INTRODUCTION

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CONSIDERING THE TYPICAL DURATION of sleep-wake states and the time constants of their regulation (minutes to hours rather than seconds), it is plausible that gene expression in the brain is subject to significant modulations across behavioral states. Our initial studies focused on the expression of the so-called immediate-early genes (IEGs), which are rapidly induced by a large number of extracellular stimuli. Our laboratory and several other laboratories (reviewed in Ref. 9) have shown that the expression of c-fos, NGFI-A, and other IEGs is powerfully modulated by behavioral state. Specifically, their expression is low or absent in most brain regions if the animals had spent most of the previous 3-8 h asleep, whereas it is high if the animals had been either spontaneously awake or sleep deprived for a few hours before death. Many IEGs function as transcription factors (22), and therefore a significant change in their expression across behavioral states could herald a widespread change in the expression of many "target" transcripts.

For this reason, over the past several years, our laboratory has been performing a systematic screening of brain gene expression across behavioral states (5- 8). The goal of these studies is to characterize specific patterns of gene expression that distinguish sleep from wakefulness, either spontaneous or forced by sleep deprivation. The identification of such patterns may help us to understand the homeostatic regulation of sleep and its functional consequences.

Our laboratory employs mRNA differential display and cDNA microarray technology to systematically establish the differences in gene expression that occur across behavioral states. We compared brain gene expression after short (3 h) and sustained (8 h) periods of sleep, spontaneous wakefulness, and sleep deprivation (5, 7, 8). More recently, our laboratory has also started to examine gene expression in the brain of rats chronically deprived of sleep for long periods of time (4-14 days; Ref. 6). Our study focuses on the cerebral cortex because several evidences suggest that higher cognitive functions are among the most affected by sleep deprivation in humans (11, 13). Moreover, according to some influential hypotheses, the cerebral cortex is the main target of the restorative effects of sleep (17). We estimated that, so far, our laboratory has examined ~10,000 transcripts. Because the number of genes expressed in the rat cerebral cortex is likely to range between 15,000 and 30,000 (19), our screening is probably extensive, but not yet exhaustive.

These studies are still in progress, but several general conclusions can already be drawn. They showed that only a small minority (<1%) of the genes expressed in the cerebral cortex are up- or downregulated (% of change ranging from ~30% to severalfold) between sleep and wakefulness or after different periods of sleep deprivation. Most of the differentially expressed genes show higher mRNA levels after a few hours (3-8) of spontaneous wakefulness and sleep deprivation than after sleep, whereas only a small minority of genes is upregulated during sleep. Most genes upregulated by 3-8 h of forced wakefulness are also upregulated by spontaneous wakefulness, although the increase in mRNA levels is generally more significant in the former condition. Most of the transcripts upregulated during wakefulness (either spontaneous or forced) correspond to known genes and can be grouped in few functional categories. On the other hand, almost all the genes whose mRNA levels are higher in sleep relative to wakefulness do not match any known sequence. The only exception so far is the gene coding for the membrane protein E25. Even in this case, however, the function of this protein is unknown (10).

	GENES RAPIDLY INDUCED BY SHORT PERIODS (3 H) OF WAKEFULNESS

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Two classes of genes are induced by 3 h of spontaneous wakefulness or sleep deprivation: IEGs/transcription factors and mitochondrial genes (Ref. 5; Table 1). The IEGs group includes Arc (Fig. 1), c-fos, NGFI-A, the rat homologue of the human Zn-15-related zinc finger (rlf) gene, which has been implicated in transcriptional regulation, and AA117313, probably similar to the human global transcription activator SNF2/SWI2. Our laboratory has also found that the transcription factor CREB is differentially phosphorylated depending on the behavioral state of the animals (8). CREB phosphorylation (P-CREB) at Ser¹³³ follows increases in the intracellular concentration of Ca²⁺ or cAMP and the activation of CREB-dependent transcription plays a crucial role in the acquisition of different forms of long-term memory in the hippocampus and the cerebral cortex (refs. cited in Ref. 7). As shown in Fig. 2, P-CREB immunolabeling is low in rats killed after a few hours of sleep and high after 3 h of either spontaneous or forced wakefulness.

View larger version (96K): [in this window] [in a new window]   Fig. 1.   Arc immunostaining in the parietal cortex (cortical layers III-VI) of rats after 3 h of sleep (S) and 3 h of spontaneous wakefulness (W). Scale bar = 100 µm.

View larger version (143K): [in this window] [in a new window]   Fig. 2.   Anti-P-CREB staining in coronal sections of parietal cortex (cortical layers II-VI) from a rat that slept for 3 h (S) and a rat that was sleep deprived for 3 h (SD). Scale bar = 100 µm.

In addition to Arc, NGFI-A, and P-CREB (which are upregulated after both 3 and 8 h of wakefulness and sleep deprivation), other IEGs/transcription factors are only induced after sustained (8 h) periods of sleep loss (7). They include CHOP, IER5, NGFI-B, N-ras, and Stat3. Many of these genes may play a role in promoting the transcription of "late" genes during wakefulness.

Although the induction of several IEGs in response to being awake for 3 h has to be expected based on our previous results (see above), the rapid regulation in the expression of mitochondrial genes is an unexpected finding. The mitochondrial genes include the subunit I of cytochrome c oxidase, the subunit 2 of NADH dehydrogenase, and the 12S rRNA. Cytochrome c oxidase is the terminal enzyme of the respiratory chain and plays a crucial role in the regulation of oxidative metabolism (24). The enzyme is made up of several subunits, some of which (e.g., subunit I) are coded by the mitochondrial genome, whereas others (e.g., subunit IV) are coded by the nuclear genome. Interestingly, we found that changes in mRNA levels between sleep and wakefulness involve only the mitochondrial genes coded by the mitochondrial genome and not those coded by the nuclear genome. Mitochondria seem to contain excess amounts of nuclear-encoded cytochrome c oxidase subunits. Changes in neuronal activity and energy demand affect the transcription of mitochondrially encoded subunits of cytochrome c oxidase more quickly and more significantly than that of the nuclear subunits (24). Thus it is the synthesis of mitochondrially encoded subunits, followed by the holoenzyme assembly, that is governed by dynamic local energy needs. Cerebral glucose is almost exclusively metabolized through mitochondrial oxidative phosphorylation, and glucose metabolism is 20-30% higher in wakefulness than in non-rapid eye movement sleep in several species, including the rat (20). The increased expression of mitochondrial genes after 3 h of wakefulness suggests a previously unsuspected mechanism by which neurons and/or glia can adapt to the increased metabolic demand of wakefulness relative to sleep. The functional role of this mitochondrial upregulation is supported by the recent finding that the expression of subunit I of cytochrome c oxidase increases after periods of wakefulness also in species, such as the fruit fly, that are phylogenetically very distant from the rat (Table 1). Indeed, in a completely independent gene screening project, our laboratory found that levels of subunit I of cytochrome c oxidase mRNA are higher after periods of wakefulness and sleep deprivation relative to comparable periods of sleeplike behavior in the brain of Drosophila melanogaster (21).

	GENES INDUCED BY 8 H OF WAKEFULNESS

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Although mitochondrial transcripts revert to baseline levels after 8 h of wakefulness, some other genes related to energy metabolism are markedly upregulated at this time (Table 1). One of these genes is Glut1, one of the major glucose transporters responsible for the transfer of glucose from blood to neurons and glia. Thus Glut1 induction may represent another mechanism by which the brain responds to the increased energy requirements of the waking state.

Several heat shock proteins and molecular chaperones such as HSP60, HSP70, and BiP show higher mRNA levels after 8 h of wakefulness. BiP, the major chaperone of the endoplasmic reticulum (ER), associates with nascent glycoproteins and secretory polypeptides during assembly in the ER and retain them in an assembly-competent shape. Increases in mRNA and/or protein levels of BiP and heat shock proteins occur during stress conditions (e.g., ischemia, severe glucose deprivation, Ca²⁺ depletion) that may cause the accumulation of unfolded or malfolded proteins in cells (see Ref. 7 for refs.). In these abnormal conditions, BiP may target unfolded proteins for degradation or participate in their refolding. The finding that molecular chaperones and heat shock proteins are induced in a completely physiological condition, i.e., during spontaneous wakefulness, is also unexpected. The events responsible for BiP induction are still unclear, but an increase in protein synthesis, notably of proteins that require assembly in the ER, could play a role. The Aplysia homologue of BiP, for instance, is induced after long-term sensitization training and has been hypothesized to play a role in the folding of newly synthesized proteins involved in synaptic plasticity (16). Despite the fact that the role of BiP induction during wakefulness is still unknown, its functional importance is once again supported by the finding that BiP mRNA levels are induced after periods of spontaneous activity or rest deprivation in the brain of Drosophila melanogaster (Ref. 21; Table 1).

Other genes upregulated after 8 h of wakefulness include components of the presynaptic and postsynaptic neurotransmission machinery, such as vesicle and synaptic-related genes, subunits of several neurotransmitter receptors, both excitatory and inhibitory, and neurotransmitter transporters. The finding that DL--amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) receptor subunits GluR2 and GluR3 mRNA levels are higher after spontaneous wakefulness and sleep deprivation relative to during sleep is particularly intriguing. The number and density of AMPA receptors at the postsynaptic membrane have been shown to be regulated by synaptic activity, and endocytosis and exocytosis of AMPA receptors regulate synaptic plasticity. Moreover, in the adult brain in vivo, long-term potentiation and long-term depression are associated with increases and decreases, respectively, of protein levels of GluR1 and GluR2 (12). Thus the upregulation of components of the presynaptic and postsynaptic neurotransmission machinery may represent a general compensatory response of the brain to the increased synaptic neurotransmission during wakefulness relative to sleep. In addition, the induction of at least some of these genes, such as GluR2, may more specifically mediate the occurrence of plastic phenomena during wakefulness.

In this regard, it is important to emphasize that, irrespective of the category in which they are listed, several of the genes upregulated in wakefulness and sleep deprivation relative to sleep have been implicated in the molecular mechanisms of neural plasticity. This is the case not only for the genes coding for GluR2, but also for those coding for several IEGs (Arc, c-Fos, CHOP, NGFI-A), as well as for the growth factor brain-derived neurotrophic factor (BDNF) and its receptor TrkB, the adhesion molecule F3, BiP, synaptotagmin IV, calmodulin, and a few others. Overall, our data suggest that the transcription (and possibly translation) of plasticity-related genes is favored during wakefulness relative to sleep. Very recently, we also found that the expression of the gene coding for the tissue inhibitor of metalloproteinase 1 (TIMP-1) increases more than twofold after 8 h of spontaneous wakefulness or sleep deprivation relative to sleep (C. Cirelli and G. Tononi, unpublished results). TIMP-1 induction is likely to be controlled by c-fos, and, although the physiological role of the metalloproteinases/TIMPs system remains largely unknown, it has been involved in neuronal circuitry formation and synaptic plasticity (15).

	ROLE OF THE NORADRENERGIC SYSTEM

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Many transcripts upregulated during wakefulness are induced diffusely in the cerebral cortex and in many other brain regions. We hypothesized that a key factor responsible for their induction might be the level of activity of neuromodulatory systems such as the noradrenergic and the serotoninergic systems. These systems project diffusely to most of the brain and regulate gene expression. Moreover, their activity is strictly state dependent.

During sleep, locus coeruleus neurons fire regularly at very low rates, whereas during wakefulness they fire at higher rates and emit phasic, short bursts of action potentials in response to salient events (1). Norepinephrine released diffusely by these neurons over large portions of the brain enhances information transmission and promotes attentive processes by increasing gamma activity in the electroencephalogram (EEG) (Ref. 3 and references therein). Norepinephrine can enable various forms of activity-dependent synaptic plasticity and can stimulate gene transcription. To assess the role of the noradrenergic system in the induction of gene expression during wakefulness, our laboratory used rats whose behavior and brain electrical activity were continually monitored and in which the left locus coeruleus was lesioned (4, 8). Thus, when these animals were awake, norepinephrine would be released only on the right side. The rats with unilateral locus coeruleus lesions behaved normally and showed normal amounts of sleep and wakefulness. Furthermore, as demonstrated by EEG analysis, brain electrical activity seemed normal and essentially indistinguishable between the right and the left side of their brains. The expression of c-fos, NGFI-A, P-CREB, Arc, and BDNF after these animals had been awake for several hours was high on the right side, as expected. However, on the left side, in which the locus coeruleus had been lesioned, the expression of these genes was abolished or greatly reduced (Fig. 3). In other words, the side of the brain depleted of norepinephrine appeared similar to that of a rat that had been asleep instead of awake. Thus, if the noradrenergic system is lesioned, waking behavior associated with a normal low-voltage fast-activity EEG is not accompanied by the induction of molecular markers of plasticity such as c-fos, NGFI-A, P-CREB, Arc, and BDNF, suggesting that the activation of the EEG can be completely dissociated from the activation of gene expression. These findings may have several implications concerning neural plasticity, learning, and memory. The reduced expression of plasticity-related genes due to the reduced firing of locus coeruleus neurons may be a key factor determining why the ability to learn and remember new material is impaired during sleep. This finding may also help explain why we do not remember most of our dreams, despite the fact that brain activity in rapid eye movement sleep, the stage of sleep more associated with vivid dreams, is very similar to that of alert wakefulness.

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Coronal sections of parietal cortex of a rat in which the left locus coeruleus was destroyed by a local injection of the neurotoxin 6-OHDA. The noradrenergic (NE) fibers are abolished on the side of the lesion, whereas they are left intact on the other side. After 3 h of sleep deprivation, Arc expression is high on the intact side (right, with NE), but it is as low as in sleep on the side where the NE innervation had been destroyed (left, without NE). Scale bar = 100 µm.

Like locus coeruleus cells, serotoninergic neurons of the dorsal raphe also fire at higher levels during wakefulness and decrease their firing during sleep (18). However, in sharp contrast to noradrenergic neurons, dorsal raphe cells are activated during repetitive motor activity such as locomoting, grooming, or feeding and are inactivated during orientation to salient stimuli (14). Concurrently, activation of the serotoninergic system inhibits information processing in various sensory pathways (14) and reduces gamma activity in the EEG (3). In rats in which dorsal raphe neurons were unilaterally lesioned, it was found that c-fos, NGFI-A, P-CREB, Arc, and BDNF expression was not affected either during wakefulness or during sleep (23). Thus the dorsal raphe does not play a crucial role in brain gene expression during wakefulness.

	GENES INDUCED BY LONG PERIODS OF SLEEP DEPRIVATION

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Most of the genes discussed above as induced by short periods of wakefulness are no longer upregulated if sleep loss is prolonged. Indeed, most of them return to baseline levels or are even downregulated after several days of sleep deprivation (C. Cirelli and G. Tononi, unpublished observations). One important exception is represented by the enzyme arylsulfotransferase (AST), which is induced more markedly after several days than after several hours of sleep deprivation (6, 7). The progressively stronger induction of AST is the first demonstration of a molecular response in the brain that is proportional to the duration of sleep loss. AST is responsible in the brain for the sulfonation of norepinephrine, dopamine, and, to a lesser extent, serotonin. AST induction during sleep deprivation may therefore constitute a homeostatic response to the uninterrupted activity of the central noradrenergic system during wakefulness. This notion is again strengthened by the evidence of converging molecular correlates recently obtained in Drosophila (21). Rest deprivation in Drosophila is associated with an increased expression of arylalkylamine N-acetyltransferase (aaNAT1b; Ref. 2), an enzyme implicated in the catabolism of monoamines and functionally related to AST (Table 1). Thus an important function of sleep may be that of counteracting the effects of continued monoaminergic discharge. If this is true, an impaired catabolism of monoamines should result in an increased need for sleep. To begin testing this hypothesis, we examined a Drosophila mutant (Dat^lo) in which the transcriptional level and activity of the aaNAT1b enzyme is deficient. The spontaneous mutation Dat^lo is a hypomorphic allele of aaNAT1b. Flies homozygous for the Dat^lo mutation do not differ from wild-type flies in the percentage and circadian distribution of rest and wakefulness and show normal amounts and patterns of activity. However, relative to wild-type flies, homozygous Dat^lo flies show an increased rest rebound after 12 h of rest deprivation (21). Moreover, Dat^lo/Df flies, in which the activity of the aaNAT1b enzyme is even lower than in Dat^lo/Dat^lo flies, show an even greater and more prolonged rest rebound after rest deprivation (21). Thus the more severely mutant the fly is at the Dat locus, the greater the rest rebound, suggesting that the accumulation of monoamines in the brain may trigger sleep homeostasis.

	CONCLUSIONS

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Distinctive categories of genes change their expression in the brain in response to the transition from sleep to wakefulness, as well as after sleep deprivation. Thus sleep and wakefulness differ not only in terms of behavior, metabolism, and neuronal activity, but also in terms of brain gene expression. Until now, almost all the genes expressed at higher levels during sleep do not correspond to known sequences. On the other hand, most of the genes that are upregulated after periods of spontaneous wakefulness and short periods of sleep deprivation are known and can be grouped into several functional categories: IEGs/transcription factors, genes related to energy metabolism, growth factors and adhesion molecules, chaperones and heat shock proteins, vesicle and synapse-related genes, neurotransmitter receptors, transporters, enzymes, and others. This suggests that several basic cellular functions are affected by the arousal state of the animal. The increase in the expression of genes regulating mitochondrial activity and glucose transport may underlie a compensatory response of the brain to the increased metabolic demand of wakefulness. The high expression during wakefulness of genes related to neurotransmission and synaptic activity can also be part of a compensatory response. Moreover, many of the genes upregulated during wakefulness and short-term sleep deprivation are involved in neural plasticity, suggesting that plastic changes, in as much as they require the induction of genes, occur during wakefulness rather than during sleep. Several heat shock proteins and chaperones involved in protein folding and endoplasmic reticulum functions also show higher expression in wakefulness than in sleep. The analysis of the genes induced by long-term sleep deprivation is still in progress, but it is clear that the genes affected by prolonged sleep loss are different from those modulated during the physiological sleep/waking cycle or after short period of sleep deprivation. One transcript whose expression increases in proportion to the duration of wakefulness has already been identified. This transcript codes for the enzyme AST involved in the catabolism of catecholamines, and its induction may signify that an important function of sleep is to prevent the uninterrupted activity of the noradrenergic system.