Sleep disruption, oxidative stress, and aging: new insights from fruit flies.
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Aging↗
Biomedical subjects
Chiara Cirelli
Publications and source records attributed to Chiara Cirelli.
Cellular consequences of sleep deprivation in the brain.
Several recent studies have used transcriptomics approaches to characterize the molecular correlates of sleep, waking, and sleep deprivation. This analysis may help in understanding the benefits that sleep brings to the brain at the cellular level. The studies are still limited in number and focus on a few brain regions, but some consistent findings are emerging. Sleep, spontaneous wakefulness, short-term, and long-term sleep deprivation are each associated with the upregulation of hundreds of genes in the cerebral cortex and other brain areas. In fruit flies as well as in mammals, three categories of genes are consistently upregulated during waking and short-term sleep deprivation relative to sleep. They include genes involved in energy metabolism, synaptic potentiation, and the response to cellular stress. In the rat cerebral cortex, transcriptional changes associated with prolonged sleep loss differ significantly from those observed during short-term sleep deprivation. However, it is too early to draw firm conclusions relative to the molecular consequences of sleep deprivation, and more extensive studies using DNA and protein arrays are needed in different species and in different brain regions.
Brain↗
Changes in brain gene expression after long-term sleep deprivation.
Long-term sleep deprivation in rats produces dramatic physiological changes including increase in energy expenditure, decrease in body weight, and death after 2-3 weeks. Despite several studies, the sleep deprivation syndrome remains largely unexplained. Here, to elucidate how prolonged sleep loss affects brain cells we used microarrays and screened the expression of > 26 000 transcripts in the cerebral cortex. Rats were sleep deprived using the disk-over-water method for 1 week. Seventy-five transcripts showed increased expression in these animals relative to controls that had been spontaneously awake or sleep deprived for a few hours. Most of them were induced as a result of chronic sleep loss and not non-specific effects of the disk stimulation. They include transcripts coding for several immunoglobulins, stress response proteins (macrophage inhibitor factor-related protein 14, heat-shock protein 27, alpha-B-crystallin), minoxidil sulfotransferase, globins and cortistatin. Twenty-eight transcripts decreased their expression in long-term sleep-deprived rats. Sixteen of them were specifically decreased as a result of chronic sleep loss, including those coding for type I procollagen and dihydrolipoamide acetyltransferase. We also compared sleeping rats to short-term and long-term sleep-deprived rats, and found that acute and chronic sleep loss led to some differences at the molecular level. Several plasticity-related genes were strongly induced after acute sleep deprivation only, and several glial genes were down-regulated in both sleep deprivation conditions, but to a different extent. These findings suggest that sustained sleep loss may trigger a generalized inflammatory and stress response in the brain.
Animals↗
Sleep function and synaptic homeostasis.
This paper reviews a novel hypothesis about the functions of slow wave sleep-the synaptic homeostasis hypothesis. According to the hypothesis, plastic processes occurring during wakefulness result in a net increase in synaptic strength in many brain circuits. The role of sleep is to downscale synaptic strength to a baseline level that is energetically sustainable, makes efficient use of gray matter space, and is beneficial for learning and memory. Thus, sleep is the price we have to pay for plasticity, and its goal is the homeostatic regulation of the total synaptic weight impinging on neurons. The hypothesis accounts for a large number of experimental facts, makes several specific predictions, and has implications for both sleep and mood disorders.
Brain↗
Sleep and wakefulness modulate gene expression in Drosophila.
In the mammalian brain, sleep and wakefulness are associated with widespread changes in gene expression. Sleep in fruit flies shares many features with mammalian sleep, but it is currently unknown to what extent behavioral states affect gene expression in Drosophila. To find out, we performed a comprehensive microarray analysis of gene expression in spontaneously awake, sleep-deprived and sleeping flies. Fly heads were collected at 4 am, after 8 h of spontaneous sleep or sleep deprivation, and at 4 pm, after 8 h of spontaneous wakefulness. As in rats, we found that behavioral state and time of day affect Drosophila gene expression to a comparable extent. As in rats, transcripts with higher expression in wakefulness and in sleep belong to different functional categories, and in several cases these groups overlap with those previously identified in rats. Wakefulness-related genes code for transcription factors and for proteins involved in the stress response, immune response, glutamatergic transmission, and carbohydrate metabolism. Sleep-related transcripts include the glial gene anachronism and several genes involved in lipid metabolism. Finally, the expression of many wakefulness-related and sleep-related Drosophila transcripts is also modulated by the time of day, suggesting an interaction at the molecular level between circadian and homeostatic mechanism of sleep regulation.
Animals↗
Locus ceruleus control of slow-wave homeostasis.
Sleep intensity is regulated by the duration of previous wakefulness, suggesting that waking results in the progressive accumulation of sleep need (Borbely and Achermann, 2000). In mammals, sleep intensity is reflected by slow-wave activity (SWA) in the nonrapid eye movement (NREM) sleep electroencephalogram, which increases in proportion to the time spent awake. However, the mechanisms responsible for the increase of NREM SWA after wakefulness remain unclear. According to a recent hypothesis (Tononi and Cirelli, 2003), the increase in SWA occurs because during wakefulness, many cortical circuits undergo synaptic potentiation, as evidenced by the widespread induction of long-term potentiation (LTP)-related genes in the brain of awake animals. A direct prediction of this hypothesis is that manipulations interfering with the induction of LTP-related genes should result in a blunted SWA response. Here, we examined SWA response in rats in which cortical norepinephrine (NA) was depleted, a manipulation that greatly reduces the induction of LTP-related genes during wakefulness (Cirelli and Tononi, 2004). We found that the homeostatic response of the lower-range SWA was markedly and specifically reduced after NA depletion. These data suggest that the wake-dependent accumulation of sleep need is causally related to cellular changes dependent on NA release, such as the induction of LTP-related genes, and support the hypothesis that sleep SWA homeostasis may be related to synaptic potentiation during wakefulness.
Analysis of Variance↗
Reduced sleep in Drosophila Shaker mutants.
Most of us sleep 7-8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3-4 h of sleep-a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.
Amino Acid Sequence↗
A molecular window on sleep: changes in gene expression between sleep and wakefulness.
Sleep is thought to be "by the brain and for the brain," but despite decades of behavioral and neurophysiologic research, we still do not know why the brain actually needs to sleep. Recently, gene expression studies have allowed researchers to investigate the molecular correlates of sleep and wakefulness and to gain new insights into the benefits that sleep may bring at the cellular level. In the latest series of studies, a genome-wide screening of brain gene expression was performed in rats that had been asleep, spontaneously awake, or sleep deprived for 8 hours. It was found that of approximately 15,000 transcripts expressed in the cerebral cortex, about 5% change their expression levels depending on behavioral state but independently of time of day. Half of the modulated genes increase in wakefulness and half in sleep. Moreover, wakefulness-related and sleep-related transcripts belong to different functional categories. Waking-related transcripts are involved in energy metabolism, excitatory neurotransmission, transcriptional activation, synaptic potentiation and memory acquisition, and the response to cellular stress. Sleep-related transcripts are involved in brain protein synthesis, synaptic consolidation/depression, and membrane trafficking and maintenance, including cholesterol metabolism, myelin formation, and synaptic vesicle turnover.
Animals↗
Chlorotoxin-mediated disinhibition of noradrenergic locus coeruleus neurons using a conditional transgenic approach.
The noradrenergic locus coeruleus (LC) has been implicated in the promotion of arousal, in focused attention and learning, and in the regulation of the sleep/waking cycle. The complex biological functions of the central noradrenergic system have been investigated largely through electrophysiological recordings and neurotoxic lesions of LC neurons. Activation of LC neurons through electrical or chemical stimulation has also led to important insights, although these techniques have limited cellular specificity and short-term effects. Here, we describe a novel method aimed at stimulating the central noradrenergic system in a highly selective manner for prolonged periods of time. This was achieved through the conditional expression of a transgene for chlorotoxin (Cltx) in the LC of adult mice. Chlorotoxin is a component of scorpion venom that partially blocks small conductance chloride channels. In this manner, the influence of GABAergic and glycinergic inhibitory inputs on LC cells is greatly reduced, while their ability to respond to excitatory inputs is unaffected. We demonstrate that the unilateral induction of Cltx expression in the LC is associated with a concomitant ipsilateral increase in the expression of markers of noradrenergic activity in LC neurons. Moreover, LC disinhibition is associated with the ipsilateral induction of the immediate early gene NGFI-A in cortical and subcortical target areas. Unlike previous gain of function approaches, transgenic disinhibition of LC cells is highly selective and persists for at least several weeks. This method represents a powerful new tool to assess the long-term effects of LC activation and is potentially applicable to other neuronal systems.
Adenoviridae↗
Sleep homeostasis in Drosophila melanogaster.
STUDY OBJECTIVES: The fruit fly Drosophila melanogaster is emerging as a promising model system for the genetic dissection of sleep. As in mammals, sleep in the fruit fly is a reversible state of reduced responsiveness to the external world and has been defined using an array of behavioral, pharmacologic, molecular, and electrophysiologic criteria. A central feature of mammalian sleep is its homeostatic regulation by the amount of previous wakefulness. Dissecting the mechanisms of homeostatic regulation is likely to provide key insights into the functions of sleep. Thus, it is important to establish to what extent sleep homeostasis is similar between mammals and flies. This study was designed to determine whether in flies, as in mammals, (1) sleep rebound is dependent on prior time awake; (2) sleep deprivation affects the intensity, in addition to the duration, of sleep rebound; (3) sleep loss impairs vigilance and performance; (4) the sleep homeostatic response is conserved among different wild-type lines, and between female and male flies of the same line. DESIGN: Motor activity of individual flies was recorded at 1-minute intervals using the infrared Drosophila Activity Monitoring System during 2 baseline days; during 6, 12, and 24 hours of sleep deprivation; and during 2 days of recovery. Sleep was defined as any period of uninterrupted behavioral immobility lasting > 5 minutes. Sleep continuity was measured by calculating the number of brief awakenings, the number and duration of sleep episodes, and a sleep continuity score. Vigilance before and after sleep deprivation was assessed by measuring the escape response triggered by 2 different aversive stimuli. SETTING: Fly sleep research laboratory at UW-Madison. PARTICIPANTS AND INTERVENTIONS: Adult flies of the Canton-S (CS) strain and 116 other wild-type lines (> or = 16 female and > or = 16 male flies per line). MEASUREMENTS AND RESULTS: In wild-type CS flies, as in mammals, the amount of sleep recovered after sleep deprivation was dependent on prior time awake. Relative to baseline sleep, recovery sleep in CS flies was less fragmented, with longer sleep episodes, and was associated with a higher arousal threshold. Sleep deprivation in CS flies also reduced performance. Sleep duration and continuity increased after 24 hour of sleep deprivation in all the other wild-type lines tested. CONCLUSION: The sleep homeostatic response in fruit flies is a stable phenotype and shares most of, if not all, the major features of mammalian sleep homeostasis, thus supporting the use of Drosophila as a model system for the genetic dissection of sleep mechanisms and functions.
Animals↗
Locus ceruleus control of state-dependent gene expression.
Wakefulness and sleep are accompanied by changes in behavior and neural activity, as well as by the upregulation of different functional categories of genes. However, the mechanisms responsible for such state-dependent changes in gene expression are unknown. Here we investigate to what extent state-dependent changes in gene expression depend on the central noradrenergic (NA) system, which is active in wakefulness and reduces its firing during sleep. We measured the levels of approximately 5000 transcripts expressed in the cerebral cortex of control rats and in rats pretreated with DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine], a neurotoxin that removes the noradrenergic innervation of the cortex. We found that NA depletion reduces the expression of approximately 20% of known wakefulness-related transcripts. Most of these transcripts are involved in synaptic plasticity and in the cellular response to stress. In contrast, NA depletion increased the expression of the sleep-related gene encoding the translation elongation factor 2. These results indicate that the activity of the central NA system during wakefulness modulates neuronal transcription to favor synaptic potentiation and counteract cellular stress, whereas its inactivity during sleep may play a permissive role to enhance brain protein synthesis.
Animals↗
Sleep deprivation and cellular responses to oxidative stress.
STUDY OBJECTIVES: It has been hypothesized that sleep deprivation represents an oxidative challenge for the brain and that sleep may have a protective role against oxidative damage. This study was designed to test this hypothesis by measuring in rats the effects of sleep loss on markers of oxidative stress (oxidant production and antioxidant enzyme activities) as well as on markers of cellular oxidative damage (lipid peroxidation and protein oxidation). DESIGN: The analyses were performed in the brain and in peripheral tissues (liver and skeletal muscle), after short-term sleep deprivation (8 hours), after long-term sleep deprivation (3-14 days), and during recovery sleep after 1 week of sleep loss. Short-term sleep deprivation was performed by gentle handling; long-term sleep deprivation was performed using the disk-over-water method. SETTING: Sleep research laboratory at University of Wisconsin-Madison. PARTICIPANTS AND INTERVENTIONS: Adult male Wistar Kyoto rats (n = 69) implanted for polygraphic (electroencephalogram, electromyogram) recording. MEASUREMENTS AND RESULTS: Aliquots of brain, liver, or skeletal muscle homogenate were used to assess oxidant production, superoxide dismutase activity, lipid peroxidation, and protein oxidation. No evidence of oxidative damage was observed at the lipid and/or at the protein level in long-term sleep-deprived animals relative to their yoked controls, nor in the cerebral cortex or in peripheral tissues. Also, no consistent change in antioxidant enzymatic activities was found after prolonged sleep deprivation, nor was any evidence of increased oxidant production in the brain or in peripheral tissues. CONCLUSION: The available data do not support the assumption that prolonged wakefulness may cause oxidative damage, nor that it can represent an oxidative stress for the brain or for peripheral tissue such as liver and skeletal muscle.
Animals↗
Extensive and divergent effects of sleep and wakefulness on brain gene expression.
Sleep is present in all species where it has been studied, but its functions remain unknown. To investigate what benefits sleep may bring at the cellular level, we profiled gene expression in awake and sleeping rats by using high-density microarrays. We find that approximately 10% of the transcripts in the cerebral cortex change their expression between day and night and demonstrate that half of them are modulated by sleep and wakefulness independent of time of day. We also show that molecular correlates of sleep are found in the cerebellum, a structure not known for generating sleep rhythms. Finally, we show that different functional categories of genes are selectively associated with sleep and wakefulness. The approximately 100 known genes whose expression increases during sleep provide molecular support for the proposed involvement of sleep in protein synthesis and neural plasticity and point to a novel role for sleep in membrane trafficking and maintenance.
Animals↗
Sleep and synaptic homeostasis: a hypothesis.
During much of sleep, the cerebral cortex is rippled by slow waves, which appear in the electroencephalogram as oscillations between 0.5 and 4.5 Hz. Slow waves are regulated as a function of previous wakefulness, being maximal at the beginning of sleep and then progressively returning to a baseline level. This paper discusses a hypothesis about the significance of slow-wave activity and its homeostatic regulation. The hypothesis is as follows: 1. Wakefulness is associated with synaptic potentiation in several cortical circuits; 2. Synaptic potentiation is tied to the homeostatic regulation of slow-wave activity; 3. Slow-wave activity is associated with synaptic downscaling; 4. Synaptic downscaling is tied to the beneficial effects of sleep on performance. The hypothesized link between sleep and synaptic homeostasis is supported by several lines of evidence and leads to testable predictions.
Animals↗
Searching for sleep mutants of Drosophila melanogaster.
The functions of sleep are still unknown, but are probably related to cellular and molecular aspects of neural function. To better understand the benefits that sleep may bring at the cellular level, recent studies have employed Drosophila melanogaster as a model system and shown that fruit flies share the fundamental features of mammalian sleep. As in mammals, sleep in Drosophila is characterized by increased arousal threshold and by changes in brain electrical activity. Fly sleep is homeostatically regulated independent of the circadian clock, is modulated by stimulants and hypnotics, and is affected by age. Also, fly sleep is associated with changes in brain gene expression similar to those observed in mammals. While Drosophila neurobiology is sufficiently complex to permit meaningful generalizations to mammals and humans, Drosophila genetics is simple enough to allow a rapid mutagenesis screening. An ongoing mutagenesis study has screened approximately 5000 mutant Drosophila lines and found that sleep amount, sleep pattern, and the homeostatic regulation of sleep are highly conserved phenotypes in flies. So far, this study has identified 10 short sleeper lines and 4 lines that show no sleep rebound after sleep deprivation. Ultimately, the characterization of these lines should help identifying crucial cellular pathways involved in the regulatory mechanisms of sleep and its functional consequences.
Animals↗
How sleep deprivation affects gene expression in the brain: a review of recent findings.
The identification of the molecular correlates of sleep and wakefulness is essential to understand the restorative processes occurring during sleep, the cellular mechanisms underlying sleep regulation, and the functional consequences of sleep loss. To determine what molecular changes occur in the brain during the sleep-waking cycle and after sleep deprivation, our laboratory is performing a systematic screening of brain gene expression in rats that have been either sleeping or spontaneously awake for a few hours and in rats that have been sleep deprived for different periods of time ranging from a few hours to several days. So far, ~10,000 transcripts expressed in the cerebral cortex have been screened. The expression of the vast majority of these genes does not change either across behavioral states or after sleep deprivation, even when forced wakefulness is prolonged for several days. A few hours of wakefulness, either spontaneous or forced by sleep deprivation, increase the expression of the same small groups of genes: immediate-early genes/transcription factors, genes related to energy metabolism, growth factors/adhesion molecules, chaperones/heat shock proteins, vesicle- and synapse-related genes, neurotransmitter/hormone receptors, neurotransmitter transporters, and enzymes. Sleep, on the other hand, induces the expression of a few unknown transcripts whose characterization is in progress. Thus, although the characterization of the molecular correlates of behavioral states is not yet complete, it is already apparent that the transition from sleep to waking can affect basic cellular functions such as RNA and protein synthesis, neural plasticity, neurotransmission, and metabolism. The pattern of changes in gene expression after long periods of sleep deprivation is unique and does not resemble that of short-term sleep deprivation or spontaneous wakefulness. A notable exception is represented, however, by the enzyme arylsulfotransferase, whose induction appears to be proportional to the duration of previous wakefulness. Arylsulfotransferase in rodents plays a major role in the catabolism of catecholamines, suggesting that an important role for sleep may be that of interrupting the continuous activity, during wakefulness, of brain catecholaminergic systems.
Animals↗
The search for the molecular correlates of sleep and wakefulness.
Knowledge of the molecular correlates of sleep and wakefulness is essential if we are to understand the restorative processes occurring during sleep and the cellular mechanisms underlying sleep regulation. In order to determine what molecular changes occur during the sleep-waking cycle, we have recently performed a systematic screening of gene expression in the brain of sleeping, sleep deprived and spontaneously awake rats. Out of the approximately 10 000 genes screened so far, a small minority ( approximately 0.5%) was differentially expressed in the cerebral cortex across behavioral states. Most genes were upregulated in wakefulness and sleep deprivation relative to sleep, while only a few had higher expression in sleep relative to wakefulness and sleep deprivation. Almost all the genes upregulated in sleep, and several genes upregulated in wakefulness and sleep deprivation, did not match any known sequence. Known genes that were upregulated in wakefulness and sleep deprivation could be grouped into functional categories: immediate early genes/transcription factors, genes related to energy metabolism, growth factors/adhesion molecules, chaperones/heat shock proteins, vesicle- and synapse-related genes, neurotransmitter/hormone receptors, neurotransmitter transporters, enzymes, and others. Although the characterization of the molecular correlates of sleep, wakefulness and sleep deprivation is still in progress, it is already apparent that the transition from sleep to waking can affect basic cellular functions such as RNA and protein synthesis, neural plasticity, neurotransmission, and metabolism.
Journal Article↗