Sleep and wake affect glycogen content and turnover at perisynaptic astrocytic processes

Astrocytic glycogen represents the only form of glucose storage in the brain, and one of the outcomes of its breakdown is the production of lactate that can be used by neurons as an alternative energetic substrate. Since brain metabolism is higher in wake than in sleep, it was hypothesized that glycogen stores are depleted during wake and replenished during sleep. Furthermore, it was proposed that glycogen depletion leads to the progressive increase in adenosine levels during wake, providing a homeostatic signal that reflects the buildup of sleep pressure. However, previous studies that measured glycogen dynamics across the sleep/wake cycle obtained inconsistent results, and only measured glycogen in whole tissue. Since most energy in the brain is used to sustain synaptic activity, here we employed tridimensional electron microscopy to quantify glycogen content in the astrocytic processes surrounding the synapse. We studied axon-spine synapses in the frontal cortex of young mice after ~7 h of sleep, 7–8 h of spontaneous or forced wake, or 4.5 days of sleep restriction. Relative to sleep, all wake conditions increased the number of glycogen granules around the synapses to a similar extent. However, progressively longer periods of wake were associated with progressively smaller glycogen granules, suggesting increased turnover. Despite the increased number of granules, in all wake conditions the estimated amount of glucose within the granules was lower than in sleep, indicating that sleep may favor glucose storage. Finally, chronic sleep restriction moved glycogen granules closer to the synaptic cleft. Thus, both short and long wake lead to increased glycogen turnover around cortical synapses, whereas sleep promotes glycogen accumulation

Higher arc nucleus-to-cytoplasm ratio during sleep in the superficial layers of the mouse cortex

The activity-regulated cytoskeleton associated protein Arc is strongly and quickly upregulated by neuronal activity, synaptic potentiation and learning. Arc entry in the synapse is followed by the endocytosis of glutamatergic AMPA receptors (AMPARs), and its nuclear accumulation has been shown in vitro to result in a small decline in the transcription of the GluA1 subunit of AMPARs. Since these effects result in a decline in synaptic strength, we asked whether a change in Arc dynamics may temporally correlate with sleep-dependent GluA1 down-regulation. We measured the ratio of nuclear to cytoplasmic Arc expression (Arc Nuc/Cyto) in the cerebral cortex of EGFP-Arc transgenic mice that were awake most of the night and then perfused immediately before lights on (W mice), or were awake most of the night and then allowed to sleep (S mice) or sleep deprived (SD mice) for the first 2 h of the light phase. In primary motor cortex (M1), neurons with high levels of nuclear Arc (High Arc cells) were present in all mice, but in these cells Arc Nuc/Cyto was higher in S mice than in W mice and, importantly, ~15% higher in S mice than in SD mice collected at the same time of day, ruling out circadian effects. Greater Arc Nuc/Cyto with sleep was observed in the superficial layers of M1, but not in the deep layers. In High Arc cells, Arc Nuc/Cyto was also ~15%–30% higher in S mice than in W and SD mice in the superficial layers of primary somatosensory cortex (S1) and cingulate cortex area 1 (Cg1). In High Arc Cells of Cg1, Arc Nuc/Cyto and cytoplasmic levels of GluA1 immunoreactivities in the soma were also negatively correlated, independent of behavioral state. Thus, Arc moves to the nucleus during both sleep and wake, but its nuclear to cytoplasmic ratio increases with sleep in the superficial layers of several cortical areas. It remains to be determined whether the relative increase in nuclear Arc contributes significantly to the overall decline in the strength of excitatory synapses that occurs during sleep. Similarly, it remains to be determined whether the entry of Arc into specific synapses is gated by sleep

Contribution of sleep to the repair of neuronal DNA double-strand breaks:evidence from flies and mice

Exploration of a novel environment leads to neuronal DNA double-strand breaks (DSBs). These DSBs are generated by type 2 topoisomerase to relieve topological constrains that limit transcription of plasticity-related immediate early genes. If not promptly repaired, however, DSBs may lead to cell death. Since the induction of plasticity-related genes is higher in wake than in sleep, we asked whether it is specifically wake associated with synaptic plasticity that leads to DSBs, and whether sleep provides any selective advantage over wake in their repair. In flies and mice, we find that enriched wake, more than simply time spent awake, induces DSBs, and their repair in mice is delayed or prevented by subsequent wake. In both species the repair of irradiation-induced neuronal DSBs is also quicker during sleep, and mouse genes mediating the response to DNA damage are upregulated in sleep. Thus, sleep facilitates the repair of neuronal DSBs

Sleep Deprivation by Exposure to Novel Objects Increases Synapse Density and Axon-Spine Interface in the Hippocampal CA1 Region of Adolescent 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.SIGNIFICANCE STATEMENT 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

Lempel-Ziv Complexity Analysis of Local Field Potentials in Different Vigilance States with Different Coarse-Graining Techniques

Analysis of electrophysiological signals recorded from the brain with Lempel-Ziv (LZ) complexity, a measure based on coarse-graining of the signal, can provide valuable insights into understanding brain activity. LZ complexity of local field potential signals recorded from the neocortex of 11 adult male Wistar-Kyoto rats in different vigilance states - waking, non-rapid-eye movement (NREM) and REM sleep - was estimated with different coarse-graining techniques (median, LZCm, and k-means, LZCkm). Furthermore, surrogate data were used to test the hypothesis that LZ complexity results reveal effects accounted for by temporal structure of the signal, rather than merely its frequency content. LZ complexity values were significantly lower in NREM sleep as compared to waking and REM sleep, for both real and surrogate signals. LZCkm and LZCm values were similar, although in NREM sleep the values deviated in some epochs, where signals also differed significantly in terms of temporal structure and spectral content. Thus, the interpretation of LZ complexity results should take into account the specific algorithm used to coarse-grain the signal. Moreover, the occurrence of high amplitude slow waves during NREM sleep determines LZ complexity to a large extent, but characteristics such as the temporal sequence of slow waves or cross-frequency interactions might also play a role. © Springer International Publishing Switzerland 2014

Ultrastructural evidence for synaptic scaling across the wake/sleep cycle.

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 6,920 synapses in mouse motor and sensory cortices using 3D-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

Metabolomic analysis of mouse prefrontal cortex reveals upregulated analytes during wakefulness compared to sleep

By identifying endogenous molecules in brain extracellular fluid metabolomics can provide insight into the regulatory mechanisms and functions of sleep. Here we studied how the cortical metabolome changes during sleep, sleep deprivation and spontaneous wakefulness. Mice were implanted with electrodes for chronic sleep/wake recording and with microdialysis probes targeting prefrontal and primary motor cortex. Metabolites were measured using ultra performance liquid chromatography-high resolution mass spectrometry. Sleep/wake changes in metabolites were evaluated using partial least squares discriminant analysis, linear mixed effects model analysis of variance, and machine-learning algorithms. More than 30 known metabolites were reliably detected in most samples. When used by a logistic regression classifier, the profile of these metabolites across sleep, spontaneous wake, and enforced wake was sufficient to assign mice to their correct experimental group (pair-wise) in 80–100% of cases. Eleven of these metabolites showed significantly higher levels in awake than in sleeping mice. Some changes extend previous findings (glutamate, homovanillic acid, lactate, pyruvate, tryptophan, uridine), while others are novel (D-gluconate, N-acetyl-beta-alanine, N-acetylglutamine, orotate, succinate/methylmalonate). The upregulation of the de novo pyrimidine pathway, gluconate shunt and aerobic glycolysis may reflect a wake-dependent need to promote the synthesis of many essential components, from nucleic acids to synaptic membranes

Evidence for sleep-dependent synaptic renormalization in mouse pups

In adolescent and adult brains several molecular, electrophysiological, and ultrastructural measures of synaptic strength are higher after wake than after sleep [1, 2]. These results support the proposal that a core function of sleep is to renormalize the increase in synaptic strength associated with ongoing learning during wake, to reestablish cellular homeostasis and avoid runaway potentiation, synaptic saturation, and memory interference [2, 3]. Before adolescence however, when the brain is still growing and many new synapses are forming, sleep is widely believed to promote synapse formation and growth. To assess the role of sleep on synapses early in life, we studied 2-week-old mouse pups (both sexes) whose brain is still undergoing significant developmental changes, but in which sleep and wake are easy to recognize. In two strains (CD-1, YFP-H) we found that pups spend ~50% of the day asleep and show an immediate increase in total sleep duration after a few hours of enforced wake, indicative of sleep homeostasis. In YFP-H pups we then used serial block-face electron microscopy to examine whether the axon-spine interface (ASI), an ultrastructural marker of synaptic strength, changes between wake and sleep. We found that the ASI of cortical synapses (layer 2, motor cortex) was on average 33.9% smaller after sleep relative to after extended wake and the differences between conditions were consistent with multiplicative scaling. Thus, the need for sleep-dependent synaptic renormalization may apply also to the young, pre-weaned cerebral cortex, at least in the superficial layers of the primary motor area