The Impact of Sleep Deprivation on Neuronal and Glial Signaling Pathways Important for Memory and Synaptic Plasticity

Cellular Signalling 24 (2012) 1251–1260

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Cellular Signalling

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Review The impact of sleep deprivation on neuronal and glial signaling pathways important for memory and synaptic plasticity

Robbert Havekes a,1, Christopher G. Vecsey b,1, Ted Abel a,⁎ a Department of Biology, University of Pennsylvania, Philadelphia, USA b Department of Biology, Brandeis University, Waltham, USA article info abstract

Article history: Sleep deprivation is a common feature in modern society, and one of the consequences of sleep loss is Received 15 December 2011 the impairment of cognitive function. Although it has been widely accepted that sleep deprivation affects Received in revised form 13 February 2012 learning and memory, only recently has research begun to address which molecular signaling pathways Accepted 14 February 2012 are altered by sleep loss and, more importantly, which pathways can be targeted to reverse the memory Available online 21 February 2012 impairments resulting from sleep deprivation. In this review, we discuss the different methods used to sleep deprive animals and the effects of different durations of sleep deprivation on learning and memory Keywords: Sleep loss with an emphasis on hippocampus-dependent memory. We then review the molecular signaling pathways Learning that are sensitive to sleep loss, with a focus on those thought to play a critical role in the memory and synaptic Hippocampus plasticity deﬁcits observed after sleep deprivation. Finally, we highlight several recent attempts to reverse the Astrocytes effects of sleep deprivation on memory and synaptic plasticity. Future research building on these studies cAMP promises to contribute to the development of novel strategies to ameliorate the effects of sleep loss on Transcription cognition. © 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction ...... 1251 2. Methods for sleep deprivation in rodents: advantages and drawbacks ...... 1252 3. Sleep deprivation and memory ...... 1253 4. Sleep deprivation and hippocampal synaptic plasticity ...... 1253 5. Sleep deprivation, cholinergic, and GABAergic signaling ...... 1255 6. Sleep deprivation and cAMP signaling ...... 1256 7. Sleep deprivation, adenosine, and astrocytes ...... 1257 8. Sleep deprivation, gene transcription, and translation ...... 1258 9. Conclusions and future directions ...... 1258 Acknowledgments ...... 1259 References ...... 1259

Abbreviations: AMPAr, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid 1. Introduction receptors; CREB, cAMP response element binding protein; ERK, extracellular signal- regulated kinase; GABAr, gamma-aminobutyric acid receptor; LTP, long-term potentia- Millions of people worldwide experience sleep deprivation on tion; mAChr, muscarinic cholinergic receptor; nAChr, nicotinic acetylcholine receptor; a daily basis [1]. The pressure to stay up longer in our modern 24/7 NMDAr, N-methyl-D-aspartate receptor; PDE, phosphodiesterase; PDE4, cyclic AMP specific phosphodiesterase-4; PKA, cyclic AMP-dependent protein kinase A; REM society impacts a growing percentage of the population [2,3].A sleep, rapid eye movement sleep; Rolipram, 4-[3-(cyclopentyloxy)-4-methoxyphenyl]- population-based study indicated that, over the past 50 years, sleep 2-pyrrolidinone; SNARE, N-ethylmaleimide-sensitive factor attachment protein receptor. duration in adult and adolescent Americans has decreased by 1.5–2h ⁎ Corresponding author at: Department of Biology, University of Pennsylvania, per night in adults and adolescents, with 30% reporting sleep of 6 h 222 Lynch Labs, 433 South University Avenue, Philadelphia, PA 19104, USA. Tel.: +1 per night or less [4]. 215 898 3100; fax: +1 215 573 1297. fi fi E-mail address: [email protected] (T. Abel). One of the rst indications that sleep might be bene cial for the 1 These authors contributed equally to this work. formation of memories came from a study by Jenkins and Dallenbach

[5] that showed that sleep attenuated the rate of forgetting. In the the researcher, with the result that gentle handling is rarely carried 1960s, Morris and colleagues found that sleep deprivation impaired out for longer than 12 h. memory processing [6]. In the decades thereafter, it became apparent Because the gentle handling method involves direct contact in both humans and animal models that specific forms of memory between the researcher and the cage or the animal itself, a concern is are affected by sleep deprivation [7–10]. To combat the effects of whether the memory deficits resulting from sleep deprivation by gentle sleep deprivation, it is critical to understand the molecular and handling are due to sleep loss or non-specific side effects of the tech- cellular mechanisms by which sleep deprivation leads to cognitive nique itself. To test this issue, Hagewoud and colleagues [21] trained deficits. Here, we review current knowledge of the intracellular animals at the beginning of the resting phase (in rodents, this is the signaling pathways that are affected by sleep deprivation, with an light phase) and recorded the amount of stimulation needed to keep emphasis on the impact of sleep deprivation on hippocampal function them awake during the following 6 h. Next, the authors trained a new (see Fig. 1 for a schematic summary). In addition, we discuss the dif- cohort of rats at the end of the resting phase and determined whether ferent approaches that have been developed to reverse memory and giving the animals the same amount of stimulation during the first 6 h plasticity deficits induced by sleep deprivation. of the active phase (in rodents, this is the dark phase, in which they spend only a short time sleeping) would also induce memory deficits. 2. Methods for sleep deprivation in rodents: advantages They found that giving the same amount of stimulation during the and drawbacks first 6 h of the active phase, in contrast to the resting phase, did not induce a memory deficit. These findings indicated that the memory To elucidate which cellular and molecular effects of sleep depriva- deficits observed after sleep deprivation in the light phase were not tion lead to memory impairments, many research laboratories have likely a consequence of the gentle handling method used to sleep utilized rodents as study objects. Three primary techniques have been deprive animals but rather a consequence of sleep loss. used to deprive laboratory rodents of sleep. Each of these methods In some other sleep deprivation studies, the introduction of novel has particular advantages and drawbacks, as discussed below. objects or new nesting material is used to keep animals awake, or The first is the platform-over-water, pedestal, or “flower pot” animals are allowed to explore novel environments [22,23]. Although method, which is the best method to selectively deprive animals of these techniques have successfully kept animals awake without ele- rapid eye movement (REM) sleep for one or multiple days with vating plasma corticosterone levels [23], they may be problematic only intermittent monitoring by the researcher [11]. Animals are when the goal is to study the effects of sleep deprivation on memory placed in a chamber with one or multiple small platforms surrounded or synaptic plasticity. For example, it has been reported that the by water. When the animals enter REM sleep, their muscle tone exploration of new environments and new objects facilitates hippo- diminishes and the animals fall into the water, waking them up and campal LTD and occludes LTP in vivo [24,25], increases the phosphor- preventing them from going into REM sleep. For control animals, ylation of NMDA and AMPA receptor subunits, and activates ERK1/2 the small platforms are replaced with larger ones, allowing them to signaling [26]. Furthermore, nest building behavior has been reported enter REM sleep without falling into the water [12]. The concern to accelerate REM sleep generation [27]. It would be useful, as sug- with this particular method is that large-platform control animals, gested by Hagewoud and colleagues [21], to separate the effects of ex- which obtain normal amounts of REM sleep, also show some alter- posure to a novel environment, novel objects, or new nesting material ations in neuronal function (see Section 4 of this review). This from the effects of prolonged wakefulness on plasticity and memory, suggests that aspects of the method other than the loss of REM sleep perhaps by applying these methods of sleep deprivation during the are responsible for some of the phenotypes observed after using the animals' active phase, in which they naturally sleep much less. platform-over-water method [13]. A recurring matter of debate in the field of sleep research is how The second method of sleep deprivation utilizes forced locomo- many of the molecular and cellular changes observed after sleep tion, in which the animal is placed in a chamber with a revolving deprivation can be attributed to stress, rather than to sleep loss itself. floor or rotating drum that forces the animal to reposition itself with We believe that the role of stress as the major factor in the memory each revolution [14,15]. This method can be tailored to achieve total and plasticity deficits observed particularly after brief sleep depriva- deprivation or selective deprivation of a particular sleep stage. Control tion is overstated. Two recent studies in which the activation of the animals can be manipulated to move just as much as deprived ani- stress signaling pathway is temporarily or permanently disrupted mals, but with longer periods of rest in between, thereby preventing have shown that sleep deprivation leads to cognitive impairments excessive sleep loss. For example, the schedule for rotation of even in the absence of stress-mediated signaling [28,29]. Also, the the chamber for a sleep-deprived animal might be 10 s on, 30 s off, changes in plasma corticosterone levels during brief sleep deprivation whereas the schedule for a control animal might be 10 min on, by gentle handling in many cases do not reach levels beyond those 30 min off. Thus, the control animal has repeated periods of 30 min observed during the stress hormone's natural circadian oscillation during which it can sleep undisturbed, but the sleep-deprived animal [30], during exploration of a novel environment containing novel ob- has a maximum of 30 s to rest before it is forced to move again (see jects [31], or due to exposure to a conspecific of the opposite sex [32]. [16]). This technique is often used to model sleep fragmentation, In some cases, the mildly increased plasma corticosterone levels ob- such as which might occur due to sleep apnea, caring for an infant served after sleep deprivation do not even reach statistical significance throughout the night, or otherwise fitful sleep. The interpretation of [18,19,21,33]. Further if brief sleep deprivation acts as a mild stressor, the effect of sleep deprivation using this technique can be challenging one might expect that this would enhance memory, as mild increases and depends on using the correct control groups, because locomotor in glucocorticoids have been shown to facilitate rather than impair activity or stress evoked using this technique may mask or reverse memory and plasticity formation [34]. Thus, although mild stress is the effects the effects of sleep deprivation. inherent to most forms of sleep deprivation in the laboratory as in The third sleep deprivation method is based on gentle handling the real world, it is unlikely that this stress component can explain the or mild stimulation. In this technique, researchers make mild noises, effects of sleep deprivation on the molecular signaling pathways that gently jostle the animal's home cage, disturb the animal's nesting result in impairments in memory and synaptic plasticity. material, and in some cases stroke the animal [17–19]. Gentle In summary, the sleep deprivation techniques described above all handling is very effective at inducing total sleep deprivation as have particular advantages, but each also has its limitations. Because determined by electroencephalography [20], and seems to be a strong of the different characteristics of each of these sleep deprivation model of typical sleep deprivation in humans. One downside of the methods, it is important to consider the technique and duration of gentle handling technique is that it requires constant vigilance by sleep deprivation used in each of the studies described below. R. Havekes et al. / Cellular Signalling 24 (2012) 1251–1260 1253

3. Sleep deprivation and memory Because the effects of sleep deprivation can be brain region-specific, the negative effects of sleep deprivation on memory can be subtle Using the platform-over-water method, Fishbein and colleagues and sometimes difficult to assess due to compensation by other less- [35] tested the effects of 24 h of REM sleep deprivation immediately impacted brain regions. For example, Hagewoud and colleagues [18], prior to training in a classical conditioning task called inhibitory showed that five hours of sleep deprivation immediately after each avoidance. Although 24 h of REM sleep deprivation had no effect on daily training session did not affect directly the rate at which mice acquisition and short-term memory, long-term memory (tested learned the location of a food-reward in a two-arm reference task. 1–7 days after training) was significantly impaired [35,36]. Follow- However, sleep deprivation did induce a shift in the behavioral strategy up studies applying REM sleep deprivation directly after conditioning used to locate the food reward: whereas non-sleep-deprived mice also attenuated memory formation [37–39], indicating that REM favored to use a hippocampus-dependent strategy, mice subjected to sleep deprivation was particularly detrimental for memory consolida- sleep deprivation switched from using a hippocampus-dependent tion (reviewed in [40]). spatial strategy to using a hippocampus-independent strategy necessi- A series of experiments conducted years later showed that mem- tating the striatum. Because this strategy could also be used to locate ory consolidation requiring the hippocampus is particularly sensitive the food-reward, the effect of sleep deprivation did not become appar- to sleep deprivation. This was first shown using the Morris water ent during training. Surprisingly, although sleep deprivation during maze, a task that can be designed to assess learning and memory for- training did not directly affect performance during the course of mation that are either hippocampus-dependent (the hidden platform training, it did impair performance during consecutive reversal training version of the task), or hippocampus-independent (the visible plat- (in which the food reward was relocated to the previously non-baited form version of the task) [41]. Twelve hours of REM sleep deprivation arm). The authors argued that the delayed effect of sleep deprivation directly after training in the hippocampus-dependent hidden plat- on performance was due to the shift from using a hippocampus- form version of the Morris water maze task impaired long-term dependent to a striatum-dependent strategy during the initial training. memory formation for platform location, tested 24 h after training. Because the striatal system is less flexible, it was more difficult for sleep- In contrast, the 12 h of REM sleep deprivation did not affect perfor- deprived mice to adapt the previously formed but now incorrect mance or memory in the hippocampus-independent visible platform memory for the location of the food reward [18]. Thus, this study con- version of the same task. REM sleep deprivation from 13 to 24 h after cluded that sleep deprivation can lead to the use of alternative learning training did not affect memory formation for either task, suggesting mechanisms and brain regions. However, there can be consequences that the consolidation period following training is specifically sensi- of using these alternative suboptimal mechanisms, such as a greater tive to sleep loss [42]. The same laboratory then elaborated on difficulty with updating outdated memories. this finding by using shorter periods of deprivation, and found that Cortical regions important for memory formation are affected REM sleep deprivation during the first 4 h directly after training in by sleep loss as well [22,54]. For example, sleep deprivation affects the water maze had a negative effect on hippocampus-dependent the consolidation of memory for objects (referred to as recognition spatial learning and memory formation, but not on hippocampus- memory) [31,55]. Object recognition is a process that, depending on independent learning and memory formation [42]. Although some the training conditions, does not critically rely on the hippocampus laboratories have confirmed the finding that REM sleep deprivation [56,57], but rather requires extra-hippocampal cortical regions in- impairs hippocampus-dependent learning in the Morris water maze cluding the perirhinal cortex [57] and insular cortex [58]. In line task [43–46], others have challenged the idea that REM sleep is essen- with these observations in rodents, studies in humans have shown tial for spatial learning in the Morris water maze [47,48]. that sleep deprivation affects cortical systems involved in a variety of Like the Morris water maze task, the fear conditioning task functions, including executive attention, working memory, and higher is a learning paradigm that enables researchers to examine both cognitive abilities (reviewed in [59]). hippocampus-dependent and hippocampus-independent learning An important finding in behavioral studies of the effects of sleep processes. The ability to associate a foot shock with the specific deprivation is that there are particular periods following learning environment, or context, in which the shock was administered is a during which sleep deprivation impacts memory consolidation. process dependent on the hippocampal formation [49,50]. In contrast, The timing of these windows differs depending on the nature of the learning to associate a shock with an explicitly paired cue, such as a training [42,60], but most studies have found that sleep deprivation tone, does not require the hippocampus (for review, see [49,50]). has the greatest detrimental effect when applied within 5–6 h after Five hours of total sleep deprivation using the gentle handling method training in the light period [21,30,31,51,55,61–64]. immediately following training caused a selective impairment in In the case of contextual fear conditioning, 5 h of sleep deprivation contextual, but not cued memory [51]. The finding that consolidation at the beginning of the resting phase (light phase in rodents) im- of contextual fear memories is sensitive to sleep deprivation has been paired memory consolidation when applied directly after training, confirmed in many other studies [21,30,52,53]. but when the period of sleep deprivation was delayed by 5 h, memory More recently, Hagewoud and colleagues [33] determined whether for contextual fear was not affected [51]. This critical time window hippocampus-dependent working memory was affected by 6–12 h of coincides with the consolidation period following learning when total sleep deprivation using the gentle handling method. Mice were transcription, translation, and cAMP-PKA signaling are required for deprived of sleep for 6 or 12 h using the gentle handling method, and memory consolidation in this task (reviewed in [65]), which has immediately at the end of the sleep deprivation period were allowed suggested that sleep deprivation might disrupt these crucial molecu- to explore 2 out of 3 accessible arms in a symmetrical Y maze for lar processes. To address this possibility, many laboratories have 10 min. After the training session in the novel arm recognition task, examined the effects of sleep deprivation on hippocampal synaptic mice remained in their home cage for 2 min followed by a 5 minute plasticity to determine which molecular mechanisms that are critical test session in which all arms were accessible. Mice deprived of sleep for memory consolidation are affected by (brief) sleep deprivation. for 12 h spent a similar time exploring the novel and familiar arm. This indicated that the sleep-deprived mice failed to discriminate 4. Sleep deprivation and hippocampal synaptic plasticity between the familiar versus novel arms of the maze, suggesting that hippocampus-dependent working memory was impaired. In summary, As described above, behavioral studies looking at the effect of the studies described above indicate that hippocampus-dependent sleep deprivation on memory indicated that the hippocampus is memory formation and working memory are particularly sensitive to particularly vulnerable to sleep loss. Therefore, electrophysiological (brief) sleep deprivation. studies were conducted to determine the effect of sleep deprivation 1254

Baseline Sleep Deprivation P P Nucleus P P Nucleus CREB CREB CREB CREB

CRE CRE TARGET GENES CRE CRE TARGET GENES MAP kinase MAP kinase

PKA PKA

Effector 1251 (2012) 24 Signalling Cellular / al. et Havekes R. molecules PDE4A5 PDE4A5

cAMP cAMP

Ca2+ Ca2+ Hippocampal Hippocampal Gs Gi Gs Gi Neuron Neuron

Adenylyl Adenylyl Ca 2+ Cyclase Ca 2+ Cyclase

Adenosine Adenosine AMPAr NMDAr mAChr GABAr AMPAr NMDAr mAChr GABAr – 1260

Glutamate ACh GABA Glutamate ACh GABA

Astrocyte Astrocyte

Neuron Neuron Neuron Neuron

Fig. 1. A schematic overview of hippocampal signaling pathways whose modulation by sleep deprivation may contribute to the effects of sleep deprivation on memory formation. (A) Signaling pathways under baseline conditions. (B) Sleep deprivation has been reported to reduce glutamatergic signaling and cholinergic signaling while increasing adenosine levels and GABAergic signaling. Sleep deprivation also attenuates cAMP signaling and CREB-mediated gene transcription. All of these molecular events are shown in a single connected pathway in order to demonstrate how the effects of sleep deprivation could potentially interact to impact learning and memory. Dashed black lines and black arrows pointing down indicate attenuation of the signaling pathway. Red lines and upward pointing arrows indicate an increase of the signaling pathway. R. Havekes et al. / Cellular Signalling 24 (2012) 1251–1260 1255 on various forms of hippocampal synaptic plasticity. One prominent to the hippocampal plasticity deficits observed after longer periods form of hippocampal synaptic plasticity is long-term potentiation of sleep deprivation [68]. (LTP), a long-lasting change in the strength of synaptic connections As mentioned above, partial LTP deficits have been observed in that is a frequently used model to study the mechanisms underlying large-platform control animals, suggesting that aspects of the treat- learning and memory. Despite using different methods to study hip- ment other than sleep deprivation may have been somewhat respon- pocampal synaptic plasticity, it is generally agreed that sleep depriva- sible for the impairment of LTP [13]. This effect could potentially also tion attenuates LTP in the hippocampus. In rats, the use of forced be mediated by the strong stress hormone induction that builds over locomotion to induce total sleep deprivation for 12 h [15], or sleep successive days of platform-over-water treatment in both control and fragmentation for 24 h [66], impaired hippocampal LTP recorded sleep-deprived animals (for a more in-depth discussion of the role of from Schaffer collateral CA1 synapses. Similar LTP deficits were also stress in long-term sleep deprivation experiments, see [76]). produced by REM sleep deprivation using the platform-over-water Although multiple studies have described changes in NMDAr technique for 24 to 75h in area CA1 both in vitro and in vivo subunit composition and function after long-term sleep deprivation, [52,67–70]. Shorter durations of REM deprivation for 3, 6, or 9 h also the debate remains whether brief sleep deprivation induces similar impaired LTP in vivo in a “dose”-dependent fashion when recordings changes in NMDAr composition and function, and whether these were performed immediately after the deprivation period [13]. Schaffer changes underlie the deficits in plasticity and memory that have collateral LTP at CA1 synapses was also attenuated by shorter periods been reported after brief periods of sleep deprivation. A study by (~4–6 h) of total sleep deprivation induced by either exposing mice Kopp et al. [23] described changes in NMDAr composition and func- to a novel environment [23] or gentle handling [30].InalloftheLTP tion after only 4 h of total sleep deprivation. In contrast with this studies described thus far, sleep deprivation was performed prior to study, a recent report by Vecsey et al. [30] that applied 5 h of sleep the induction of plasticity. Two in vivo studies using recordings from deprivation did not observe any changes in NMDAr function in area the dentate gyrus in non-anesthetized rats found that REM-specific CA1. It is important to note that these studies used different methods sleep deprivation by gentle handling performed after the induction of of sleep deprivation, which may explain this discrepancy. Kopp and LTP caused significant deficits in LTP maintenance [71,72]. Importantly, colleagues [23] used exposure to a novel environment and ad libitum when the same method was used to specifically deprive animals of access to nesting material to keep mice awake, whereas Vecsey et al. non-REM sleep, no LTP deficits in the dentate gyrus were observed [30] used the gentle handling method. [72], suggesting that REM sleep plays a critical role in the maintenance NMDA receptors are only required during LTP induction, but not of LTP in the dentate gyrus. during the maintenance of LTP [77] (reviewed in [78]). As described In contrast to the abundance of studies looking at the effects of above, changes in NMDAr composition and function, affecting the in- sleep deprivation on LTP, there are only three studies to our knowl- duction of LTP, have been particularly prominent in studies applying edge that examine the effect of sleep deprivation on LTD. Tadavarty long-term sleep deprivation [52,68,75]. To determine whether sleep et al. [73] deprived rats of sleep for 12 h using the gentle handling deprivation impairs the maintenance of LTP in the dentate gyrus, method and then induced LTD in rat CA1 slices with 30 s of 20 Hz Ishikawa et al. [72] deprived rats of REM sleep for 24–48 h, in this stimulation to the Schaffer collaterals. They found that LTD was case by stroking with a brush starting directly after LTP induction. enhanced in hippocampal slices from sleep-deprived rats. In a They found that LTP in the dentate gyrus was still impaired, suggesting follow-up study, the authors showed that the enhancement of LTD that the molecular processes underlying the maintenance of LTP are was accompanied by elevated expression and heterodimerization sensitive to sleep loss. In line with the latter in vivo study, several of γ-aminobutyric acid (GABA)B and metabotropic glutamate 1α sleep deprivation studies using hippocampal slices have shown that receptors in the hippocampus [74]. 4–5 h of sleep deprivation can impair the maintenance phase of LTP In line with these observations, Kopp and colleagues [23] reported without affecting its induction [30,71,79]. In conclusion, although that 4 h of sleep deprivation attenuated LTP induced by 2×100-Hz modulation of NMDAr function has been found to be modulated by trains, while it facilitated LTD induced by stimulations in the theta sleep deprivation, additional cellular signaling mechanisms are likely frequency range (5 Hz). No effect of sleep deprivation was observed to be altered by sleep loss, particularly in the case of brief periods of on LTD induced by 1-Hz stimulation [23]. Together, these studies sleep deprivation, as described below. examining the effects of sleep deprivation on synaptic plasticity suggest that sleep deprivation inhibits synaptic potentiation in the hip- 5. Sleep deprivation, cholinergic, and GABAergic signaling pocampus (but see [22]), while it facilitates certain forms of synaptic depression. In the paragraphs below, we will discuss the role of specific The cholinergic system plays a critical role in memory formation signaling pathways that may contribute to the plasticity changes ob- (reviewed in [80,81]) and is a major modulator of neuronal activity served after sleep deprivation. (reviewed in [82]). Ninety-six hours of REM sleep deprivation increases While many studies have assessed the effect of sleep deprivation acetylcholinesterase (the enzyme that breaks down acetylcholine) on synaptic plasticity, only a few studies have attempted to identify in the pons, thalamus, and medulla oblongata, but not in other brain which specific molecular signaling pathways mediate the changes in regions including the hippocampus [83]. It is important to note that synaptic efficacy observed after sleep deprivation. Two studies from the pons contains cholinergic cells involved in the generation of the same laboratory suggested that 24 h of REM sleep deprivation REM, while the thalamus and medulla oblongata receive cholinergic in mice disrupted the function of N-methyl-D-aspartate receptors input from the pons. The higher levels of acetylcholinesterase suggest (NMDAr) in the dentate gyrus [75] and 72 h of REM deprivation in that there is a higher turnover of acetylcholine in these regions as a rats disrupted NMDAr function in CA1 [68]. Specifically, both reports consequence of sleep deprivation. Receptor expression studies have found decreased NMDAr/2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4- indicated that REM sleep deprivation reduces muscarinic M2 choliner- yl) propanoic acid receptor AMPAr current ratios that were attributed gic receptors in the pons and hippocampus [84]. to decreased surface expression of NR1 subunits. Reduced hippocam- There have been efforts to boost cholinergic activity with the pal NR1 expression was also reported in a later study in which aim to reverse the effect of sleep deprivation on memory formation. rats were deprived of REM sleep for 75 h [67]. The most intriguing Activation of nicotinic acetylcholine receptors, in particular the α7 finding in the experiments by McDermott et al. [68] was that applica- nACh receptor, through systemic application of nicotine, was found tion of glycine, which enhances NMDAr function, reversed the to rescue the memory deficits in the hippocampus-dependent radial effects of 24–48 h of sleep deprivation on LTP in area CA1. Thus, arm maze task observed after 24 and 48 h of REM sleep deprivation attenuated NMDAr function appears to be a functional contributor using the platform-over-water method [85,86]. In addition to rescuing 1256 R. Havekes et al. / Cellular Signalling 24 (2012) 1251–1260 hippocampus-dependent memory consolidation, nicotine treatment handling method caused specific disruptions of the cAMP-PKA path- during REM sleep deprivation reversed the impairment in hippocam- way in the hippocampus, without affecting NMDA receptor function pal LTP in vivo in area CA1 and in the dentate gyrus [86]. The authors in area CA1 as determined using whole-cell recordings. offered two hypotheses for how nicotine treatment might be revers- Other studies looking at downstream targets of the cAMP-PKA ing the effects of sleep deprivation on memory and plasticity: 1) nico- pathway have also suggested that this pathway is sensitive to sleep tine treatment activates pre-synaptic nicotinic receptors, leading to deprivation. Although longer periods of sleep deprivation were ap- elevated glutamate release from pre-synaptic terminals [87], thereby plied, both Ravassard et al. [67] and Hagewoud et al. [33] found that increasing the activity of excitatory neurons, and 2) nicotine treat- AMPA receptor GluR1 (or GluA1) serine 845 phosphorylation, a target ment could facilitate activity of excitatory neurons through desensiti- substrate of the cAMP-PKA pathway that controls AMPA receptor zation of α7 nACh receptors in GABAergic neurons reducing the function [98], was reduced after 75 h of REM sleep deprivation using release of GABA [88]. Finally, Aleisa et al. [89] showed that chronic the platform-over-water method and 12 h of total sleep deprivation nicotine treatment reversed stress-induced reductions in protein respectively. However, Vyazovskiy et al. described opposite results levels of the brain-derived neurotrophic factor (BDNF), a key protein [22]. They found that 4 h of spontaneous or enforced wakefulness in hippocampal synaptic plasticity [90]. Like stress, sleep loss can led to elevated levels of AMPA receptor GluR1 S845 phosphorylation lead to reductions in hippocampal BDNF protein levels in the hippo- in both hippocampus and cortex. These different observations may campus [91] and more specifically in the dentate gyrus [92]. However, be due to the length of the sleep deprivation period. to our knowledge, no experiments have been conducted to assay A second PKA target that has been studied in relation to the effects whether nicotine treatment reverses the sleep deprivation-induced of sleep deprivation on plasticity and memory is the cAMP response decrease in BDNF protein levels, or whether changes in BDNF content element binding protein (CREB), a transcription factor playing a cru- play a critical role in the rescue of sleep deprivation-mediated memory cial role in memory and plasticity (for review see [99]). Vecsey et al. impairments. In conclusion, although the rescue of sleep deprivation- [30] showed that brief sleep deprivation reduced hippocampal CREB induced memory deficiencies by nicotine is exciting, much work phosphorylation of the serine 133 site in area CA1 and the dentate remains to ascertain the molecular mechanisms by which nicotine gyrus of the hippocampus. Other studies have also found decreased treatment ameliorates sleep loss-induced cognitive deficits. hippocampal CREB phosphorylation at serine 133, although longer Fast synaptic inhibition in the adult brain is primarily mediated by periods of sleep deprivation were applied [70,100]. A recent study

γ-aminobutyric acid receptors (GABArs). Regulation of GABAAr sur- by Hagewoud et al. [18] looked at the effect of daily sleep deprivation face expression at synapses – a process that is critical for maintaining for 5 h on learning-induced changes in hippocampal and striatal the correct level of synaptic inhibition – is important for memory phosphorylation of CREB at serine 133. The authors found that perfor- consolidation [93]. Wang and Li [94] reported higher GABA levels in mance in a Y-maze reference memory task was not affected by 5 h of cortex, hypothalamus, and brain stem after 72 h of sleep deprivation sleep deprivation after each daily training session. However, sleep- in mice. This suggested that sleep deprivation might increase GABA deprived mice avoided using a hippocampus-dependent strategy tone, leading to increased GABAergic signaling and a suppression and preferred to use a striatum-dependent response strategy to locate of activity of excitatory neurons. Modirrousta et al. [95] showed that the food reward. In line with the shift in behavioral strategy used to expression of the GABAr β2–3 subunit is enhanced in cholinergic locate the reward, the training-induced increase in CREB serine 133 cells in the basal forebrain after sleep deprivation, suggesting that phosphorylation shifted from hippocampus to striatum indicating one way through which prolonged wake reduces cholinergic activity that daily brief sleep deprivation attenuated CREB function in the hip- is through higher GABAergic activity. Increases in GABABr receptor pocampus and shifted it to the striatum. A question that remains to be protein levels in hippocampal lysates after 12 h of sleep deprivation answered is whether sleep deprivation induces a shift in the behavioral using the gentle handling method have also been reported by others strategy used, thus leading to a change in CREB function, or alternative- [74]. It remains to be elucidated whether other neurotransmitter ly, whether sleep deprivation directly limits CREB signaling leading to systems besides the cholinergic system are also inhibited after sleep the change in behavioral strategy used. deprivation through elevation of GABAergic activity. It should be noted that these reductions in CREB serine 133 phosphorylation are not necessarily uniform for the entire brain. Cirelli and 6. Sleep deprivation and cAMP signaling Tononi [54] reported that keeping animals awake for 1–9 h through the introduction of novel objects in their recording cage elevated As mentioned previously, sleep deprivation can impair the main- CREB phosphorylation in the cortex. Furthermore, Vecsey et al. [30] tenance of LTP without affecting LTP induction [30,71,72,79]. These reported that CREB phosphorylation in the amygdala was not affected observations suggest that specific intracellular signaling pathways by 5 h of sleep deprivation using the gentle handling method. important for the maintenance of LTP are affected by brief sleep Both the cAMP-PKA and the extracellular signal-regulated kinase deprivation. To determine which pathways are impacted, the Abel (ERK, also known as MAPK) pathway critically regulate changes in laboratory conducted a set of electrophysiological experiments with synaptic efficacy important for memory formation [101–103], and differing molecular requirements [30]. The authors found that 5 h of crosstalk between both pathways has been described [104] through sleep deprivation impaired spaced four-train LTP, theta-burst LTP, the exchange protein activated by cAMP (Epac) and Ras [105]. Because and forskolin-induced potentiation [30]. These forms of LTP have sleep deprivation attenuates hippocampal cAMP levels [30], one the common feature that they are all dependent on the cAMP-PKA would hypothesize that sleep deprivation would also indirectly affect pathway, a pathway generally acknowledged to play a critical role the ERK pathway. Indeed, Ravassard et al. [67] showed that 75 h of in memory consolidation [96] (reviewed in [97]). Importantly, one- sleep deprivation using the platform-over-water method reduced train LTP and massed 4-train LTP, both PKA-independent forms of ERK P44/P42 phosphorylation in area CA1. Likewise, in an earlier plasticity, were not affected by 5 h of sleep deprivation. Biochemical study, Guan et al. [44] found reductions in ERK P44/P42 phosphoryla- analysis of hippocampal CA1 mini slices indicated that both basal tion after 6 h of total sleep deprivation. and forskolin-induced cAMP levels were reduced after 5 h of sleep Because the sole means to inactivate cAMP is through degradation deprivation. Thus, in contrast to studies, using longer periods of by cAMP phosphodiesterases (PDEs) (reviewed in [106,107]), Vecsey sleep deprivation allowing exploration of novel environments and et al. [30] determined whether the impairments in hippocampal ad libitum access to nesting material to achieve sleep deprivation in Schaffer collateral LTP observed after 5 h of sleep deprivation could which changes in NMDA receptor function were observed, Vecsey be reversed by bath application of the general PDE inhibitor IBMX et al. [30] showed that brief 5 hour sleep deprivation using the gentle or the PDE4-specific inhibitor rolipram. Indeed, blocking PDE4 R. Havekes et al. / Cellular Signalling 24 (2012) 1251–1260 1257 activity rescued the LTP deficits induced by sleep deprivation. To neostriatum [119]. Adenosine levels have also been reported to decline determine whether PDE4-mediated degradation of cAMP induced during sleep [120]. A direct link between increased adenosine levels and by sleep deprivation was also the cause of contextual fear memory elevated sleep drive came from a study by Porkka-Heiskanen et al. deficits, the authors repeated the contextual fear conditioning exper- [120], which showed that sleep deprivation or pharmacologically iment with systemic administration of the PDE4 inhibitor rolipram elevating adenosine levels, both led to similar changes in sleep/wake during the 5 h of sleep deprivation. In line with the LTP findings, patterns: a reduction in wakefulness and an increase in slow wave rolipram treatment rescued the sleep deprivation-induced memory sleep. With prolonged wakefulness, elevation of extracellular adeno- deficit for contextual fear. sine levels leads to activation of the adenosine A1 receptor, which in The rescue of plasticity and behavior in sleep-deprived animals turn attenuates transmitter release of surrounding excitatory presyn- with PDE inhibitors suggested that elevated degradation of cAMP aptic terminals by increasing potassium currents [121]. Conditional de- might be responsible for the effects of SD on cAMP signaling. Indeed, letion of the adenosine A1 receptor from forebrain neurons reduces biochemical analysis of hippocampal tissue indicated that 5 h of sleep the rebound in slow wave activity observed in wild-type animals deprivation led to the upregulation of both PDE4 enzymatic activity [122]. The adenosine A1 receptor primarily couples to Gi proteins, and the protein levels of one specific PDE4 isoform, PDE4A5, rather which inhibit the production of cAMP [114,115]. Therefore higher levels than an upregulation of all of the 20 described PDE4 isoforms [30]. of adenosine, as a consequence of sleep loss, may lead to increased Gi Each PDE4 isoform has a unique N-terminal region resulting in distinct signaling, thereby attenuating cAMP levels. In addition to affecting the intracellular localization and compartment-specific local degradation cAMP pathway, changes in adenosine levels may also alter the activity of cAMP (reviewed in [106,107]). Therefore, the finding that sleep of the cholinergic neurons in the basal forebrain, which appear to deprivation specifically increases protein levels of the PDE4A5 isoform be central in the regulation of recovery sleep after sleep deprivation may indicate that sleep deprivation reduces cAMP in specific PDE4A5- [120,123,124]. containing intracellular compartments rather than degrading cAMP in One intriguing topic that has only recently received attention is to a global fashion. The next challenge will be to determine the molecular identify which brain cells are responsible for the build up of adeno- consequences of sleep deprivation-mediated changes in PDE4A5 ac- sine after sleep deprivation. In addition to neurons, astrocytes have tivity in the hippocampus. A first step will be to determine the activity been shown to release transmitter molecules such as glutamate, ATP, of proteins known to interact with PDE4A5, which will help identify and D-serine, thereby altering neuronal function [125–128]. These which PDE4A5-containing signaling complexes and pathways are have been termed ‘gliotransmitters’, although their role in vivo re- altered by sleep loss. These proteins include MK2 (also known as mains a topic of debate [129]. Transmitter release from astrocytes is MAPK-activated protein kinase 2), which phosphorylates PDE4A5 triggered by elevation of intracellular calcium levels, and is suggested thereby reducing its activity [108], caspase-3, which causes N- to require the formation of a soluble N-ethylmaleimide-sensitive fac- terminal cleavage of PDE4A5 [109], and the immunophilin XAP2, tor attachment protein receptor (SNARE) complex between the target which inhibits PDE4A5 activity [110]. Another challenge will be to de- membrane and vesicles [130]. Pascual et al. [128] overexpressed the velop pharmacological agents that specifically target PDE4A5 (or its cytosolic portion of the SNARE domain of synaptobrevin 2 (DN- human ortholog PDE4A4 [106]) and determine whether the cognitive SNARE) selectively in astrocytes, thereby preventing release of glio- impairments induced by sleep loss can be reversed by targeting this transmitters from astrocytes. Using the same mouse model, Halassa particular PDE4 isoform without affecting the other PDE4 isoforms et al. [55] determined whether attenuating release of gliotransmitters in both mice and humans. This approach of developing drugs that prevented the effect of brief sleep deprivation on memory formation. target specific PDE4 isoforms is currently underway with the hope of They found that mice expressing the DN-SNARE protein did not have providing novel therapeutic strategies for the treatment of inflammato- any rebound sleep after brief sleep deprivation and also showed no ry diseases including asthma, chronic obstructive pulmonary disease, memory deficits as a consequence of sleep deprivation immediately psoriasis, and depression [111]. following training in a hippocampus-independent learning task. Treatment with caffeine has also been reported to reverse the These findings indicate that astrocytes are potentially implicated in effects of sleep deprivation on hippocampal synaptic plasticity and the build-up of adenosine after prolonged wakefulness. The authors memory [70,92]. It is important to note that one of the many biological hypothesized that the crucial gliotransmitter mediating the effects actions of caffeine is the inhibition of several PDE families (e.g. PDE1, of sleep deprivation on memory was ATP, which is quickly converted PDE4 and PDE5) (reviewed in [112]). Thus, one potential mechanism to adenosine after release [131] and acts through the adenosine A1 through which caffeine treatment could reverse the effect of sleep receptor. Chronic infusion of the adenosine A1 receptor antagonist deprivation is through inhibition of PDE4 activity, thereby facilitating (8-cyclopentyl-1,3-dimethylxanthine (CPT)) into the brain prevented cAMP-PKA signaling [30]. Another hint that caffeine may indeed act on sleep deprivation-induced deficits in hippocampus-independent the cAMP-PKA pathway came from recent work in flies [113], which memory formation. Subsequent research has demonstrated that found that caffeine reduced and fragmented sleep in flies, and sleep deprivation-induced impairments in hippocampal LTP and that those effects were mitigated in flies with reduced PKA activity. memory are also rescued by DN-SNARE expression or by targeted Caffeine could also prevent the effects of sleep deprivation on memory CPT infusion into the hippocampus [79]. Therefore, astrocyte-derived and plasticity by antagonizing adenosine receptors (reviewed in adenosine induced by sleep deprivation may impact synaptic plastici-

[112]), which inhibit cAMP signaling through coupling to Gi proteins ty underlying memory formation in multiple brain areas. Because [114,115]. This is a particularly interesting possibility, given the role the DN-SNARE mutant mice exhibit reductions in sleep rebound and of adenosine signaling in the impairments in plasticity and memory a resistance to the cognitive impact of sleep deprivation, these studies induced by sleep deprivation as discussed in detail below. raise the intriguing question of what the role of recovery sleep is and how it might impact cognitive function. 7. Sleep deprivation, adenosine, and astrocytes Together, the studies using CPT and mice expressing the DN- SNARE protein have suggested that the build-up of extracellular Adenosine, a degradation product of ATP whose extracellular adenosine through gliotransmitter release from astrocytes plays a levels increase with brain metabolism, plays a critical role in sleep key role in the plasticity and memory deficits observed after brief regulation through the modulation of slow wave activity [116–118]. sleep deprivation, and suggest that the A1 receptor may be a potential In rats, extracellular adenosine levels have been reported to be higher therapeutic target to combat the cognitive deficits observed after during the circadian active period (the dark phase) than during sleep deprivation. An important question that remains to be answered the resting period (the light phase) in both the hippocampus and is how overstimulation of the adenosine A1 receptor – as observed 1258 R. Havekes et al. / Cellular Signalling 24 (2012) 1251–1260 after sleep deprivation – leads to memory impairments. Based on the changes at the RNA level do not automatically mean that protein levels known signaling pathways downstream of the A1 receptor, Florian and function are affected in parallel or that these changes are uniform and colleagues [79] have hypothesized that sleep deprivation- throughout the brain. Thus, in order to fully understand how sleep induced elevations in adenosine levels may activate presynaptic A1 deprivation affects both neuronal and glial function, future research receptors, inhibiting adenylyl cyclase activity and resulting in reduc- should compare effects of sleep deprivation on gene expression in tions in cAMP levels and ERK pathway signaling. Although the studies specific brain regions and determine whether these changes reflect by Florian et al. [79] and Halassa et al. [55] indicate that sleep loss may functional alterations at the protein level. Such studies may be useful result in memory deficits through stimulation of the adenosine A1 to determine why particular brain functions such as memory are receptor, potentially attenuating the production of cAMP, it does not disrupted by sleep loss. explain how sleep loss leads to elevated hippocampal PDE4A5 protein levels and increased PDE4 activity as described by Vecsey et al. [30]. 9. Conclusions and future directions Thus, it may very well be that multiple signaling pathways in parallel attenuate the cAMP-PKA pathway, leading to plasticity and memory One of the hallmarks of our modern society is to work longer each impairments as a consequence of sleep loss. day and for more days each year, usually at the expense of sleep time, which can lead to cognitive impairments. Over the last few decades, 8. Sleep deprivation, gene transcription, and translation significant advances have been made in unraveling the mechanisms underlying the memory and plasticity deficits observed after both In addition to looking at specific signaling pathways to identify the brief and longer periods of sleep deprivation. The role of specific mechanisms underlying the memory deficits caused by sleep loss, genes and signaling pathways responsible for sleep deprivation- many laboratories have used gene expression studies to identify induced deficits have been elucidated and efforts have been under- the molecular targets of sleep deprivation. In particular, microarray taken to reverse the effects of sleep deprivation on memory and plas- studies allowing for the simultaneous analysis of thousands of tran- ticity by targeting these specific molecular substrates. Still, many scripts have led to the identification of many genes whose expression important questions remain unanswered and methodological issues change after sleep deprivation (for in depth review of the gene have not been addressed. As described in this review, depending on expression studies see [132–136]). Based on a computational analysis the method used for sleep deprivation, opposite effects of sleep dep- of multiple previously published gene expression studies assessing rivation on specific signaling pathways have been described. Thus, it the effect short-term sleep deprivation on gene expression, Wang et remains to be determined which sleep deprivation method is best al. [135] concluded that almost all genes affected by sleep deprivation suited to study the effects of sleep deprivation on cognitive perfor- are either directly or indirectly regulated by the cAMP-responsive mance as observed in humans. One exciting approach, which may element. In addition, they found that across studies, expression levels avoid many of the potential side effects of the currently used sleep of several immediate early genes were elevated with sleep depriva- deprivation methods, would be to use optogenetic strategies – the tion, including the Egr family (Egr1, Egr2, Egr3), Homer1a, Nr4a1, and use of light-sensitive receptors to modulate the activity of genetically Arc. Consistently down-regulated genes include cold-induced RNA defined cells – to manipulate sleep. A recent set of studies showed binding proteins such as Rbm3 and Cirbp, which are involved in that this may very well be feasible in the near future. Adamantidis RNA/lipid metabolic processing. et al. [142] developed an optogenetic approach to increase the occur- Gene expression studies are well-suited to determine which gene rence of transitions from a sleep state to wakefulness in freely moving families, gene clusters, and molecular pathways are affected by ma- mice, and a follow-up study showed that an optogenetic approach nipulations such as sleep deprivation. However, one caveat is that could be successfully used to disrupt sleep, which led to impaired meta-analysis has shown that changes in gene expression after memory consolidation [143]. sleep deprivation are not uniform throughout the brain. For example, One interesting but yet unanswered question is why certain brain Arc mRNA expression was elevated in most brain subregions, but was regions are more vulnerable to sleep deprivation than others. As de- reduced in the lateral hypothalamus (in the vicinity of hypocretin scribed above, mice that were sleep deprived directly after training neurons and in the suprachiasmatic nucleus) [135]. Thus, gene ex- for 6 h per day shifted from using a hippocampus-dependent learning pression alterations in one region of cortex may not be representative strategy to a strategy that requires the striatum [18], suggesting that of the changes taking place in another area. This may be an interesting the hippocampus in comparison with the striatum is more sensitive phenomenon to probe further, by comparing the effects of sleep to sleep loss. In addition, although most studies have reported that deprivation across multiple brain regions. sleep deprivation hampers hippocampal function, some evidence Another important issue with gene expression studies is that a suggests that sleep loss facilitates plasticity in the medial prefrontal change in mRNA expression is not necessarily indicative of functional cortex [71]. Gene expression studies showing varied effects of sleep changes at the protein level. An example of differently regulated tran- deprivation also emphasize that sleep deprivation does not impact scription and translation came from a study of genes in the Egr family all brain regions identically. Future studies will have to determine by Cheval and colleagues [137], which determined the effect of LTP the molecular mechanisms that underlie the different sensitivity to induction on changes in gene expression and protein levels for the sleep loss. Egr family. Although LTP induction led to an increase in Egr1, Egr2 Another challenge for the field of memory and sleep research is to and Egr3 mRNA expression, protein levels were enhanced only for determine whether the molecular signaling mechanisms underlying Egr1 but not for Egr2 and Egr3. Unfortunately, none of the microarray memory consolidation are different during the animals' resting studies looking at the effect of sleep deprivation on gene transcription phase and active phase. Many studies have shown that 5 to 6 h of also examined protein levels in parallel. Several studies have sug- sleep deprivation directly following training in the animals resting gested that sleep promotes translation [138–140], and microarray phase leads to long-term memory deficits. This sleep deprivation- analysis has shown that gene clusters important for protein synthesis sensitive period coincides with the time-window in which pharmaco- are altered by sleep deprivation [133,141]. 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