synapses. These results support the hypothesis that a core function of sleep is to renormalize overall synaptic strength increased by wake.
We previously found that
and its ligand
, astrocytic genes involved in phagocytosis, are upregulated after acute sleep deprivation. These results suggested that astrocytes may engage in phagocytic ...activity during extended wake, but direct evidence was lacking. Studies in humans and rodents also found that sleep loss increases peripheral markers of inflammation, but whether these changes are associated with neuroinflammation and/or activation of microglia, the brain's resident innate immune cells, was unknown. Here we used serial block-face scanning electron microscopy to obtain 3D volume measurements of synapses and surrounding astrocytic processes in mouse frontal cortex after 6-8 h of sleep, spontaneous wake, or sleep deprivation (SD) and after chronic (∼5 d) sleep restriction (CSR). Astrocytic phagocytosis, mainly of presynaptic components of large synapses, increased after both acute and chronic sleep loss relative to sleep and wake. MERTK expression and lipid peroxidation in synaptoneurosomes also increased to a similar extent after short and long sleep loss, suggesting that astrocytic phagocytosis may represent the brain's response to the increase in synaptic activity associated with prolonged wake, clearing worn components of heavily used synapses. Using confocal microscopy, we then found that CSR but not SD mice show morphological signs of microglial activation and enhanced microglial phagocytosis of synaptic elements, without obvious signs of neuroinflammation in the CSF. Because low-level sustained microglia activation can lead to abnormal responses to a secondary insult, these results suggest that chronic sleep loss, through microglia priming, may predispose the brain to further damage.
We find that astrocytic phagocytosis of synaptic elements, mostly of presynaptic origin and in large synapses, is upregulated already after a few hours of sleep deprivation and shows a further significant increase after prolonged and severe sleep loss, suggesting that it may promote the housekeeping of heavily used and strong synapses in response to the increased neuronal activity of extended wake. By contrast, chronic sleep restriction but not acute sleep loss activates microglia, promotes their phagocytic activity, and does so in the absence of overt signs of neuroinflammation, suggesting that like many other stressors, extended sleep disruption may lead to a state of sustained microglia activation, perhaps increasing the brain's susceptibility to other forms of damage.
Astrocytes can mediate neurovascular coupling, modulate neuronal excitability, and promote synaptic maturation and remodeling. All these functions are likely to be modulated by the sleep/wake cycle, ...because brain metabolism, neuronal activity and synaptic turnover change as a function of behavioral state. Yet, little is known about the effects of sleep and wake on astrocytes.
Here we show that sleep and wake strongly affect both astrocytic gene expression and ultrastructure in the mouse brain. Using translating ribosome affinity purification technology and microarrays, we find that 1.4 % of all astrocytic transcripts in the forebrain are dependent on state (three groups, sleep, wake, short sleep deprivation; six mice per group). Sleep upregulates a few select genes, like Cirp and Uba1, whereas wake upregulates many genes related to metabolism, the extracellular matrix and cytoskeleton, including Trio, Synj2 and Gem, which are involved in the elongation of peripheral astrocytic processes. Using serial block face scanning electron microscopy (three groups, sleep, short sleep deprivation, chronic sleep restriction; three mice per group, >100 spines per mouse, 3D), we find that a few hours of wake are sufficient to bring astrocytic processes closer to the synaptic cleft, while chronic sleep restriction also extends the overall astrocytic coverage of the synapse, including at the axon-spine interface, and increases the available astrocytic surface in the neuropil.
Wake-related changes likely reflect an increased need for glutamate clearance, and are consistent with an overall increase in synaptic strength when sleep is prevented. The reduced astrocytic coverage during sleep, instead, may favor glutamate spillover, thus promoting neuronal synchronization during non-rapid eye movement sleep.
During non-rapid eye-movement (NREM) sleep, cortical and thalamic neurons oscillate every second or so between ON periods, characterized by membrane depolarization and wake-like tonic firing, and OFF ...periods, characterized by membrane hyperpolarization and neuronal silence. Cortical slow waves, the hallmark of NREM sleep, reflect near-synchronous OFF periods in cortical neurons. However, the mechanisms triggering such OFF periods are unclear, as there is little evidence for somatic inhibition. We studied cortical inhibitory interneurons that express somatostatin (SOM), because ∼70% of them are Martinotti cells that target diffusely layer I and can block excitatory transmission presynaptically, at glutamatergic terminals, and postsynaptically, at apical dendrites, without inhibiting the soma. In freely moving male mice, we show that SOM+ cells can fire immediately before slow waves and their optogenetic stimulation during ON periods of NREM sleep triggers long OFF periods. Next, we show that chemogenetic activation of SOM+ cells increases slow-wave activity (SWA), slope of individual slow waves, and NREM sleep duration; whereas their chemogenetic inhibition decreases SWA and slow-wave incidence without changing time spent in NREM sleep. By contrast, activation of parvalbumin+ (PV+) cells, the most numerous population of cortical inhibitory neurons, greatly decreases SWA and cortical firing, triggers short OFF periods in NREM sleep, and increases NREM sleep duration. Thus SOM+ cells, but not PV+ cells, are involved in the generation of sleep slow waves. Whether Martinotti cells are solely responsible for this effect, or are complemented by other classes of inhibitory neurons, remains to be investigated.
Cortical slow waves are a defining feature of non-rapid eye-movement (NREM) sleep and are thought to be important for many of its restorative benefits. Yet, the mechanism by which cortical neurons abruptly and synchronously cease firing, the neuronal basis of the slow wave, remains unknown. Using chemogenetic and optogenetic approaches, we provide the first evidence that links a specific class of inhibitory interneurons-somatostatin-positive cells-to the generation of slow waves during NREM sleep in freely moving mice.
Sleep has been hypothesized to rebalance overall synaptic strength after ongoing learning during waking leads to net synaptic potentiation. If so, because synaptic strength and size are correlated, ...synapses on average should be larger after wake and smaller after sleep. This prediction was recently confirmed in mouse cerebral cortex using serial block-face electron microscopy (SBEM). However, whether these findings extend to other brain regions is unknown. Moreover, sleep deprivation by gentle handling was reported to produce hippocampal spine loss, raising the question of whether synapse size and number are differentially affected by sleep and waking. Here we applied SBEM to measure axon-spine interface (ASI), the contact area between pre-synapse and post-synapse, and synapse density in CA1 stratum radiatum. Adolescent YFP-H mice were studied after 6-8 h of sleep (S = 6), spontaneous wake at night (W = 4) or wake enforced during the day by novelty exposure (EW = 4; males/females balanced). In each animal ≥425 ASIs were measured and synaptic vesicles were counted in ~100 synapses/mouse. Reconstructed dendrites included many small, nonperforated synapses and fewer large, perforated synapses. Relative to S, ASI sizes in perforated synapses shifted toward higher values after W and more so after EW. ASI sizes in nonperforated synapses grew after EW relative to S and W, and so did their density. ASI size correlated with presynaptic vesicle number but the proportion of readily available vesicles decreased after EW, suggesting presynaptic fatigue. Thus, CA1 synapses undergo changes consistent with sleep-dependent synaptic renormalization and their number increases after extended wake.
Sleep benefits learning, memory consolidation, and the integration of new with old memories, but the underlying mechanisms remain highly debated. One hypothesis suggests that sleep's cognitive benefits stem from its ability to renormalize total synaptic strength, after ongoing learning during wake leads to net synaptic potentiation. Supporting evidence for this hypothesis mainly comes from the cerebral cortex, including the observation that cortical synapses are larger after wake and smaller after sleep. Using serial electron microscopy, we find here that sleep/wake synaptic changes consistent with sleep-dependent synaptic renormalization also occur in the CA1 region. Thus, the role of sleep in maintaining synaptic homeostasis may extend to the hippocampus, a key area for learning and synaptic plasticity.
The activity of the noradrenergic system of the locus coeruleus (LC) is high in wake and low in sleep. LC promotes arousal and EEG activation, as well as attention, working memory, and cognitive ...flexibility. These functions rely on prefrontal cortex and are impaired by sleep deprivation, but the extent to which LC activity changes during wake remains unclear. Moreover, it is unknown whether noradrenergic neurons can sustain elevated firing during extended wake. Recent studies show that relative to LC neurons targeting primary motor cortex (M1), those projecting to medial prefrontal cortex (mPFC) have higher spontaneous firing rates and are more excitable. These results suggest that noradrenaline (NA) levels should be higher in mPFC than M1, and that during prolonged wake LC cells targeting mPFC may fatigue more, but direct evidence is lacking.
We performed in vivo microdialysis experiments in adult (9-10 weeks old) C57BL/6 mice implanted for chronic electroencephalographic recordings. Cortical NA levels were measured during spontaneous sleep and wake (n = 8 mice), and in the course of sleep deprivation (n = 6).
We found that absolute NA levels are higher in mPFC than in M1. Moreover, in both areas they decline during sleep and increase during wake, but these changes are faster in M1 than mPFC. Finally, by the end of sleep deprivation NA levels decline only in mPFC.
Locus coeruleus (LC) neurons targeting prefrontal cortex may fatigue more markedly, or earlier, than other LC cells, suggesting one of the mechanisms underlying the cognitive impairment and the increased sleep presure associated with sleep deprivation.
A commentary on this article appears in this issue on page 11.
Insufficient sleep is a serious public health problem in modern society. It leads to increased risk of chronic diseases, and it has been frequently associated with cellular oxidative damage and ...widespread low-grade inflammation. Probiotics have been attracting increasing interest recently for their antioxidant and anti-inflammatory properties. Here, we tested the ability of probiotics to contrast oxidative stress and inflammation induced by sleep loss.
We administered a multi-strain probiotic formulation (SLAB51) or water to normal sleeping mice and to mice exposed to 7 days of chronic sleep restriction (CSR). We quantified protein, lipid, and DNA oxidation as well as levels of gut-brain axis hormones and pro and anti-inflammatory cytokines in the brain and plasma. Furthermore, we carried out an evaluation of microglia morphology and density in the mouse cerebral cortex.
We found that CSR induced oxidative stress and inflammation and altered gut-brain axis hormones. SLAB51 oral administration boosted the antioxidant capacity of the brain, thus limiting the oxidative damage provoked by loss of sleep. Moreover, it positively regulated gut-brain axis hormones and reduced peripheral and brain inflammation induced by CSR.
Probiotic supplementation can be a possible strategy to counteract oxidative stress and inflammation promoted by sleep loss.
Recent work has demonstrated that behavioral manipulations targeting specific cortical areas during prolonged wakefulness lead to a region-specific homeostatic increase in theta activity (5-9 Hz), ...suggesting that theta waves could represent transient neuronal OFF periods (local sleep). In awake rats, the occurrence of an OFF period in a brain area relevant for behavior results in performance errors. Here we investigated the potential relationship between local sleep events and negative behavioral outcomes in humans. Volunteers participated in two prolonged wakefulness experiments (24 h), each including 12 h of practice with either a driving simulation (DS) game or a battery of tasks based on executive functions (EFs). Multiple high-density EEG recordings were obtained during each experiment, both in quiet rest conditions and during execution of two behavioral tests, a response inhibition test and a motor test, aimed at assessing changes in impulse control and visuomotor performance, respectively. In addition, fMRI examinations obtained at 12 h intervals were used to investigate changes in inter-regional connectivity. The EF experiment was associated with a reduced efficiency in impulse control, whereas DS led to a relative impairment in visuomotor control. A specific spatial and temporal correlation was observed between EEG theta waves occurring in task-related areas and deterioration of behavioral performance. The fMRI connectivity analysis indicated that performance impairment might partially depend on a breakdown in connectivity determined by a "network overload." Present results demonstrate the existence of an association between theta waves during wakefulness and performance errors and may contribute explaining behavioral impairments under conditions of sleep deprivation/restriction.
Even modest sleep restriction, especially the loss of sleep slow wave activity (SWA), is invariably associated with slower electroencephalogram (EEG) activity during wake, the occurrence of local ...sleep in an otherwise awake brain, and impaired performance due to cognitive and memory deficits. Recent studies not only confirm the beneficial role of sleep in memory consolidation, but also point to a specific role for sleep slow waves. Thus, the implementation of methods to enhance sleep slow waves without unwanted arousals or lightening of sleep could have significant practical implications. Here we first review the evidence that it is possible to enhance sleep slow waves in humans using transcranial direct-current stimulation (tDCS) and transcranial magnetic stimulation. Since these methods are currently impractical and their safety is questionable, especially for chronic long-term exposure, we then discuss novel data suggesting that it is possible to enhance slow waves using sensory stimuli. We consider the physiology of the K-complex (KC), a peripheral evoked slow wave, and show that, among different sensory modalities, acoustic stimulation is the most effective in increasing the magnitude of slow waves, likely through the activation of non-lemniscal ascending pathways to the thalamo-cortical system. In addition, we discuss how intensity and frequency of the acoustic stimuli, as well as exact timing and pattern of stimulation, affect sleep enhancement. Finally, we discuss automated algorithms that read the EEG and, in real-time, adjust the stimulation parameters in a closed-loop manner to obtain an increase in sleep slow waves and avoid undesirable arousals. In conclusion, while discussing the mechanisms that underlie the generation of sleep slow waves, we review the converging evidence showing that acoustic stimulation is safe and represents an ideal tool for slow wave sleep (SWS) enhancement.
Multiple evidence in rodents shows that the strength of excitatory synapses in the cerebral cortex and hippocampus is greater after wake than after sleep. The widespread synaptic weakening afforded ...by sleep is believed to keep the cost of synaptic activity under control, promote memory consolidation, and prevent synaptic saturation, thus preserving the brain's ability to learn day after day. The cerebellum is highly plastic and the Purkinje cells, the sole output neurons of the cerebellar cortex, are endowed with a staggering number of excitatory parallel fiber synapses. However, whether these synapses are affected by sleep and wake is unknown. Here, we used serial block face scanning electron microscopy to obtain the full 3D reconstruction of more than 7000 spines and their parallel fiber synapses in the mouse posterior vermis. This analysis was done in mice whose cortical and hippocampal synapses were previously measured, revealing that average synaptic size was lower after sleep compared to wake with no major changes in synapse number. Here, instead, we find that while the average size of parallel fiber synapses does not change, the number of branched synapses is reduced in half after sleep compared to after wake, corresponding to ~16% of all spines after wake and ~8% after sleep. Branched synapses are harbored by two or more spines sharing the same neck and, as also shown here, are almost always contacted by different parallel fibers. These findings suggest that during wake, coincidences of firing over parallel fibers may translate into the formation of synapses converging on the same branched spine, which may be especially effective in driving Purkinje cells to fire. By contrast, sleep may promote the off-line pruning of branched synapses that were formed due to spurious coincidences.
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