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European Journal of Neuroscience

European Journal of Neuroscience, Vol. 29, pp. 1810–1819, 2009

doi:10.1111/j.1460-9568.2009.06719.x

REVIEW

Consequences of sleep deprivation on neurotransmitter receptor expression and function Fabio Longordo,1 Caroline Kopp2 and Anita Lu¨thi1 1

Department of Cell Biology and Morphology, Faculty of Biology and Medicine, Rue du Bugnon 9, University of Lausanne, CH-1005 Lausanne, Switzerland 2 University of Basel, Birmannsgasse 8, CH-4009 Basel, Switzerland Keywords: hypothalamic-pituitary-adrenocortical axis, learning and memory, sleep loss, sleep-promoting systems, wake-promoting systems

Abstract Several pieces of evidence suggest that sleep deprivation causes marked alterations in neurotransmitter receptor function in diverse neuronal cell types. To date, this has been studied mainly in wake- and sleep-promoting areas of the brain and in the hippocampus, which is implicated in learning and memory. This article reviews findings linking sleep deprivation to modifications in neurotransmitter receptor function, including changes in receptor subunit expression, ligand affinity and signal transduction mechanisms. We focus on studies using sleep deprivation procedures that control for side-effects such as stress. We classify the changes with respect to their functional consequences on the activity of wake-promoting and ⁄ or sleep-promoting systems. We suggest that elucidation of how sleep deprivation affects neurotransmitter receptor function will provide functional insight into the detrimental effects of sleep loss.

Introduction When waking is prolonged beyond its natural duration, several selective disruptions in brain sensory processing and cognitive performance occur (Banks & Dinges, 2007; Lim & Dinges, 2008). In particular, there is accumulating evidence that insufficient sleep negatively affects memory formation and consolidation (Walker & Stickgold, 2004; Boonstra et al., 2007; Marshall & Born, 2007). Moreover, prolonged waking leads to the intrusion of sleep into the waking state, and to a homeostatically regulated intensification of the subsequent sleep episode (Borbe´ly & Achermann, 2005). The sleep deprivation (SD)-associated cellular mechanisms that promote sleepiness and induce physiological and cognitive deficits are largely unknown, although several molecular and biochemical correlates of sleep loss have been identified (Cirelli, 2006; Guzman-Marin et al., 2006; Maret et al., 2007; Landolt, 2008; Scharf et al., 2008). The decreased cognitive performance and the instability of the waking state during SD probably reflect altered neuronal excitability and synaptic communication in neuronal networks implicated in cognition and wakefulness. Hence, the identification of neuronal components that are modified by sleep loss could advance our understanding of the roles for sleep in neuronal functions. The waking state is controlled by neurochemically diverse sets of ascending fibers that release neurotransmitters into diencephalon and telencephalon (Jones, 2005; Datta & MacLean, 2007). During prolonged waking associated with SD, neurotransmitter levels resemble those of the natural waking state. When stimulated continuously or

Correspondence: Dr A. Lu¨thi, as above. E-mail: [email protected] Received 16 November 2008, revised 8 January 2009, accepted 13 January 2009

repeatedly, a common feature of neurotransmitter receptors is their desensitization, which attenuates or alters their activity (Grady et al., 1997; Gainetdinov et al., 2004). Moreover, chronically elevated activity in neural circuits provokes adaptive regulations in receptor number and function that reset excitability to within a working range (Turrigiano, 1999; Turrigiano & Nelson, 2004; Marder & Goaillard, 2006). Such cellular and receptor-intrinsic adaptive processes, occurring over hours to days, could be recruited during neurochemical conditions associated with SD. This would mean that sleep loss eventually leads to a functional weakening, if not a drop-out, of neurotransmitter receptors involved in maintaining waking, with probable consequences for the stability of wakefulness. Additionally, changes involving receptors of the sleep-promoting systems might modulate the impact of these on the brain. The goal of this article is to give a survey of studies providing evidence for changes in neurotransmitter receptor functionality related to SD (Table 1). It aims to show that these, sometimes surprisingly pronounced, changes are widespread in brain regions related to the control of sleep and waking (Table 1). We also present converging evidence that glutamate receptor trafficking and function, which is central to cellular forms of learning and memory, is affected by SD. Although research into these effects of SD is only beginning, the data suggest that altered activity of neurotransmitter receptors could represent a primary functional correlate of sleep loss in the brain. The article summarizes studies that were carried out in rodents and carnivores, in which SD can be combined with cellular examination. To date, in humans, the consequences of SD on neurotransmitter receptors have been investigated in only a few studies, mainly using positron emission tomography. We emphasize articles that consider two important aspects of experimental approaches linking sleep to

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1810–1819

Upregulated protein for adenosine receptor in prefrontal cortical areas

Upregulated protein for adenosine receptor in BF

Enhanced expression of GABAA receptor subunits on BF neurons

Altered effects of agents acting on muscarinic receptors on REM sleep and REM sleep rebound

Potentiation of glutamatergic responses

Switch from depolarizing to hyperpolarizing actions of noradrenaline

Adenosine A1 receptor

Adenosine A1 receptor

GABAA receptor subunit b2–3

Unknown

AMPAR upregulation

Could attenuate activity in prefrontal cortex

Could promote inhibition of BF

Could promote inhibition of cholinergic BF neurons

Not determined

Could promote enhanced activity of orexinergic neurons

Could promote inhibition of orexinergic neurons

Effects correlate with subjective measure of tiredness

Dopamine D2 ⁄ D3 receptors

Possibly a1-and a2adrenergic receptors

Could promote activation of monoaminergic nuclei

Serotonin 5HT1A receptor

Receptor subtype involved

Possible impact on wake- ⁄ sleep-promoting systems

No, elevated receptor levels in spite of maintained neurotransmitter receptor concentrations Not determined

Yes, elevated BF activity demonstrated through c-Fos expression, can lead to adaptive upregulation of inhibitory receptors

Not determined

Mechanisms more compatible with experience-dependent synaptic plasticity

Yes, chronically elevated levels of noradrenaline may produce a switch in receptor function

Yes, chronically elevated levels of serotonin may lead to receptor desensitization or homeostatic downregulation Not determined

Consistent with adaptive regulation of neurotransmitter receptors?

Elmenhorst et al., (2007)

Basheer et al., (2007)

Modirrousta et al., (2007)

Salıń-Pascual et al., (1989, 1992)

Rao et al., (2007)

Grivel et al., (2005); Modirrousta et al., (2005)

Pre´vot et al., (1996); Maudhuit et al., (1996); Gardner et al., 1997; Evrard et al., (2006) Volkow et al., (2008)

References

For each neurotransmitter system, the SD method and species used are given in column 1, SD effects in column 2, and receptor subtype involved in column 3. These SD-associated alterations are classified with respect to whether they affect wake-or sleep-promoting systems (column 4), and ⁄ or are consistent with adaptive changes in receptor function (column 5). The most representative references are reported in column 6.

Human, total SD (24 h)

Adenosinergic system Rat, total SD, gentle handling (3–24 h)

GABAergic system Rat, gentle handling (3 h)

Cholinergic system Human, overnight, REM SD

Rat, gentle SD (4 h) or modafinil treatment

Orexinergic system Rat, gentle SD (2 h)

Decreased receptor binding of radioligand

Receptor desensitization

Monoaminergic system Rat, chronic partial SD (2–8 days)

Human, overnight, total SD

SD effect

System and SD method

Table 1. Summary of the main alterations in neurotransmitter receptor function described after SD

Sleep deprivation and neurotransmitter receptors 1811

1812 F. Longordo et al. cellular functions. The application of SD protocols that take care to minimize side-effects, such as stress and exposure to unfamiliar environments, is one important criterion. Furthermore, we highlight cases in which several, ideally complementary, techniques have been used to show altered receptor function, such as electrophysiological recordings, immunostaining and radioligand binding.

Methodological issues of sleep deprivation research in rodents SD has become a useful approach in assessing the consequences of insufficient sleep on neuronal functions. This section discusses experimental procedures that have been commonly applied to achieve SD in rodents, and that have been instrumental in elucidating the consequences of sleep loss on cellular and molecular aspects of brain function. We will also discuss how SD procedures have been progressively improved to better isolate the effects of sleep loss from potential confounding factors. All forms of SD interfere with spontaneous behavior of the animal such as locomotion, sensory stimulation, food intake, exploration and learning. If brain areas involved in controlling these aspects of rodent behavior show differences after SD, these might not result from sleep loss per se but instead from otherwise altered neural activity while waking was enforced. Furthermore, a number of widely used SD procedures are accompanied by elevated stress levels which, by themselves, affect neuronal and synaptic function (Kim & Diamond, 2002; Pittenger & Duman, 2008). The time window over which SD is carried out is a further parameter of substantial variability. Acute total SD usually lasts a few hours to maximally 12–48 h, while partial SD is often applied chronically for days to weeks. Whereas short-term SD may thus preferentially influence sleep-related mechanisms, long-term procedures may be additionally intrusive for non-sleep-related neural functions. For all these reasons, understanding the specific effects of sleep loss on the brain requires a careful assessment of how to dissociate the influence of non-sleep-related variables from sleep loss (see also Meerlo et al., 2008). Finding adequate control experiments has thus remained a major challenge for widely used methods of SD. Early experimentation on SD in animals was centered on the consequences of loss of rapid-eye-movement (REM) sleep, which led to the development of tools to preferentially interfere with REM sleep. The inverted platform technique (inverted ‘flower-pot’), originally proposed by Jouvet (Jouvet et al., 1964), involves the use of a small platform emerging from a water bath, which is large enough for rodents to sit on when awake, but from which their limbs will touch the water during REM sleep-associated loss of muscle tone, thereby leading to awakening. The procedure is typically carried out for 72–96 h (Mendelson et al., 1974; Coenen & van Luijtelaar, 1985; van Luijtelaar & Coenen, 1986). Control animals are placed on similar platforms that are, however, large enough for undisturbed sleep. This technique has also been expanded to multiple platforms to alleviate the stress resulting from forced immobility on a single platform. However, even under these conditions, stress levels remain elevated as assessed by adrenal hypertrophy and body weight loss (Coenen & van Luijtelaar, 1985). Moreover, a loss of REM sleep also occurs in largeplatform control animals (Mendelson et al., 1974), and even the amount of non-REM (NREM) sleep is reduced compared to homecage controls (van Luijtelaar & Coenen, 1986; Ruskin et al., 2004). Similar side-effects were also reported for the disk-over-water method for total SD, in which both control and test animals are forced to escape the water on a rotating platform as soon as the test animal shows signs of REM sleep (Bergmann et al., 1989; Rechtschaffen &

Bergmann, 1995). Some studies, however, report differences in cellular alterations after SD on the small platform and those found on either the large platform (sleep-allowed) or multiple platforms (SD with less stress) experiments (e.g. Porkka-Heiskanen et al., 1995; Maloney et al., 1999; McDermott et al., 2003; Meerlo et al., 2008). In addition, cellular effects of SD by the method of multiple platforms can be observed independently of adrenal stress hormones (Mueller et al., 2008) although the specific role of REM sleep remains unclear due to a concomitant and less pronounced reduction in NREM sleep in deprived animals. An alternative to these automated REM SD methods can be applied in animals implanted with electroencephalogram and electromyogram electrodes and kept in their home cage (Endo et al., 1997). As soon as REM sleep is recognized on the polygraphic recordings, the animals are gently stimulated. The stimulations used here were mild and induced cortical desynchronisation without behavioral awakening, leading to a more selective REM SD. The so-called ‘gentle handling’ method is widely used to achieve total SD in rodents. In this case, the animals are continuously observed and maintained awake by gentle sensory stimulations as soon as behavioral and ⁄ or electrophysiological signs of sleep appear (Franken et al., 1991; Tobler et al., 1997). A modified version of this method consists of maintaining wakefulness by touching the animals with a brush when they show signs of drowsiness (Graves et al., 2003; Modirrousta et al., 2005). However, isolating the effect of sleep loss from other sensory-motor modulation generated by these methods remains challenging. The introduction of novel objects or a novel environment may be accompanied by learning effects (Kopp et al., 2007). Direct manipulations of the animal have to be minimized to prevent elevation of stress levels (Kopp et al., 2006). Activation of the hypothalamic-pituitary-adrenocortical axis has also been reported after acute SD (Campbell et al., 2002; Sgoifo et al., 2006) or chronic sleep restriction (Roman et al., 2006) achieved by forced locomotion. While the biological effects of sleep loss and of stress can be separated using control adrenalectomized animals (Roman et al., 2006), additional experiments are needed to control for the possible impact of the wake-promoting manipulations on sensory processing and learning. In a recent study, mice were maintained awake by giving them free access to an enlarged environment; touching the animals was strictly avoided and the procedure was additionally tested in whisker-trimmed animals (Kopp et al., 2006). Notably, mice exposed to this SD procedure showed unaltered levels of the stress hormone corticosterone compared to control undisturbed animals, yet presented altered neurotransmitter receptor function that could most parsimoniously be attributed to sleep loss.

Wake-promoting systems The initiation and maintenance of wakefulness is a coordinated effort of multiple, neurochemically specific arousal systems in the brainstem, hypothalamus and basal forebrain (BF). These systems provide ascending projections into the forebrain and descending afferents into spinal cord (Jones, 2005; Datta & MacLean, 2007) to promote cortical activation and behavioral arousal, mainly through tonically active monoaminergic, cholinergic and orexinergic inputs. The wakepromoting monoaminergic system includes projections from the serotonergic raphe nuclei (RN), the noradrenergic locus coeruleus (LC), the histaminergic tuberomammillary nucleus and dopaminergic neurons, presumably predominantly those in the ventral periaqueductal gray matter (Lu et al., 2006). Cholinergic neurons involved in the control of wakefulness are located in the pedunculopontine tegmentum


Sleep deprivation and neurotransmitter receptors 1813 (PPT), the lateral dorsal tegmentum and the BF. The orexinergic neurons are located in the perifornical lateral hypothalamus. Typically, SD is accompanied by maintained activity in wakepromoting structures and thus creates a neurochemical environment in the projection areas that is closest to that of the waking state (see below). Amongst the most consistently observed effects across various types of SD is a persistence, or an increase in, the levels of wakepromoting neurotransmitters in projection areas, often combined with alterations of enzymatic activities that promote neurotransmitter turnover. Paralleling the evidence for a maintenance of neurotransmitter levels during SD, there are a number of studies showing altered neurotransmitter receptor functionality as a result of SD.

Monoaminergic projections The impact of SD on the neuronal discharge pattern and neurotransmitter concentration in monoaminergic nuclei has been studied with different techniques. Serotonergic RN neurons display ongoing or increased firing rate in response to different types of SD in cat and rat (Maudhuit et al., 1996; Pre´vot et al., 1996; Gardner et al., 1997). Enhanced serotonin release and turnover in numerous brain areas of hamster and rat have also been described after SD (Asikainen et al., 1995, 1997; Grossman et al., 2000; Bjorvatn et al., 2002; LopezRodriguez et al., 2003; Penãlva et al., 2003). The increased serotonin release during SD occurs in a manner independent of stress (Penãlva et al., 2003). In the noradrenergic LC, 1 day of REM SD revealed a small decrease in single-unit activity recorded in cat (Mallick et al., 1990) and in the concentration of noradrenaline measured in rat (PorkkaHeiskanen et al., 1995), whereas a longer REM SD (72 h) led to an increased noradrenaline concentration and turnover in the rat LC (Porkka-Heiskanen et al., 1995; Perez & Benedito, 1997; Basheer et al., 1998). Furthermore, the waking-induced expression of transcription factors and neurotrophins in rat cerebral cortex, which depends on noradrenergic input, is maintained during 3–8 h of total SD (Cirelli & Tononi, 2000). During gentle SD, the histamine concentration in hypothalamic areas of cat remained elevated at levels seen during waking (Strecker et al., 2002). An augmented c-Fos expression level, indicative of neuronal activity, was also found in rat tuberomammillary nucleus after treatment with the wake-promoting agent modafinil (Scammell et al., 2000). In the dopaminergic ventral tegmental area, REM SD elevated c-Fos expression in rodents (Maloney et al., 2002). Altogether, SD appears to be accompanied by high monoaminergic levels, which represents a condition favoring alterations in neurotransmitter receptor function. Indeed, in rat, chronic partial SD (2–8 days) achieved by forced locomotion (20 h ⁄ day) gradually diminished the hypothermic response mediated by the serotonergic 5-HT1A receptor subtype (Roman et al., 2005, 2006). This effect was not due to elevated stress or forced activity, and persisted even after a prolonged period of recovery sleep, suggesting that this SD paradigm could lead to longterm decreases in 5-HT1A receptor ligand affinity and ⁄ or signal transduction efficacy. Moreover, the decreased functionality appeared only gradually, and was found not after 2 days but after 8 days of chronic partial SD. Similar chronic sleep restriction blunted the pituitary-induced release of adrenocorticotropin through a mechanism involving 5-HT1A receptor desensitization (Novati et al., 2008). Eighteen to 24 h of total SD and repeated sessions of REM SD in rodents led to a reduction in the 5-HT1A autoreceptor-mediated inhibition of RN neuron firing (Maudhuit et al., 1996; Pre´vot et al.,

1996; Gardner et al., 1997; Evrard et al., 2006) which seemed to be mediated, however, by stress hormones (Evrard et al., 2006). These data suggest that the 5-HT1A system could be comparatively sensitive to insufficient sleep. The serotonergic 5-HT1A receptor is located both pre- and postsynaptically and is implied in the negative feedback control of serotonergic neurons (Sharp et al., 2007). Decreased signaling via this receptor subtype has been linked to mood disorders and anxiety (Nutt & Stein, 2006), suggesting that chronic sleep loss could predispose to susceptibility to neuropsychiatric disorders associated with decreased 5-HT1A receptor function. Altered noradrenergic receptor expression has been studied in the context of selective REM SD and represents one of the first attempts to link sleep function to neurotransmitter receptors (Siegel & Rogawski, 1988). However, several types of SD experiments (3 h to 10 days) failed to support consistently the original hypothesis of an SD-induced downregulation of these receptors (Mogilnicka & Pilc, 1981; Radulovacki & Micovic, 1982; Abel et al., 1983; Mogilnicka et al., 1986; Tsai et al., 1993; Hipo´lide et al., 1998; Pedrazzoli & Benedito, 2004). In humans, overnight total SD led to decreased radioligand binding to D2 ⁄ D3 dopaminergic receptors in the thalamus and striatum, possibly resulting from elevated dopamine levels but also from decreased receptor expression (Volkow et al., 2008). In rodents, although changes in dopamine receptor density have been reported, these effects have not, so far, been dissociated from the stress associated with SD (Gillin et al., 1993; Nunes Junior et al., 1994a).

Orexinergic projections Measurement of c-Fos expression revealed that orexinergic neurons in rat lateral hypothalamus remain active during gentle SD (Modirrousta et al., 2004, 2005); however, the amount of neurons expressing c-Fos was in the range of a few per cent (Modirrousta et al., 2005). Much stronger activation was seen during stressful SD paradigms (Espanã et al., 2003). Moreover, levels of orexinergic peptides were increased in the cerebrospinal fluid after SD (Wu et al., 2002; Pedrazzoli & Benedito, 2004). Prolonged REM SD using the inverted platform technique left largely unaltered the expression of both orexinergic receptor isoforms (OX1R and OX2R) throughout major brain areas with orexinergic innervation (D’Almeida et al., 2005). However, the orexinergic neurons themselves underwent alterations in receptor function upon mild total SD (Grivel et al., 2005). In undisturbed young rats, killed during the first half of the light phase, orexinergic neurons in the lateral hypothalamic and perifornical areas showed an excitatory response to noradrenaline. In contrast, when slices were taken from young rats gently sleep-deprived for 2 h the response to noradrenaline was predominantly inhibitory, and spontaneous action potential discharge was arrested. These findings indicate that orexinergic neuronal activity may become attenuated during prolonged waking due to a switch in noradrenergic receptor function from excitation to inhibition, which could be due to altered receptor subtype expression. For example, a1- and a2-adrenergic receptors are known to induce depolarization or hyperpolarization of orexinergic neurons, respectively (Bayer et al., 2005). So far, immunohistochemistry assessing the expression of the a1- and a2-adrenergic receptor subtypes in orexinergic neurons has been carried out in adult rats but revealed no major differences after SD (Modirrousta et al., 2005). A mild SD (4 h) or modafinil-induced wakefulness caused a persistent potentiation of glutamatergic synaptic transmission on orexinergic neurons, mediated through an enhanced expression of the


1814 F. Longordo et al. glutamatergic a-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) subtype (Rao et al., 2007). In the case of modafinil, this potentiation was also accompanied by a greater number of asymmetric synapses on orexinergic cells. Such mechanisms could potentiate orexinergic neuronal activity and thereby maintain wakefulness. Together, these findings show that orexinergic neurons are a site of potentially high susceptibility to SD. Given the critical role of orexinergic neurons in promoting waking (Fuller et al., 2006), the resulting imbalance in their excitability could contribute to SD-induced short-term alterations in arousal.

of these at the wake-to-sleep transition. GABAergic cells of the BF also project to the cerebral cortex and locally on codistributed wakeactive cholinergic neurons (Jones, 2005). Several endogenous sleep-promoting factors accumulate in the brain in proportion to the duration and quality of wakefulness and contribute to sleep homeostasis (Datta & MacLean, 2007; Szymusiak et al., 2007). A prominent representative of these is adenosine, a wellestablished somnogenic compound, whose levels progressively and diffusely increase during wakefulness and decrease during sleep (Chagoya de Sanchez et al., 1993; Huston et al., 1996; PorkkaHeiskanen et al., 1997, 2000; Strecker et al., 2000).

Cholinergic projections Since the cholinergic system has been implicated in REM sleep generation (Sakai et al., 2001), it has been proposed that changes in the amount of REM sleep are associated with altered cholinergic activity. Prolonged REM SD (50 h) in rat failed to change c-Fos expression in three of the four subdivisions of the cholinergic PPT nuclei, whereas REM sleep recovery augmented c-Fos levels in the total cholinergic neuron population (Maloney et al., 1999). Moreover, brief total SD (3 h) revealed increased c-Fos expression in cholinergic BF neurons compared to the control condition (Modirrousta et al., 2004), suggesting a largely maintained, if not enhanced, cholinergic output during SD. Finally, 96 h of REM SD enhanced the activity of acetylcholinesterase, the enzyme responsible for the inactivation of released acetylcholine, in the brainstem of rat (Thakkar & Mallick, 1991). In REM sleep-deprived humans, the cholinesterase inhibitor physostigmine induced REM sleep whereas an awakening effect was observed in control subjects (Salıń-Pascual et al., 1989). These authors also reported that low doses of the muscarinic receptor antagonist biperiden reduced REM sleep rebound after REM SD, but left REM sleep unaffected in undisturbed controls (Salıń-Pascual et al., 1992). Both these observations suggest that acetylcholine receptor coupling is altered after REM SD. Subtype-specific autoradiography, in situ hybridization and pharmacological approaches yielded, however, largely unaltered muscarinic receptor expression after REM SD in rat (Kushida et al., 1995; Moreira et al., 2003; Murugaiah & Ukponmwan, 2003), but there is some converging evidence for a decreased density of the M2 muscarinic subtype in the pontine cholinergic nuclei (Nunes Junior et al., 1994b; Kushida et al., 1995; Salıń-Pascual et al., 1998). Most recently, a study using chronic SD of rat (20 h ⁄ day of slow forced locomotion) found that hypothermic body temperature responses induced by stimulating cholinergic receptors were unaffected, arguing against an alteration of muscarinic receptors in response to SD (Roman et al., 2006). However, these responses were potentiated when SD was associated with strong forced locomotion causing stress. Thus, at present, there is some evidence for changes in the functional sensitivity of cholinergic muscarinic receptors after REM SD but not after chronic partial sleep restriction, and the changes may result more from stress than from sleep loss.

Sleep-promoting systems Sleep onset and maintenance are principally controlled by GABAergic neurons located in the preoptic area of the hypothalamus and in the BF (Jones, 2005; Datta & MacLean, 2007; Szymusiak et al., 2007). Projections arising from these neuronal populations provide dense innervation of multiple arousal systems, in particular to the monoaminergic and orexinergic neurons, and induce a coordinated inhibition

GABAergic projections Sleep-active neurons are found at high density in the ventrolateral preoptic area (vlPOA; Szymusiak et al., 1998) and in the median preoptic nucleus (Suntsova et al., 2002), and a substantial proportion of these (50–75%) is GABAergic (Gallopin et al., 2000; Gong et al., 2004; Modirrousta et al., 2004). Total SD in rat caused the sleepactive vlPOA neurons to enhance their discharge rate relative to undisturbed sleep during the wake-to-sleep transition and subsequent early and late NREM sleep (Szymusiak et al., 1998). Consistently, c-Fos expression in sleep-active GABAergic neurons in vlPOA, median preoptic nucleus and BF was increased during recovery sleep following SD (Gong et al., 2004; Modirrousta et al., 2004). Subsequent studies demonstrated that vlPOA neurons may be predominantly implicated in promoting sleep during the recovery phase whereas median preoptic nucleus neurons may be responsive to increased sleep pressure [for review, see (Szymusiak & McGinty, 2008)]. Double immunostaining experiments in adult rat revealed that 3 h of total SD dramatically enhanced the expression of the b2–3 subunit of GABAA receptors on BF cholinergic cells (Modirrousta et al., 2007). These cells poorly express this GABA receptor subunit when derived from undisturbed control animals that slept 75% of the time, or after sleep recovery, suggesting that prolonged waking promoted its expression. The effect was most pronounced in c-Fos-positive cholinergic cells and was reversed after 3 h of recovery sleep. The data indicate that SD could potentiate the inhibitory impact of the GABAergic systems onto wake-active cholinergic neurons within the BF. As the newly expressed GABAA receptor subunits were homogeneously distributed along the somatic membrane, both synaptic and extrasynaptic forms of inhibition could be augmented following SD. This could result in a markedly enhanced inhibition and could contribute to the loss of cortical and behavioral activation with prolonged waking.

Adenosinergic effects Microdialysis experiments performed in cat and rat showed that 3–6 h of SD cause a progressive increase in extracellular adenosine in the cholinergic BF (Porkka-Heiskanen et al., 1997; Basheer et al., 1999; Kalinchuk et al., 2003; Murillo-Rodriguez et al., 2004). In cats, a significant but less pronounced increase was also detected in the cerebral cortex but not in thalamus, preoptic area, serotonergic RN or cholinergic PPT (Porkka-Heiskanen et al., 2000). During recovery sleep, BF adenosine levels showed a slow decline yet remained significantly elevated after 3 h of recovery, whereas in cortex adenosine levels had already started to decrease during the SD procedure and reached baseline levels during recovery sleep (PorkkaHeiskanen et al., 2000).


Sleep deprivation and neurotransmitter receptors 1815 A short total SD of 3–6 h induced a selective increase in A1 adenosine receptor mRNA levels in the BF but not in cingulate cortex (Basheer et al., 2001), but 24 h of SD were required for membrane protein levels of A1 adenosine receptors to be elevated in the BF (Basheer et al., 2007). Consistently, brief (1–6 h) SD did not alter A1 receptor-dependent G-protein activity in the BF (Alanko et al., 2004) but the same SD protocol induced a rapid and transient increase in A1 receptor-dependent G-protein activity in frontal and cingulate cortex, reaching a maximal level after 2 h of SD and being completely negligible after 6 h. A positron emission tomography study found an increased A1 receptor expression throughout major brain regions in humans, with most prominent increases in prefrontal cortical areas (Elmenhorst et al., 2007). Although the exact role of the A1 receptor is still debated (see Landolt, 2008) it may be implicated in sleep regulation (Thakkar et al., 2003). Notably, its upregulation by SD in the face of enhanced adenosine levels goes against a process of desensitization, rather pointing to facilitated recruitment of receptors during SD. Such maintenance of receptor function has been described for G-protein-coupled receptors for hormones (Souaze´, 2001).

Systems involved in learning and memory Both human and animal studies increasingly document detrimental effects of SD on learning and memory processes (Graves et al., 2001; Peigneux et al., 2001; Benington & Frank, 2003; Walker & Stickgold, 2004; Boonstra et al., 2007; Marshall & Born, 2007). Here we focus on hippocampus-dependent learning and memory, for which the impact of sleep loss has been studied in greatest detail. Paralleling numerous in vitro studies showing altered synaptic plasticity after different SD procedures in rodents (see Kopp et al., 2006, and references therein), an increasing number of biochemical and electrophysiological studies supports the idea that SD alters the molecular composition of glutamatergic ionotropic N-methyl-Daspartate receptors (NMDARs), key players in the induction of synaptic plasticity. Western blot analyses performed on proteins from whole hippocampus showed that 24 h of REM SD, obtained with the single platform technique in adult mice, resulted in an increase in the intracellular pool of the NR1 subunit of NMDARs, whereas no changes were detected in the intracellular levels of NR2A and NR2B subunit proteins (Chen et al., 2006). A similar analysis performed on the hippocampus of adult rats sleep-deprived for 72 h by using the multiple platforms method showed increased intracellular NR1 and NR2A subunit protein levels (McDermott et al., 2006), suggesting that SD altered NMDAR turnover. Moreover, electrophysiological recordings revealed that 24–72 h of REM SD reduced the synaptic NMDAR:AMPAR ratio in the CA1 and dentate gyrus areas of the hippocampus (McDermott et al., 2003; Chen et al., 2006), probably due to an altered density of NMDARs but not AMPARs (McDermott et al., 2006). Substantiating the notion of an SD-induced trafficking of NMDAR subtypes, a stress-free total SD of adult mice resulted in an enhanced ratio of the NR2A ⁄ NR2B subunit composition of synaptic NMDARs in adult mice (Kopp et al., 2006). This enhancement was due to augmented membrane levels of the NR2A subunit as revealed by Western blot analysis on purified synaptosomal membranes, a subcellular fraction rich in synaptic proteins. In addition, a recent study using synaptoneurosomes, a tissue fraction rich in both membrane-bound and cytosolic synaptic proteins, showed that a substantial number of postsynaptic components of the excitatory synapse vary as a function of the recent sleep-wake behavior (Vyazovskiy et al., 2008). In particular, periods of spontaneous wakefulness were associated with higher levels of the NR2A

subunit protein in the hippocampus and with a widespread increase in the GluR1 subunit of AMPARs, a well established molecular correlate of synaptic strength (Collingridge et al., 2004). An SD lasting 8 h or spontaneous wakefulness also altered expression of genes encoding for AMPAR and NMDAR subunit proteins. In particular, in the cortex of adult rats, genes encoding for the NR2A subunit of NMDAR and for the GluR2 ⁄ 3 subunits of AMPARs were expressed at higher levels (Cirelli & Tononi, 2001). Altogether, essential elements of the central glutamatergic synapse, in particular ionotropic receptor subunits, appear to be strongly regulated by the sleep-wake behavior.

Conclusions Both molecular and electrophysiological studies described here begin to indicate that, throughout the brain, neurotransmitter receptor expression and ⁄ or function are sensitive to sleep loss. Some cases demonstrate that neurotransmitter receptors are affected rather dramatically by SD, for example those switching polarity in their signal transduction properties or those becoming more strongly expressed in neurons. In other cases more gradual effects are observed, for instance receptor desensitization. An important question in the future will be how the diversity of effects observed can be classified in terms of adaptive regulation of receptor functions and of their consequences for the activity of wake- and sleep-promoting centers (Table 1). While the exact functional implications of these alterations thus remain to be assessed, it is likely that they will influence excitability of the major brain areas involved in sleep-wake control and in memory processes. Such altered excitability has immediate implications for the maintenance of the arousal state and for cognitive performance. As already highlighted, a current limitation to the interpretation of SD-related alterations in neuronal properties is the diversity of SD procedures used, which often hampers the dissociation of procedurespecific effects from those caused by sleep loss. Although recent experiments increasingly use mild SD protocols that limit exposure to stress and unfamiliar environments, further standardization could be helpful. In particular, careful control for stress and activation of the hypothalamic-pituitary-adrenocortical axis should be applied. The manipulations adopted to maintain wakefulness should be specifically described. The duration of the SD should be physiologically relevant and should take species and age features into account. The reversibility of SD effects on the time scale of sleep-homeostatic processes should be a further important criterion. Such standardization should help significantly in reconciling studies focusing on similar brain areas and ⁄ or similar receptor types. Adaptive regulation of synaptic transmission and neuronal excitability is required to maintain adequate network performance in the face of constantly changing neurochemical and physiological environments (Turrigiano & Nelson, 2004). Although the mechanistic underpinnings of the receptor changes reported here are unknown, some of them are well consistent with an adaptive mechanism to counteract excessive activity. For example, the upregulation of inhibitory GABAA receptor subunits in the wake-active BF could be a result of prolonged discharge in the BF, which would be antagonized through enhanced synaptic inhibition. Furthermore, attenuation of 5HT1A autoreceptor function could be explained by receptor desensitization or downregulation. By contrast, the upregulation of A1 adenosine receptors in the BF that parallels increasing adenosine levels goes against the predictions of adaptive regulation to counteract excessive adenosinergic impact. It is interesting to note, however, that molecules implicated in cellular forms of adaptive plasticity, such as brain-derived neurotrophic factor, retinoic acid and tumor necrosis factor-a (Rutherford et al., 1998; Stellwagen & Malenka, 2006; Aoto


1816 F. Longordo et al. et al., 2008) are implicated in the homeostatic regulation of sleep intensity (Yoshida et al., 2004; Maret et al., 2005; Huber et al., 2007). Therefore, cellular and synaptic adaptations in response to SD, such as those adjusting neurotransmitter receptor function, could be related to the intensification of sleep after SD. As both wake-promoting (Ouyang et al., 2004; Cirelli et al., 2005) and sleep-promoting (Szymusiak et al., 2007) systems have been implicated in sleep homeostasis, altered neurotransmitter receptor function in these areas could possibly represent one aspect of their role in sleep regulation. In contrast to adaptive attenuation in sleep-wake control centers, glutamatergic synapses in the hippocampus show features of synaptic strengthening and, in some cases, long-term potentiation. In particular, the NR2A subunit of the NMDAR is repeatedly described to be present in increased amounts at synapses, although its synaptic integration has not been verified in all cases. NR2A-containing NMDARs can enhance trafficking of AMPARs (Kim et al., 2005) but may also limit further plasticity (Kopp et al., 2006; Yashiro & Philpot, 2008). From studies based on mild, stress-free SD (Kopp et al., 2006) or spontaneous waking (Vyazovskiy et al., 2008), the level of NR2A protein at synapses most consistently emerges as sensitive to the absence of sleep per se, and has recently been implicated in the SDinduced consequences on bidirectional synaptic plasticity (Longordo et al., 2007). An important question that remains to be clarified is the exact role of the NR2A-containing NMDAR subtype in plasticity, in particular in brain areas with different basal NMDAR composition. The rather short time span on which it appears at synaptic sites argues against a de novo synthesis of NR2A, but in favor of altered synaptic targeting of preexisting receptor subunits. The mechanisms underlying this redistribution, which could be influenced by wake-promoting monoaminergic or orexinergic receptors (Kopp et al., 2007), also await further study. Altogether, the links between sleep loss and neurotransmitter receptor function are just beginning to be recognized. Much further work is needed to evaluate their functional implications for sleep homeostasis and for learning processes. However, it goes without saying that neurotransmitter receptors are at center position to set a neuron’s readout of incoming stimuli and are thus well positioned to help us further in understanding what causes tiredness and how sleep alleviates it.

Acknowledgements This work is supported by the Swiss National Science Foundation (No. 3100A0-116006) and by the E´tat de Vaud. We thank all members of the laboratory and Dr M. A. Di Castro for useful discussions. We are grateful to Professor P. Clarke for constructive comments on the manuscript.

Abbreviations AMPAR, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor; BF, basal forebrain; LC, locus coeruleus; NMDAR, N-methyl-D-aspartate receptor; NREM, non-REM; PPT, pedunculopontine tegmenteum; REM, rapid-eyemovement; RN, raphe nuclei; SD, sleep deprivation; vlPOA, ventrolateral preoptic area.

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