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- Materials and methods
- Supporting Information
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, α-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.
Sleep and wakefulness are associated with widespread changes in brain gene expression in both vertebrates (Cirelli et al. 2004; Terao et al. 2005) and invertebrates (Cirelli et al. 2005a). In the rat cerebral cortex, ∼ 5% of the transcripts are differentially expressed after 8 h of spontaneous sleep compared with 8 h of either spontaneous wakefulness or sleep deprivation (Cirelli et al. 2004). Sleep-related transcripts code for glial proteins, proteins involved in protein synthesis, cholesterol synthesis, membrane trafficking, synaptic down-regulation and memory consolidation. Transcripts expressed at higher levels during spontaneous wakefulness or short-term sleep deprivation, on the other hand, include those coding for several mitochondrial proteins, chaperones and heat-shock proteins, and proteins involved in synaptic potentiation and glutamatergic transmission. These findings suggest that continuous wakefulness not only increases brain energy demand, but also represents a cellular stress for neurons and/or glial cells. Indeed, a recent study found that, in the mouse cerebral cortex, as little as 6 h of forced wakefulness triggers the unfolded protein response, which includes the slowing down of protein synthesis and the induction of immunoglobulin heavy chain-binding protein (BiP) (Naidoo et al. 2005).
Long-term sleep deprivation in rats produces a series of dramatic physiological changes that invariably culminate in death after 2–3 weeks (Rechtschaffen and Bergmann 2002). Within the first 1–2 days, sleep-deprived rats show an increase in food intake, energy expenditure and heart rate, followed 1–3 weeks later by a decrease in body weight and in body and brain temperature. The sleep deprivation syndrome and its lethal consequences have also been observed after selective rapid eye movement (REM) sleep deprivation, although the pathology associated with the loss of sleep takes longer to appear, the survival time is longer, and body and brain temperature are not significantly decreased (Rechtschaffen and Bergmann 2002). Despite extensive studies, the long-term sleep deprivation syndrome has not been fully explained (Rechtschaffen 1998). No major organ pathology has been documented, and systemic infections, although a frequent and early event in sleep-deprived animals (Everson and Toth 2000), have been prevented without reversing the time course of the syndrome (Bergmann et al. 1996). In the brain, signs of brain cell death have either not been found (Cirelli et al. 1999) (Hipolide et al. 2002) or found only in the supraoptic nucleus of the hypothalamus (Eiland et al. 2002). Moreover, markers of oxidative stress are either absent (e.g. in the cerebral cortex; Gopalakrishnan et al. 2004) or restricted to a few brain areas (e.g. the hippocampus; D'Almeida et al. 1998; Ramanathan et al. 2002). Finally, even when markers of oxidative stress are induced, there is no sign of oxidative damage, including protein oxidation, lipid peroxidation and nucleic acid oxidation (D'Almeida et al. 1997, 1998; Gopalakrishnan et al. 2004). By contrast, episodic hypoxia without sleep loss is sufficient in rodents to cause cortico-hippocampal apoptosis and cognitive impairment, at least partially by inducing oxidative stress (Xu et al. 2004). This suggests that any potentially irreversible damage in the brain of patients with obstructive sleep apnea is more likely the result of abnormalities in blood gas composition than to sleep loss per se.
- Top of page
- Materials and methods
- Supporting Information
In this study, as in all previous array studies that aimed to identify transcripts affected by sleep and wakefulness (Cirelli et al. 2004; Terao et al. 2005), arrays were hybridized with pooled RNA. Although pooling is required to reduce the cost of the experiment, it is important to assess for biological variability using independent methods. To this end, we used qPCR and RPA in independent groups of animals not used in the array analysis. In our previous rat (Cirelli et al. 2004) and fly (Cirelli et al. 2005a) studies, PCR confirmed ∼ 80% of the array results, suggesting that less than 20% of the genes identified as differentially expressed in the arrays were false positives. In this study, all but one of the 16 transcripts identified by the array analysis as not changing, increased or decreased, were confirmed using PCR. Similarly, the four genes tested using RPA were confirmed as changing in the same direction as indicated by the arrays. The higher success rate may be partly due to the bigger fold change of some of these transcripts, especially those up-regulated in l-SD rats. However, we previously found that high expression levels (i.e. a reliable detection), rather than fold changes as indicated by the array analysis, are the best predictors of whether or not the results for a gene will be confirmed by another method. In this study, up to eight array replicas were used for the l-SD and YC groups and, most importantly, transcripts were identified using a restrictive conjunction analysis. These stringent criteria may explain both the high confirmation rate, as well as the short list of transcripts.
The largest category of genes specifically up-regulated in the cerebral cortex because of chronic sleep loss codes for immunoglobulins, in most cases κ chains. This group also includes two autoantibodies, against AchR and NGF. These genes were not previously identified after short-term sleep deprivation. Moreover, all but one were not significantly up-regulated in YCs relative to sleeping animals, and in all cases were significantly up-regulated in l-SD rats relative to their YCs. Thus, it seems that the up-regulation of these immunoglobulins requires a sleep deprivation that is both chronic and extreme, and thus cannot be triggered by 8 h of sleep loss or by chronic sleep restriction.
It has been suggested that sleep and sleep deprivation are associated with changes in immune function. Whether sleep loss actually results in an impairment of immune functions, however, remains unclear (Benca et al. 1997; Bryant et al. 2004). Both animal and human studies have shown that prolonged sleep deprivation results in activation of the immune response. In humans, 3 days of sleep deprivation produce increases in natural killer cell activity and in granulocyte and monocyte counts (with no change in lymphocytes) (Dinges et al. 1994). In a recent study Everson found that rats deprived of sleep for up to 20 days with the DOW show leukocytosis with increased counts of neutrophils and monocytes (and a trend toward decreased counts of lymphocytes), induction of pro-inflammatory cytokines and chemokines, and increased production of serum IgM, IgG and IgA, consistent with polyclonal activation of B lymphocytes (Everson 2005). The author suggests that this broad-based antibody production may be in response to the endotoxemia and the presence of opportunistic pathogens in internal tissues of rats sleep deprived with the DOW, both of which are detected at an early stage of sleep deprivation (Everson and Toth 2000). The present study and the Everson study used the DOW, and this method is currently the only one available to enforce prolonged sleep loss in animals with an adequate stimulation control. Thus, until novel methods are designed, it may be difficult to establish whether chronic sleep deprivation in animals always results in increased expression of immunoglobulins. In humans, very few studies have analyzed the effects of sleep deprivation on immunoglobulin levels, and with contrasting results. Boyum et al. (1996) found a decrease in Ig levels in subjects enrolled in 5–7 days of a military training course, but in that study sleep restriction was associated with continuous physical exercise and caloric restriction. Ozturk et al. (1999) found no change in serum IgG and IgM, but their subjects were deprived of sleep for only 48 h.
In a recent study in rats subjected to long-term sleep deprivation Everson mentioned that polyclonal B responses, such those associated with chronic antigenic stimulation, have the potential to induce autoimmune response (Everson 2005). Interestingly, we found in this study that the expression of two autoantibodies was increased after 1 week of sleep deprivation. They are directed against NGF and the AchR. Anti-NGF antibodies, when injected into rat cerebral cortex, can produce degeneration of cortical cholinergic boutons (Debeir et al. 1999) and disrupt learning (Gutierrez et al. 1997). Moreover, transgenic mice overexpressing anti-NGF antibodies develop an age-dependent neurodegenerative pathology with dementia-like symptoms (Capsoni et al. 2000). AchR antibodies, on the other hand, when infused in the rat cerebral cortex, produce fatigue and ataxia (e.g. Gomez et al. 1984). Thus, it cannot be excluded that these antibodies may contribute to the sleepiness, decreased attention and fatigue associated with chronic sleep deprivation. Autoantibodies have also been described in a few cases of severe insomnia, but never as a consequence of relatively acute sleep deprivation. Liguori et al. (2001) described a patient with Morvan's syndrome whose severe insomnia, associated with the presence of autoantibodies against voltage-dependent potassium channels, improved after plasma exchange. (Batocchi et al. (2001) reported a case of multiple cranial nerve palsy, recurrent episodes of total insomnia, and respiratory crises that responded to plasma exchange and immunosuppressive treatment. In that case the serum was negative for antibodies against voltage-dependent potassium channels, but positive for antibodies against GABAergic synapses. In the case described by Liguori et al. it is possible that the presence of the autoantibodies against potassium channels was at least partially responsible for the insomnia (Cirelli et al. 2005b). However, the autoantibodies were associated with a paraneoplastic syndrome, and thus it remains unclear whether prolonged sleep loss per se can trigger an autoimmune response.
Among other genes up-regulated by chronic sleep loss there were several stress response genes. One codes for the MRP14 (also called S100 calcium-binding protein A9 or calgranulin B). MRP14 is detected on microglial cells in bacterial encephalitis, Alzheimer's disease and after cerebral infarction (Postler et al. 1997;Staba et al. 2002). Others code for the small heat-shock proteins Hsp27 (Hspb1) and Cryab. These genes are strongly induced in glial cells (astrocytes and oligodendrocytes) after different forms of stress, and their induction may have a protective effect (Goldbaum and Richter-Landsberg 2001). Among the stress response genes not up-regulated in l-SD rats is BiP. BiP is the major chaperone of the endoplasmic reticulum and is involved in the degradation of misfolded proteins. BiP mRNA levels increase during short-term sleep deprivation in the brain of rats (Cirelli et al. 2004), hamsters (Cirelli C., DeBoer T., Tobler I., unpublished results) and sparrows (Jones S., Benca R., Cirelli C., unpublished results). In mouse cerebral cortex BiP expression is increased by as little as 6 h of sleep deprivation, which also causes a decrease in protein synthesis, another sign of the cellular stress response called the unfolded protein response (Naidoo et al. 2005). Our array analyses, confirmed by RPA experiments, showed that in the cerebral cortex BiP mRNA levels do not increase after long-term sleep deprivation as much as after short-term sleep deprivation. Interestingly, RPA experiments also showed that BiP expression is similarly induced after short-term and long-term sleep deprivation in skeletal muscle, whereas in the liver the most significant induction occurs in animals housed in the DOW apparatus, including YCs. Thus, BiP mRNA levels do not seem to reflect the duration of sleep loss.
Other genes specifically up-regulated in long-term sleep deprived rats code for minoxidil sulfotransferase, the α and β chains of hemoglobin, and cortistatin. These genes are also induced after short periods of spontaneous or forced wakefulness (Cirelli et al. 2004; Spier and de Lecea 2000), but more so after prolonged sleep deprivation, and more significantly in l-SD rats than in their YCs. The minoxidil (aryl) sulfotranserase belongs to the phenol sulfotransferase family and was originally identified in the liver (Hirshey et al. 1992). It is a class II sulfoconjugation enzyme involved in the catabolism of catecholamines and in the detoxification of drugs (Yeh and Yen 2005). In humans, two forms of phenol sulfotransferases have been described, a phenol-preferring form and a monoamine-preferring form; work in our laboratory is currently characterizing the substrate specificity of the enzyme induced by sleep deprivation. Globins are a family of heme proteins that can bind, transport and scavenge O2, CO and NO. RNAs for both the α and β chains of hemoglobin cycle robustly in the SCN, with peak expression at the end of the dark period. In mouse brain, the same genes are also strongly induced by a light pulse given at night (Ben-Shlomo et al. 2005). In our previous study, we found that these genes are induced in the rat cerebral cortex during spontaneous wakefulness at night, but also after short-term sleep deprivation during the day, suggesting that behavioral state per se can induce their expression independently of circadian time. Because these genes are expressed at a higher level after long-term than after short-term sleep deprivation, their induction is unlikely to reflect the energy demand of waking per se, because prolonged sleep deprivation in animals is accompanied by a decrease, rather than an increase, in cerebral glucose utilization (Everson et al. 1994). Moreover, in Djungarian hamster cerebral cortex they are up-regulated, relative to sleep, during short-term sleep deprivation as well as during torpor, when brain metabolism is reduced (DeBoer T., Tobler I., Cirelli C., manuscript in preparation). Thus, the up-regulation of globin RNAs may be more directly related to their role as extracellular scavengers of NO and CO, and thus be part of a cellular stress response. Cortistatin is a neuropeptide mainly expressed in GABAergic interneurons of the cerebral cortex; when infused into rat brain ventricles it increases the time spent in slow wave sleep (Spier and de Lecea 2000). Its pronounced up-regulation in long-term sleep deprivation may reflect increased sleep pressure.
Intriguingly, two transcripts up-regulated in l-SD rats code for Dbp and squalene synthase, which are also up-regulated during spontaneous sleep. Among the factors that could control the expression of these genes in both conditions, one intriguing possibility is that Dbp and squalene synthase expression somehow reflects a decrease in brain metabolism, a condition shared by both sleep (Maquet 1995) and long-term sleep deprivation (Everson et al. 1994).
Twenty-eight transcripts were down-regulated in l-SD rats. One transcript specifically down-regulated because of chronic sleep loss codes for procollagen type I. Three more transcripts coding for procollagen type I were also down-regulated in l-SD rats, but they also showed decreased expression in YC rats, and did not show a further decrease in l-SD rats relative to YC rats. Thus, it remains unclear whether changes in collagen expression are related to DOW effects or to chronic sleep loss. Another transcript down-regulated because of chronic sleep loss codes for dihydrolipoamide acetyltransferase. This enzyme is a component of the pyruvate dehydrogenase complex involved in the synthesis of acetyl-CoA, and therefore its decreased expression is consistent with the decrease in brain metabolism in l-SD rats.
Many of the stress response genes up-regulated after prolonged sleep loss relative to short-term sleep deprivation and spontaneous wakefulness are preferentially expressed in glial cells, either in microglia (MRP14) or in astrocytes and oligodendrocytes (Hsp27, metallothionein 1–2). On the other hand, several genes down-regulated after both short-term and long-term sleep deprivation relative to sleep are also expressed in glial cells (this paper; Cirelli et al. 2004). In this study, for instance, we confirmed with qPCR that long-term sleep deprivation down-regulated the expression of two myelin-related genes, one coding for plasmolipin, which constitutes 50% of myelin protein, and the other for CD9, a membrane protein normally expressed in the mature myelin sheath and a gene up-regulated during sleep in the cerebellum (Cirelli et al. 2004). Overall, these findings suggest that sustained sleep loss may trigger a generalized inflammatory and stress response in the brain. Glial cells may help to protect neurons against this cellular insult, but may also suffer some of its negative consequences. Future studies will determine whether sleep loss may be detrimental to the maintenance of cellular membranes and more specifically to myelin.