University of Groningen Neurobiological and functional consequences of chronic partial sleep deprivation Román, Viktor

Neurobiological and functional consequences of chronic partial sleep deprivation Román, Viktor

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The role of the serotonergic system

Viktor Román

RIJKSUNIVERSITEIT GRONINGEN

Neurobiological and functional consequences of chronic partial sleep deprivation The role of the serotonergic system

Proefschrift

ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

vrijdag 16 februari 2007 om 16.15 uur

door

Viktor Román geboren op 15 juli 1978 te Kecskemét, Hongarije

Copromotor: Dr. P. Meerlo

Beoordelingscommissie: Prof. dr. J. Adrien

Prof. dr. D.G.M. Beersma

Prof. dr. J.A. den Boer

ISBN printed version: 90-367-2917-3

ISBN e-version: 90-367-2916-5

„The best of the rest is sleep.”

William Shakespeare: Measure for Measure

School of Behavioral and Cognitive Neurosciences (BCN) and the Netherlands Organisation for Scientific Research (NWO grant 864.04.002 to Peter Meerlo).

he printing of the present thesis was financially supported by the Dutch Society for Sleep - Wake

over: François Boucher - The Interrupted Sleep (1750)

rs Ipskamp B.V., Enschede, The Netherlands T

Research (Nederlandse vereneging voor Slaap - Waak Onderzoek; NSWO), the University of Groningen and the BCN.

C

Design: Viktor Román Printed by: PrintPartne

Chapter 1 General introduction 9 Chapter 2 Too little sleep gradually desensitizes

the serotonin-1A receptor system 21 Chapter 3 Differential effects of chronic partial sleep

deprivation and stress on serotonin-1A and

muscarinic cholinergic receptor sensitivity 31 Chapter 4 Altered serotonergic and corticotropin releasing

hormone regulation of the hypothalamo-pituitary-

adrenal axis in chronic partial sleep deprivation 45 Chapter 5 Losing too much sleep alters serotonin signalling

in the amygdala 55

Chapter 6 Chronic partial sleep deprivation diminishes fear and alters fear-related hippocampal neuronal

activation patterns 65

Chapter 7 No evidence for cross-talk between the adenosine and the serotonin-1A receptor system after chronic

adenosine receptor stimulation 73

Chapter 8 Sleep restriction by forced activity reduces

hippocampal cell proliferation 83

Chapter 9 General discussion 93

References 103

Nederlandse samenvatting 121

Summary in Hungarian 127

Acknowledgements 133

Curriculum vitae 135

List of publications 136

List of abbreviations 137

Notes 138

General introduction

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1. Why do we sleep?

2. Chronic partial sleep loss

3. Chronic partial sleep loss and mood 4. Chronic partial sleep loss and stress 5. The serotonergic system, sleep and mood 6. Sleep loss and neuronal plasticity

7. The concept of the thesis 8. The outline of the thesis

1. W

?

The fact that we spend every day approximately eight hours asleep which, added up, is the third of our lives, suggests that sleep must have an important physiological function. Similar to humans, all animals show cycles of behavioural rest and activity of varying length, and the resting phase often proves to be a sleep-like condition comparable to our sleep (Shaw et al., 2000; Horne, 2002). Since sleep is specific for animals with complex nervous structures, the generally accepted idea is that sleep is primarily for the brain. This idea is supported by the effects of sleep deprivation: the loss of sleep leads to clear changes in the brain’s electrophysiological activity (Borbély and Neuhaus, 1979, Dijk et al., 1987). On top of such a fundamental neurobiological change, the first symptoms of sleep loss concern the higher executive functions of the brain comprising mood, cognition and motor control (Maquet, 2001; Durmer and Dinges, 2005). Next to its effects on the nervous system, sleep restriction also influences peripheral functions including adverse circulatory, metabolic, endocrine and immunological effects (Spiegel et al., 1999; Vgontzas et al., 2004; Gangwisch et al., 2006). Since sleep deprivation leads to complex changes in several systems, and since we do not know which of these is more important than the other, the exact function of sleep is still a hot and fiercely debated topic in the life sciences.

Hypotheses on the function of sleep are numerous and many of them try to explain the function of sleep as a homeostatic process. Some of the hypotheses focus on different aspects of brain homeostasis such as restoration, energy balance, temperature regulation or detoxification (Walker and Berger, 1980; McGinty and Szymusiak, 1990; Inoué et al., 1995; Maquet, 1995). The restorative hypothesis has been further developed by Benington and Heller (1995), who suggested that sleep is a state that allows the replenishment of the brain’s glycogen stores, which serve as an important energy buffer supporting neuronal activity during waking (Gruetter, 2003; Brown, 2004).

Another leading hypothesis champions sleep as a state that supports neuronal plasticity and synaptic organization (Benington and Frank, 2003; Tononi and Cirelli, 2006), which would support basic neuronal function and communication but also help learning and memory processes (Crick and Mitchinson, 1983; Walker and Stickgold, 2004).

What makes the quest for the function of sleep difficult is that sleep is not a homogenous state. The brain’s electrical activity changes throughout sleep following a cyclic pattern, where non- rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep alternate. As sleep has these two forms, it may be that sleep has multiple and stage-specific functions (Benington and Heller, 1995; Siegel, 1995; Walker and Stickgold, 2004). A recent hypothesis discusses NREM sleep in particular as a state which primarily serves synaptic homeostasis (Tononi and Cirelli, 2006). According to this hypothesis, high neuronal activity during waking promotes synaptic potentiation and increases the strength, size and number of synapses. This prolonged waking- induced synaptic potentiation would reduce available space and energy reserves in the brain and conversely, sleep would serve to downscale overall synaptic strength and bring it down to lower levels. Importantly, synaptic downscaling during NREM sleep would decrease the overall synaptic

strength but conserve the relative differences in synaptic strength between specific neuronal populations with different levels of activity.

REM sleep, on the other hand, may function to support neuronal plasticity and promote synaptic strength, particularly in neuronal circuits involved in memory formation (Walker and Stickgold, 2004). Such a role of REM sleep is supported by the fact that after waking, which is associated with information processing and learning, the subsequent REM sleep stages are characterized by the re-activation of learning-specific neuronal firing patterns and re-expression of genes involved in synaptic plasticity and memory formation (Graves et al., 2001; Benington and Frank, 2003).

Acting like this, NREM sleep and REM sleep together may serve to maintain overall synaptic homeostasis and at the same time promote the relative synaptic strength in specific circuits that are actively used for memory traces and cognitive functions. Thereby, the two sleep states together might increase synaptic efficacy and signal-noise ratio.

Whatever the exact function(s) of sleep may be, sleep is certainly needed for a proper and well-balanced mental functioning. Indeed, without sleep, mood, cognitive function, and motor performance are the most prominently impaired functional outputs in humans (Pilcher and Huffcutt, 1996; Dinges et al., 1997; Van Dongen et al., 2003).

2. C

Among humans living in modern society, chronic partial sleep deprivation is increasingly common (Bonnet and Arand, 2003; Alvarez and Ayas, 2004; Malik and Kaplan, 2005). Sleep loss is due to several factors comprising lifestyle, work-related factors and stress. (1) Lifestyle-like reasons include the element of personal choice, which is influenced by the unlimited access to television and the internet (Vioque et al., 2000; Ohida et al., 2001; Thompson and Christakis, 2005). Next to the personal choice element, the pressure of the 24/7 society, where everything is available at any time during the day and where circadian rhythms and geographical time zones are ignored, further threatens normal and healthy sleep schedules (Rosekind, 2005). (2) Shiftwork is also a prominent factor that leads to chronic loss of sleep in a large segment of modern society (Rajaratnam and Arend, 2001). Not only does doing shifts lead to the loss of sleep, but it also seriously and persistently damages the timing and architecture of sleep that people working in such schedules are allowed (Dumont et al., 1997; Akerstedt, 2003). Finally, (3) stress and sleep loss make up a kind of vicious circle, where the one is able to induce the other and vice versa (Riemann and Voderholzer, 2003; Taylor et al., 2005). In other words, daytime feelings associated with everyday stressors are likely to continue as unsettling thoughts in bed making it difficult to fall asleep, and conversely, insomnia is mostly experienced as feeling stressed. The inability to fall asleep is common among humans: insomnia has been reported in 10-35% of the population (Ohayon, 2002;

Ohayon and Partinen, 2002). All in all, these factors, mostly in combination, have led to major changes in human sleep hygiene, illustrated by the fact that during the last century our average

sleep time has been reduced by approximately 20%, which means hours of sleep lost (Ferrara and De Gennaro, 2001).

Importantly, the incidence of sleep loss and excessive daytime sleepiness are high not only among working adults but are becoming an important issue among children and adolescents as well (Fredriksen et al., 2004; Teixeira et al., 2004; Amschler and McKenzie, 2005; Thompson and Christakis, 2005; Gibson et al., 2006).

Interestingly, humans adapt to increasing sleepiness which is inherent to chronic partial sleep deprivation (Carskadon and Dement, 1981), and tend to disregard the profound cognitive deficits that accumulate over time and view sleep as a luxurious item in their daily routine (Durmer and Dinges, 2005). However, the question remains whether chronic loss of sleep results in gradual and persistent neurobiological changes that may, on the long-term, have consequences for physical and mental well-being.

3. C

Several human studies show that sleep deprivation has detrimental effects on mood and emotionality, which may even exceed the impact on cognition and motor performance (Pilcher and Huffcutt, 1996; Dinges et al., 1997; Lieberman et al., 2002; Bonnet and Arand, 2003; Haack and Mullington, 2005; Scott et al., 2006). A meta-analytical study has demonstrated that chronic partial sleep deprivation (less than 5 hours sleep deprivation per day) has even more profound functional effects on cognitive function than either short or long-term total sleep deprivation (less than 45 hour continuous sleep deprivation or more, respectively) (Pilcher and Huffcutt, 1996). This finding underscores the importance of the cumulative feature of changes caused by sleep loss.

In the long run, chronically restricted or disrupted sleep may even play a role in the development of psychopathologies. According to the traditional view, sleep loss or insomnia is a symptom of depressive episodes or mood disorders (Adrien, 2002). The inability to sleep is possibly caused by increased anxiety, disturbed circadian rhythms or by the neurobiological impairments linked to depression (Benca, 2000). On the other hand, an increasing number of studies suggest that sleep problems may in some cases be primary to mood disorders (for review see Riemann and Voderholzer, 2003). It still may be that insomnia is both cause and symptom of depression however, the exact causal relationship between insomnia and mood disorders remains yet unclear (Abad and Guilleminault, 2005; Taylor et al., 2005; Turek, 2005). Nevertheless, the idea of sleep loss being primary to mood disorders is supported by mounting evidence showing that insufficient sleep may precede mood disorders including depressive or manic episodes, clinical depression and anxiety (Wehr et al., 1987; Wright, 1993; Taylor et al., 2003; Taylor et al., 2005;

Kaneita et al., 2006). Sleep loss or disturbances in adolescents may even signal an increased risk for future suicidal behaviour (Liu and Buysse, 2005). Epidemiological studies have shown that insomnia may be a risk factor for depression over a few years (follow-up period ranging from 6

1993; Breslau et al., 1996; Foley et al., 1999; Perlman et al., 2006). These mood changes not only occur within a few years, but also on much longer time scales: the sensitivity to or occurrence of sleep disturbances can predict depression even over several decades. One particular longitudinal study with a follow-up time of 30 years, the Johns Hopkins Precursors Study, showed that insomnia in young men indicates a significantly greater risk for clinical depression later in life (Chang et al., 1997).

Next to depression, sleep loss can also be a predictor of other mood disorders such as mania or anxiety. This issue has been explored in a study where individuals were asked to identify the prodromes of their mood disorder, and almost 80% of the subjects identified sleep disturbance as the most robust predictor of mania (Jackson et al., 2003). Another study with a follow-up period of 10-15 years demonstrated that childhood sleep problems are associated with anxiety disorders in adulthood (Gregory et al., 2005).

A further psychopathology for which sleep disturbances have been suggested as a mediatory factor, is the posttraumatic stress disorder (PTSD). PTSD is caused by a major life event (such as war combat, car accident, catastrophe, rape, etc.) and one of its main symptoms is insomnia along with other sleep disturbances (Singareddy and Balon, 2002). However, sleep changes prior to or shortly after the stressful event can also predict the development of the disorder. In one study, 77% of rape victims who developed PTSD reported sleep problems before the sexual assault (Krakow et al., 2001). This suggests that disturbed sleep may change the brain in a way that makes it more vulnerable to PTSD, should a major life event occur. Another study showed that sleep complaints within one month of a vehicle accident can predict which of the victims will develop PTSD over a year (Koren et al., 2002). All these results indicate that the relationship between sleep and stress is complex and probably bidirectional (Krakow et al., 2001;

Abad and Guilleminault, 2005).

4. C

Animal studies indicate that sleep loss activates the hypothalamo-pituitary-adrenal axis (HPA-axis) and results in increased plasma levels of the adenocorticotropic hormone (ACTH) and corticosteroids. The extent of HPA-axis activation depends on the amount of stress involved in the sleep deprivation protocol: more stressful techniques such as the flower pot technique used for REM sleep deprivation result in a more profound activation of the HPA-axis (Patchev et al., 1991;

Suchecki et al., 1998; Andersen et al., 2005), while forced locomotion and gentle handling which are less stressful lead to a milder activation (Tobler et al., 1983; Meerlo et al., 2001a; Meerlo et al., 2002; Sgoifo et al., 2006). In humans however, the relationship between sleep loss and HPA-axis activation is not so clear-cut as it is in rodents. A few human studies have shown that sleep deprivation results only in a mild activation of the HPA-axis (Leproult et al., 1997; Vgontzas and Chrousos, 2002; Alexander, 2003; Voderholzer et al., 2004). A study found a burst in cortisol and ACTH secretion at the beginning of a sleep fragmentation schedule, but then plasma cortisol levels

decreased below baseline sleep levels (Spath-Schwalbe et al., 1991). Also, even a decrease or no significant change have been described in the awakening-linked cortisol response after disturbed sleep (Backhaus et al., 2004; Williams et al., 2005). In a further study, no increase has been found in cortisol levels at the end of a 24-hour sleep deprivation experiment (Gonzalez-Ortiz et al., 2000).

While data concerning effects of sleep loss on basal activity of the HPA-axis remains inconclusive, virtually nothing is known regarding effects of sleep loss on the reactivity of this system to subsequent new stressors. A number of animal studies suggest that the reactivity of the HPA-axis is changed in chronically sleep restricted rats (Meerlo et al., 2002; Sgoifo et al., 2006).

The study by Meerlo et al. (2002) demonstrated that rats subjected to chronic partial sleep loss have a blunted ACTH response to a heterotypic stressor. In contrast, other stressors including immobilization, cold or noise exposure and exercise lead to the sensitization of the ACTH response to subsequent new stressors (Bhatnagar et al., 1995; White-Welkley et al., 1995; Van Raaij et al., 1997; Ma et al., 1999). The contrast between these findings underscores the fact that sleep loss is a special kind of stressor (Meerlo et al., 2002).

Similarly to sleep deprivation, in PTSD the responsiveness of the HPA-axis to a novel stressor is blunted (Heim et al., 2000). Also, damage to the amygdala, reduces the ACTH response to immobilization stress (Beaulieu et al., 1986). The limbic system is of great importance in the modulation of HPA-axis function, since many nervous structures within the limbic system project to the central regulatory nuclei of the HPA-axis and are highly involved in the regulation of the stress system (Herman et al., 1996; Herman et al., 2005).

One of the systems that might mediate effects of sleep loss on the HPA-axis is the serotonergic system. This idea is supported by the fact that the HPA-axis is under the stimulatory regulation of this neurotransmitter system (Calogero et al., 1990; Feldman and Weidenfeld, 1991;

Dinan, 1996b). The interaction between the two systems is not exclusively unidirectional (that is, the serotonergic modulation of the stress system), since stress hormones can induce changes in the serotonin system too (Dinan, 1996a; Porter et al., 2004). Beside the stress systems, the serotonergic system is also intimately linked to sleep (Adrien, 2002; Ursin, 2002): this interaction will be discussed in the next section.

5. T

,

The serotonergic system originates in the raphe nuclei of the brain stem (Hornung, 2003), and its fibres extend to almost all structures of the central nervous system including the limbic system, the cortical mantle, the cerebellum, and the spinal cord (Tohoyama and Takatsuji, 1998). This widespread innervation pattern ensures an exquisite position for the serotonergic system to modulate other systems (Hensler, 2006). The modulatory role is further supported by the presence of 14 different serotonin receptor subtypes which are linked to various signal transduction pathways (Barnes and Sharp, 1999; Raymond et al., 2001). On this basis, the serotonergic system effectively

modulates the central regulation of many physiological and behavioural functions including mood, stress, and sleep (Adrien, 2002; Neumeister and Charney, 2002; Ursin, 2002).

The release of serotonin changes across the day (Portas et al., 1998; Park et al., 1999;

Portas et al., 2000). The serotonergic neurons show highest firing and release rates during wakefulness. These rates decrease during NREM sleep and the serotonergic neurons almost completely cease to fire and stop releasing serotonin during REM sleep. It is believed that different amounts of serotonin released in the projection areas (for example in the thalamus) are in part responsible for the differential sleep stage-specific electroencephalographic activity of the brain (Ursin, 2002). In depression, which is characterized by a diminished serotonin production, sleep architecture is abnormal and sleep is disrupted. The abnormalities include decreased amounts of deep NREM sleep, decreased REM sleep latency and increased amounts of REM sleep, frequent or early awakenings (Benca et al., 1992).

However, the interaction between the serotonergic system and sleep is not a one-way process: the loss of sleep has a profound effect on the functioning of the serotonergic system too (Seifritz, 2001). In line with the findings on normal wakefulness, sleep deprivation activates serotonergic neurons and increases serotonergic neuronal firing rates (Santos and Carlini, 1983;

Maudhuit et al., 1996; Prevot et al., 1996; Gardner et al., 1997; Adell et al., 2002; Evrard et al., 2006). This increased serotonin transmission (Lopez-Rodriguez et al., 2003; Penalva et al., 2003;

Senthilvelan et al., 2006) is believed to counteract the diminished functioning of the serotonergic system which is one aspect in the pathophysiology of affective disorders, and contribute to the improvement of mood (Cryan and Leonard, 2000; Sobczak et al., 2002; Stockmeier, 2003).

Decreased serotonergic function in mood disorders is supported by a large body of evidence.

Firstly, a decrease in serotonin-1A receptor-mediated signalling in depressed patients has been shown by pharmacological challenges (Lesch, 1991; Mann et al., 1995; Shapira et al., 2000) and PET studies (Drevets et al., 1999; Sargent et al., 2000). Secondly, a number of studies on postmortem brain material of suicide victims are consistent with a decrease in serotonin-1A receptor function in depression (Stockmeier, 2003). Finally, antidepressant medication such as the selective serotonin reuptake inhibitor-based treatment, is based on drugs that enhance serotonergic neurotransmission by increasing the amount of serotonin present in the synaptic cleft (Blier and De Montigny, 1994; Middlemiss et al., 2002).

Restricted sleep might affect the brain and its functional outputs directly on the level of serotonin receptors but it might also modulate intracellular signalling pathways downstream the receptors. The majority of different serotonin receptor subtypes are G-protein-coupled metabotropic receptors, with the exception of one ionotropic subtype, the serotonin-3 receptor (Barnes and Sharp, 1999; Raymond et al., 1999; Raymond et al., 2001). The activated G-proteins associated to the receptors may either activate or inhibit intracellular signalling cascades, depending on the receptor subtype. The most widely studied serotonin-1 receptors are coupled to a multitude of signalling pathways including the cAMP, phosphoinositol, calcium, and arachidonic adic second messenger systems (Raymond et al., 2001). The production of second messengers leads to the activation of ion channels and protein kinases, and the latter ones are able to phosphorylate a

number of substrates. These intracellular changes are later translated into genomic changes by transcription factors such as phospho-CREB, c-Fos and zif-268 (Tilakaratne and Friedman, 1996;

Kovács, 1998; Knapska and Kaczmarek, 2004; Josselyn and Nguyen, 2005).

Important in this context is the increasing awareness that affective disorders may not just be related to alteration on the level of receptors for serotonin or other neurotransmitters but, rather, may at least in part be due to impairments in the signal transduction pathways beyond the neurotransmitter receptors (Duman, 1998). Potential candidates for impairments in mood disorders are the G-proteins (Donati and Rasenick, 2003), the enzymes adenylyl cyclase and protein kinase A (Duman, 1998) as well as the transcription factor CREB (Nestler et al., 2002; Berton and Nestler, 2006). In depressed suicide victims the amount of inhibitory G-proteins and the activity of adenylyl cyclase in the brain is diminished in comparison with postmortem material obtained from non- suicidal humans (Pacheco et al., 1996; Dwivendi et al., 2002; Dowlatshahi et al., 1999). Similarly, in the same condition, both the expression and the activity of PKA are altered (Dwivedi et al., 2003;

Dwivedi et al., 2004). Other studies have shown that the expression of CREB and its phosphorylation are decreased in limbic areas in mood disorders (Dwivedi et al., 2003; Akin et al., 2005) and antidepressant treatment can counteract this decrease (Dowlatshahi et al., 1998; Thome et al., 2000). Given these new insights in the neurobiology of psychopathologies, it is an important question how these intracellular signalling mechanisms may be affected by restricted and disrupted sleep. Yet, the effects of sleep loss on these elements of signalling pathways, except a few studies, have not been extensively examined yet (Cirelli and Tononi, 2000; Alanko et al., 2004).

6. S

One neurobiological process that is not only under serotonergic control but also forms a potential link between restricted sleep and alterations in cognitive function and mood is hippocampal plasticity, in particular, adult hippocampal neurogenesis (Gould, 1999; Radley and Jacobs, 2002;

Banasr et al., 2004; Huang and Herbert, 2005). It has been found in depressed humans or subjects with PTSD that the volume of the hippocampus decreases (Bremner et al., 2000; Manji et al., 2001), but it is not clear yet whether this is due to dendritic atrophy, cell loss or decreased neurogenesis. Nevertheless, chronic stress and corticosterone treatment, two animal models of depression, result in diminished hippocampal cell proliferation and neurogenesis (Gould et al., 1997; Gould et al., 1998). Moreover, pro-serotonergic antidepressant treatments restore the rate of neurogenesis (Malberg et al., 2000; Malberg and Duman, 2003; Malberg, 2004). Other animal studies have demonstrated that also the beneficial behavioural effects of antidepressants are paralleled by restored neurogenesis (Santarelli et al., 2003).

With respect to possible effects of restricted and disturbed sleep, a few studies have shown that sleep deprivation leads to a diminished production of new cells in the hippocampus (Guzman- Marin et al., 2003; Guzman-Marin et al., 2005; Hairston et al., 2005; Tung et al., 2005). Such

acute and short sleep deprivation during a normal single resting phase (Van der Borght et al., 2006). The results of the long-term sleep deprivation studies suggest that sleep loss-induced reductions in hippocampal plasticity may contribute, along with other factors, to impaired hippocampal functions such as learning and mood (Graves et al., 2001; McDermott et al., 2003;

Marks and Wayner, 2005).

7. T

While frequently restricted and disrupted sleep is a rapidly increasing problem in modern society, very little is known on the long-term consequences of chronic sleep loss for brain function and health. This thesis describes a series of studies that examined the neurobiological consequences of restricted sleep is rats, with special emphasis on changes in serotonergic signalling and brain systems that are involved in the regulation of stress and emotionality. The background of these studies was the hypothesis that restricted and disrupted sleep may gradually alter the brain and sensitize individuals for mood disorders such as depression. If sleep loss were indeed a potential causal factor in the development of psychopathologies, we expected that chronic sleep restriction would gradually induce changes in the brain that are similar as what is seen in such psychopathologies.

Figure 1. The conceptual background of the present thesis. Sleep loss is a commonly occurring and growing problem in human society. It is mostly related to work, stress and other factors associated with an overactive modern life style (top pictures). A number of studies have indicated that sleep loss may be a risk factor for psychopathologies including depression. Mood disorders are often linked to alterations in neurotransmitter systems. In particular, an impaired serotonergic signalling has been implicated as an underlying mechanism for depression (the figure includes two positron emission tomography scans: the right is a depressed brain with a lower serotonin function, the left is a healthy brain with a normal serotonergic system). The present work aims to establish whether sleep loss leads to neurobiological changes which are also characteristic to mood disorders. Therefore, the major questions of this project are: does sleep loss affect the brain’s serotonergic system, and does it eventually lead to impairments in the functional outputs of the brain in a manner that is similar to mood disorders? In this framework, we studied how sleep loss affects the serotonin-1A receptor system; our findings about this system and its interactions with other systems (muscarinic cholinergic and adenosinergic) are discussed in Chapters 2, 3, 5 and 7. As functional outputs of the brain, we examined neuroendocrine stress reactivity, emotionality (illustrated by a rat in a fear conditioning box) and neuronal plasticity (depicted by a newly produced – lighter in colour – granule cell of the hippocampal dentate gyrus). The results of these studies are discussed in Chapters 4, 6 and 8, respectively. The numbers next to the pictures in the figure represent the chapters of the thesis related to the respective topics.

Although indirect evidence and epidemiological studies in humans suggest that disrupted sleep may be a risk factor for psychopathologies, few attempts have been made to study the relationship between chronic sleep loss and brain function with a realistic experimental model.

Since experiments with human beings have various limitations, we have chosen a rat model of chronic partial sleep deprivation as our experimental approach to mimic sleep loss as it often occurs in human society. Although there have been numerous animal studies on the effects of sleep deprivation, most of these studies were based on total sleep deprivation for a short period of time, unlike the situation that occurs in real life. Instead of using total sleep deprivation which is not typical for human individuals, we used a novel approach including animals subjected to a protocol of partial sleep deprivation over a longer period of time. The protocol allowed the rats 4 hours of sleep every day, which is less than the 10 to 12 hours that they normally sleep, and presumably would not be sufficient for full recovery. Our question then was how such insufficient recovery over the course of many days would affect the brain.

8. T

In this paragraph, I will shortly introduce the contents and rationale of each chapter in the present thesis. The study described in Chapter 2 examined the effects of chronic sleep restriction on the functional sensitivity of the serotonin-1A receptor system, a receptor system that has long been implicated in mood disorders. The sensitivity of the receptors in sleep restricted and control rats was studied by subjecting them to pharmacological challenges with a serotonin-1A receptor agonist and measuring body temperature responses with a radio telemetry set-up. We assessed how changes in receptor sensitivity develop over time and how persistent these changes are. Since glucocorticoid stress hormones are capable of affecting the serotonin-1A receptor system, in Chapter 3, we investigated whether the sleep loss-induced decreased serotonin-1A receptor sensitivity found in the previous chapter might be due to elevated adrenal stress hormone levels.

To this end, we used adrenalectomized animals. Next to this, we studied whether the effects of chronic partial sleep loss are specific for the serotonergic receptors or they concern a further receptor system which also has implications in mood disorders; the muscarinic cholinergic system.

In Chapter 4, we studied how sleep loss changes the reactivity of the HPA-axis if experimentally challenged. We did this by measuring pituitary hormone (ACTH) and corticosterone responses to pharmacological challenges with a serotonergic agonist and corticotropin releasing hormone.

Based on our earlier findings of a sleep loss-induced desensitization of the serotonin-1A receptor system, in Chapter 5, we investigated whether sleep loss alters the number of serotonin-1A receptors in a way so that it could explain the diminished 1A receptor-related physiological responses. Additionally, we examined the inhibitory G-proteins coupled to the 1A receptors as potential molecular substrates of receptor sensitivity. Our investigations included a number of

behaviour of rats upon re-exposure to a fearful environment in a contextual fear conditioning paradigm. Furthermore, we tried to correlate emotional behaviour with neuronal activation in the amygdala and the hippocampus. In Chapter 7, we set out to examine whether cross-talk between different receptor populations could be the cause of decreased serotonin-1A receptor sensitivity.

Here, our question was whether chronic pharmacological activation of the adenosine receptors, as it may occur under conditions of chronic sleep restriction, would affect the functional sensitivity of the serotonin-1A receptors. The rationale for this particular study was based on data suggesting that adenosine accumulates in the brain during wakefulness as well as during sleep deprivation.

Also, a number of studies indicate that there a cross-talk between the adenosinergic and serotonergic receptors exists. Chapter 8 describes the effects of chronic partial sleep loss on a form of adult neuronal plasticity; cell proliferation in the dentate gyrus of the hippocampus. It is known that sleep loss impairs cognitive functions and mood, and some studies link adult neurogenesis to both of these functional outputs of the brain. However, data on the impact of sleep loss on hippocampal cell production has been scarce. Finally, in Chapter 9 the results of all experiments are summarized and discussed.

Too little sleep gradually desensitizes the serotonin-1A receptor system

Viktor Román, Irene Walstra, Paul G. M. Luiten, Peter Meerlo

Department of Molecular Neurobiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands

Sleep (2005) 28(12):1505-1510

A

In our 24h society, frequently disrupted and restricted sleep is a rapidly increasing problem that may contribute to the development of diseases such as depression. One of the proposed neurobiological mechanisms underlying depression is a disturbance in the brain’s serotonergic neurotransmission, particularly a desensitization of the serotonin-1A receptor system. However, a relationship between chronic sleep loss and changes in serotonin receptors has not been established yet. Therefore, in the present study we experimentally tested the hypothesis that chronic sleep restriction leads to desensitization of the serotonin-1A receptor system. Rats were subjected to a schedule of restricted sleep allowing them 4h of sleep per day. Sleep restriction was achieved by placing the animals in slowly rotating wheels. The sensitivity of the 1A receptor system was examined by measuring the hypothermic response to a standard injection of a 1A agonist. The results show that 2 days of restricted sleep had not yet affected the sensitivity of the serotonin-1A receptor system whereas the system was desensitized after 8 days of sleep restriction. Control experiments indicated that the effect of sleep restriction was not due to forced activity or stress.

The effect of sleep loss persisted for many days even with unlimited recovery sleep. The desensitization of the 1A system was still present after 1, 2, and even 7 days of recovery. These findings provide a link between chronic sleep loss and sensitivity for disorders that are associated with deranged serotonergic neurotransmission.

I

A rapidly increasing number of people in our modern society experiences regular sleep loss due to our modern around-the-clock lifestyle. Concerns have been raised that, in the long run, chronically restricted sleep may have serious repercussions for health and well being (Rajaratnam and Arendt, 2001). Controlled studies have provided evidence that acute sleep deprivation strongly affects cognitive performance and emotionality (Pilcher and Huffcutt, 1996). Also, recent experiments in healthy subjects showed that successive nights of restricted sleep result in a gradually accumulating decline in cognitive function (Dinges et al., 1997; Van Dongen et al., 2003). Whereas subjects may initially recover from these effects after subsequent sleep, frequent or chronic sleep loss may induce neurobiological changes that are not immediately evident but accumulate over time, ultimately with serious health consequences. One long-term prospective study that clearly suggested such a link between inadequate sleep and sensitivity to disease showed that insomnia and sleeping problems in otherwise healthy young people were associated with an increased risk for clinical depression 20 to 40 years later (Chang et al., 1997).

Several lines of evidence indicate that the neurotransmitter serotonin is involved in the regulation of mood and that serotonergic neurotransmission is impaired in affective disorders (Cryan and Leonard, 2000; Sobczak et al., 2002; Stockmeier, 2003). A decrease in serotonin-1A receptor-mediated signalling in depressed patients has been shown by pharmacological challenges (Lesch, 1991; Mann et al., 1995; Shapira et al., 2000) and PET studies (Drevets et al., 1999;

Sargent et al., 2000). Although postmortem studies have yielded various results, some of them are consistent with a decrease in serotonin-1A receptor function in depression (Stockmeier, 2003).

Finally, antidepressant medication is often based on drugs that enhance serotonergic neurotransmission (Blier and De Montigny, 1994; Middlemiss et al., 2002).

Given the evidence for a role of the serotonergic system in clinical depression, a gradual alteration in this system seems a candidate mechanism by which disrupted and restricted sleep might increase the risk for this disease. However, this potential link between sleep loss and sensitivity to psychopathology has not been established. Therefore, the aim of our study was to experimentally test the hypothesis that restricted sleep gradually causes a desensitization of the serotonin-1A receptor system and thereby changes the brain in a direction that makes it more vulnerable to psychopathology.

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Animals and housing

In a series of 3 experiments, we used 76 adult male Wistar rats (± 250 g at the start of the experiments) bred at the local animal facility of the University of Groningen, Haren, The Netherlands. Animals were housed under a 12h light/12h dark cycle, with lights on from 09.00 h to 21.00 h. The average temperature of the room was 21 ± 1^oC. Rats were provided with food and water ad libitum in all experiments. The experiments were approved by the Ethical Committee of Animal Experiments of the University of Groningen.

Experiment 1. Sleep restriction

Rats were subjected to a sleep restriction protocol allowing them 4h of undisturbed rest per day at the beginning of the light phase (09.00-13.00 h), their normal resting phase. The remainder of the time, the animals were kept awake by placing them in slowly rotating wheels (40 cm in diameter) driven by an engine at constant speed (0.4 m/min). Since rats normally sleep about 10 to 12h per day (Borbély and Neuhaus, 1979) the 4h of rest would not be sufficient to fully recover from the 20h of wakefulness. Animals had free access to food and water inside the wheels. A total of 28 rats was used: 8 rats were subjected to 2 days of sleep restriction and 8 rats underwent 8 days of sleep restriction. Six rats in both sessions served as controls and remained in their home cage.

Figure 1. Experimental set-up of sleep restriction protocol and forced activity control. Top bar: rats in Experiment 1 were sleep restricted by forced locomotion for 20h each day (grey section of the bar) and were allowed 4h of rest in their home cage (first 4 hours of the light phase). Middle bar: rats in Experiment 2 were subjected to a protocol of forced activity at double speed for half the time. The forced activity was divided in 5 blocks of 2h (dark sections of the bar) separated by 2.5h of rest (white sections of the bar). Lower bar depicts the 24h LD cycle. (*) Serotonin 1A agonist injections on day 2 and day 8 took place between the third and fourth hour of the light phase. (●) Blood samples after 1 or 7 days of sleep restriction / forced activity were collected at the beginning of the light phase (the end of the daily sleep deprivation or forced activity session) and after the 4th hour of the light phase (after the daily 4h recovery phase).

Experiment 2. Forced activity control

Since the procedure of sleep deprivation included mild forced locomotion, we performed an additional experiment to establish whether effects of sleep restriction were partly due to forced activity rather then sleep loss per se. A second group of rats was subjected to a schedule of forced activity in the same drums that were used for the sleep restriction. However, these new animals were forced to walk at double speed for half the time (0.8 m/min for 10h per day). In other words, the housing conditions were the same and the animals covered the same distance as the sleep restricted rats in Experiment 1, however, they had to walk at a higher intensity and had more time to sleep (14h of rest per day versus 4h in the sleep restricted animals). The 10h of daily forced activity was divided in 5 blocks of 2h separated by 2.5h of rest, thus covering a time frame of 20h that corresponded to the 20h time frame of sleep restriction in the first experiment (see Fig. 1). For the 20h period of alternating rest and activity, the rotation of the wheels was controlled by a timer. Only for the first 4h of the light phase were the animals returned to their standard home cage, similar to the sleep restricted animals in Experiment 1. In the forced activity experiment, 32 rats were used. Eight rats were subjected to a 2- day and 8 rats to an 8-day forced activity schedule. Eight animals served as controls in both sessions.

Experiment 3. Recovery

After establishing sleep loss-induced changes in serotonin-1A sensitivity, another important question was how long such changes would persist with unrestricted recovery. In a third group of rats, we examined the hypothermic response to 8-OH-DPAT after 8 days of sleep restriction followed by different durations of unrestricted recovery sleep: 1, 2 and 7 days of recovery. The protocol of sleep restriction was similar to that in Experiment 1. After 8 days, the animals were returned to their home cage for undisturbed recovery. In this third experiment a total of 32 rats were used. In one group of rats, the sensitivity of the serotonin-1A receptor system was measured after 8 days of sleep restriction and after 2 days of recovery (8 sleep restricted and 8 control rats). In a second series of rats the 1A sensitivity was measured after 1 and 7 days of recovery (8 sleep restricted and 8 control rats). Thus, in this experiment, each individual rat received 2 pharmacological challenges to test the serotonin-1A sensitivity. We did not perform more than two challenges in each animal to prevent desensitization of the receptors as a consequence of the pharmacological challenges themselves.

Serotonergic challenge

To examine the effect of chronic sleep loss on the sensitivity of the serotonin-1A receptor system, we measured the physiological response to a subcutaneous injection with the serotonin-1A agonist (±)-8-hydroxy- 2-(di-n-propyl-amino) tetralin hydrobromide (8-OH-DPAT; 0.25 mg/kg body weight; Sigma, St. Louis, MO, USA). This drug causes an acute hypothermic response that can be used as an indicator of central serotonin- 1A neurotransmission, as has been shown in rats (Hjorth, 1985) as well as humans (Blier et al., 2002).

Importantly, in depressed patients, this serotonin-1A mediated hypothermia is attenuated, in accordance with other evidence of decreased serotonin-1A signalling (Lesch, 1991; Mann et al., 1995; Shapira et al., 2000).

The pharmacological challenges were performed between 11.00 h and 13.00 h, the third and fourth hour of the light phase, when all animals were in their home cage see Fig. 1). The sensitivity to the drug was determined by measuring the acute hypothermic response by means of radio telemetry.

Radio telemetry of body temperature

To record the serotonin-1A receptor mediated drop in body temperature we applied radio telemetry with chronically implanted transmitters (model TA10TA-F40; Data Sciences, St. Paul, MN, USA). Implantation of the transmitters in the abdominal cavity was performed under full anaesthesia (inhalation anaesthesia with a mixture of N2O, O2, and isoflurane). After surgery, the animals were allowed at least 10 days of recovery. The transmitters measured core body temperature and transformed temperature values into frequency coded radio signals. These radio signals were relayed to a PC by receivers placed under home cages (model RPC-1; Data Sciences, St. Paul, MN, USA). Body temperature was sampled for 5 sec every 5 min and processed with Dataquest Labpro^TM system (Data Sciences).

Blood sampling and corticosterone measurements

It has been reported that serotonin-1A receptor sensitivity can be attenuated by stress and elevated levels of glucocorticoids (Meijer and De Kloet, 1994; Bush et al., 2003; Leitch et al., 2003). We therefore sought to determine whether our sleep restriction protocol might attenuate serotonin-1A receptor sensitivity by increased levels of stress hormones. In Experiment 1 and Experiment 2, blood samples were collected to measure effects of sleep restriction and forced activity on plasma levels of corticosterone. The blood samples were taken on the first and the seventh day of the protocol, thereby not interfering with the 8-OH-DPAT challenges on day 2 and 8. On both days, 0.3 ml blood samples were collected by a making a small incision in the tail, one sample at 09.00 h (the end of the daily sleep deprivation or forced activity session) and another sample at 13.00 h (after the daily 4h recovery phase; see Fig. 1). The blood was collected in pre-chilled Eppendorf tubes containing EDTA as anti-coagulant. The samples were centrifuged at 2600 g for 15 min and the supernatant was stored at -80 ^oC for later analysis. Corticosterone levels were determined by radioimmunoassay (ICN Biomedicals, Costa Mesa, CA, USA).

Data analysis and statistics

To test for effects of sleep restriction or forced activity on the hypothermic response to 8-OH-DPAT injection, body temperature data were subjected to analysis of variance (ANOVA) with repeated measures. When appropriate, post hoc t-test was applied to establish at which time points after injection the experimental and control groups differed. Plasma levels of corticosterone were analyzed with ANOVA.

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Experiment 1: Sleep restriction

The subcutaneous injection of 8-OH-DPAT caused an immediate hypothermia that reached its lowest value within 20-30 min, approximately 2^oC below baseline temperature. Body temperature values returned to baseline 90 min after the injection (Fig. 2). In rats that were sleep restricted for 2 days, the hypothermic response was not different from control animals that were allowed unrestricted sleep (Fig. 2A). However, after 8 days of restricted sleep the serotonin-1A receptor- mediated response was significantly attenuated (Fig. 2B).

No differences were observed in serum corticosterone levels between sleep restricted and home cage control rats, neither after 1 day nor after 7 days of sleep restriction (Fig. 3A and B).

Experiment 2: Forced activity control

Contrary to the sleep restricted animals in Experiment 1, the rats subjected to the protocol of forced activity at higher intensity did not show significant changes in the temperature response to 8-OH- DPAT (Fig. 2C and D). Whereas sleep restricted rats did not show significantly elevated levels of the stress hormone corticosterone, the animals that were subjected to the forced activity protocol had increased levels of corticosterone at the end of the activity session, which returned to baseline levels during the rest periods (Fig. 3C and D). The elevation in corticosterone in these animals was similar after the first and seventh day of the protocol.

Figure 2. Chronic sleep restriction gradually desensitizes serotonin-1A receptors in the brain. Rats received injections of the serotonin-1A agonist 8-OH-DPAT (0.25 mg/kg). The sensitivity to the drug was measured by recording the acute hypothermic response by means of radio telemetry with implanted transmitters. [A and B]

The hypothermic response to 8-OH-DPAT after 2 or 8 days of restricted sleep. After 8 days of restricted sleep the serotonin-1A receptor-mediated hypothermic response was significantly attenuated (repeated measures ANOVA: treatment effect: F(1,12) = 7.615, p = 0.017; treatment x time interaction: F(20, 240) = 3.78, p<0.001). On each day, n=8 for sleep restriction, n=6 for control). [C and D] The hypothermic response to 8-OH-DPAT after 2 or 8 days of forced activity at double speed for half the time. No significant differences were found between animals subjected to forced activity and home cage controls. On each day, n=8 for sleep restriction and n=8 for control.

Figure 3. Plasma levels of the stress hormone corticosterone in rats subjected to sleep restriction and forced activity. Blood samples were collected by tail bleeding after the first and after the seventh day of the sleep restriction or forced activity protocol. [A and B] No differences were observed in serum corticosterone levels between sleep restricted and home cage control rats, neither at the end of the daily sleep deprivation session (SR), nor at the end of the daily 4h resting phase (R). [C and D] Animals subjected to a protocol of forced activity at double speed had significantly elevated corticosterone levels compared to home cage rats, both after 1 day (F(1,14)=14.326, p<0.01) and after 7 days (F(1,14)=20.506, p<0.01). On each day, the elevations that occurred immediately after the forced activity (FA) had disappeared after 4h of rest (R).

Figure 4. The reduced hypothermic response to serotonin 1A stimulation after 8 days of sleep restriction persists for several days even with unlimited recovery sleep. (A) 8 days of sleep restriction (treatment effect:

F(1,13) = 8.462, p = 0.012; treatment x time interaction:

F(20, 260) = 5.351, p<0.001). (B) 8 days of sleep restriction followed by 1 day of unlimited recovery sleep (treatment effect: F(1,13) = 5.440, p = 0.036; treatment x time interaction: F(20, 280) = 3.274, p<0.001). (C) 8 days of sleep restriction followed by 2 days of unrestricted recovery sleep (treatment x time interaction: F(20, 260) = 3.351, p<0.001). (D) 8 days of sleep restriction followed by 7 days of unrestricted recovery sleep (treatment x time interaction: F(20, 280) = 2.721, p<0.001). On each day, n=8 for sleep restriction and n=8 for control.

Experiment 3: Recovery

Confirming the results of the first experiment, rats had a significantly attenuated response to 8-OH- DPAT after 8 days of restricted sleep (Fig. 4A). The attenuated serotonin-1A response did not rapidly normalize with unrestricted recovery sleep but persisted for many days (Fig. 4B-D). Even after 7 days, the serotonin 1A mediated response had not fully normalized (Fig. 4D).

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The present study aimed to make a link between two sets of observations: one, the observation that sleep problems may be associated with increased sensitivity to psychopathology; and two, the observation that mood disturbances are associated with decreased serotonergic neurotransmission.

The data confirm that chronic sleep restriction gradually alters serotonin-1A receptor sensitivity in a direction that is similar to what is seen in affective disorders. Along with other evidence of attenuated serotonergic neurotransmission, depressed patients show a blunted temperature response to serotonin-1A receptor stimulation similar to our chronically sleep restricted rats (Lesch, 1991; Mann et al., 1995; Shapira et al., 2000).

In the present study, sleep restriction was achieved by forced locomotion. Therefore, changes in serotonin-1A receptor sensitivity might have been partly due to physical activity or to stress associated with the protocol. We performed a control experiment with rats that were forced to walk at double speed for half the time. These rats covered the same distance as the sleep restricted rats, however, they walked at a higher intensity and had more time to sleep (14h of rest per day versus 4h in the sleep restricted animals). Contrary to the sleep restricted animals, these rats did not show significant changes in the serotonin-1A response. The latter finding is in accordance with other studies showing that neither acute exercise nor chronic training affected postsynaptic serotonin-1A receptor sensitivity measured by behavioural responses such as forepaw treading and flat body posture in rats (Chauoloff, 1994).

We also measured plasma levels of the stress hormone corticosterone to examine the possible involvement of stress in the effects of sleep loss. In the sleep restricted animals, corticosterone levels were not significantly elevated compared to home cage control rats, suggesting that the sleep disruption procedure was not particularly stressful for these rats. These data are in line with other studies showing that sleep deprivation by forced locomotion does not or only mildly increase corticosterone levels (Tobler et al., 1983; Meerlo et al., 2002). In contrast, the animals that were subjected to forced activity at double speed for half the time had elevated levels of corticosterone at the end of their activity sessions. Together these results suggest that sleep restriction attenuates serotonin-1A receptor sensitivity by a mechanism that does not involve glucocorticoids and that is independent of stress and forced activity.

A potential explanation for the gradual desensitization of the serotonin-1A receptor system in the sleep restricted rats is a direct effect of serotonin itself, that is, a chronically enhanced serotonergic load on the serotonin-1A receptors. Microdialysis studies have shown that the release of serotonin during wakefulness and sleep deprivation is higher than during sleep (Park et al., 1999;

Lopez-Rodriguez et al., 2003; Penalva et al., 2003), and it is a common phenomenon that continued or frequent stimulation of receptors gradually diminishes their functional reactivity.

Indeed, it has been shown that repeated injections of an agonist result in 1A receptor desensitization (Kreiss and Lucki, 1992). Also, in serotonin transporter knock-out mice with

a condition with chronically elevated levels of serotonin which, in the long run, may be responsible for the receptor desensitization here reported.

Alternatively, the desensitization of the serotonin-1A receptor population after chronic sleep restriction may be an indirect consequence of cross-talk between this receptor system and other neurotransmitter systems, for example, the adenosine system. Adenosine in particular is an important homeostatic molecule that signals neuronal activity and wakefulness (Porkka-Heiskanen et al., 1997; for review see Basheer et al., 2004). Adenosine is a metabolite of ATP, the main source of fuel in our body, and is thereby directly coupled to cellular energy use, including neuronal energy use in a highly active waking brain. Release of adenosine, via stimulation of its widespread G-protein-coupled A1 receptors, inhibits neuronal activity and protects the brain against overactivity.

However, chronic stimulation of these receptors may result in desensitization (Olah and Stiles, 2000). Such desensitization may not be restricted to the receptors themselves but may involve downstream elements of the signalling pathway, which could eventually also affect the serotonin- 1A receptor system. In various brain regions, adenosine A1 and serotonin-1A receptors are co- localized and share elements of their signal transduction pathways, including the G-proteins via which these receptors act on intracellular signalling cascades (Zgombick et al., 1989). A number of studies have demonstrated that G-protein levels associated with adenosine A1 receptors may decrease in response to chronic agonist exposure (Zgombick et al., 1989). It might be that increased adenosine turn-over and frequent stimulation of adenosine receptors under conditions of chronic prolonged wakefulness ultimately affects intracellular signalling pathways associated with the serotonin 1A receptor.

In our experiment, desensitization of the serotonin-1A receptor system developed gradually.

The sleep restricted rats displayed normal temperature responses to 8-OH-DPAT after two days, but after 8 days of restricted sleep a significant attenuation of the response had developed. This finding of an accumulated effect of sleep loss is in line with studies in humans showing that successive nights of restricted sleep cause a gradually accumulating decline in cognitive function (Dinges et al., 1997; Van Dongen et al., 2003). Importantly, in our experiment the serotonin-1A receptor desensitization persisted for several days, despite unrestricted recovery sleep. In fact, after 8 days of restricted sleep, complete normalization of serotonin 1A receptor sensitivity almost required a similar period of recovery. The important implication of these data is that, sleep loss- induced changes in the brain not only accumulate over time but are also far more persistent than is generally assumed. Chronically restricted sleep causes gradual and persistent alterations in the serotonergic system, thereby providing a mechanism via which disrupted and restricted sleep may alter the sensitivity to psychopathologies such as depression (Chang et al., 1997).

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The authors thank Herman van Hengelaar for his help with the sleep deprivation drums used in the present study and Jan Bruggink for his excellent technical assistance in the corticosterone analysis.

Differential effects of chronic partial sleep deprivation and stress on serotonin-1A and muscarinic acetylcholine receptor sensitivity

Viktor Román, Roelina Hagewoud, Paul G. M. Luiten, Peter Meerlo

Department of Molecular Neurobiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands

Journal of Sleep Research (2006) 15(4):386-394

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Disrupted sleep and stress are often linked to each other, and considered as predisposing factors for psychopathologies such as depression. The depressed brain is associated with a reduced serotonergic and an enhanced cholinergic neurotransmission. In an earlier study, we showed that chronic sleep restriction by forced locomotion caused a gradual decrease in postsynaptic serotonin-1A receptor sensitivity, whilst chronic forced activity alone, with sufficient sleep time, did not affect receptor sensitivity. The first aim of the present study was to examine whether the sleep loss-induced change in receptor sensitivity is mediated by adrenal stress hormones. The results shows that the serotonin-1A receptor desensitization is independent of adrenal hormones since it still occurs in adrenalectomized rats. The second aim of the study was to establish the effects of sleep restriction on cholinergic muscarinic receptor sensitivity. While sleep restriction only slightly affected muscarinic receptor sensitivity, forced activity significantly hypersensitized the muscarinic receptors. This hyper-sensitization is due to the stressful nature of the forced activity protocol as it did not occur in adrenalectomized rats. Taken together, these data confirm that sleep restriction may desensitize the serotonin-1A receptor system. This is not a generalized effect since sleep restriction did not affect the sensitivity of the muscarinic cholinergic receptor system, but the latter was hypersensitized by stress. Thus, chronic stress and sleep loss may, partly via different pathways, change the brain into a direction as it is seen in mood disorders.

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Chronic partial sleep deprivation is an increasing problem in modern society, often related to workload and stress (Drake et al., 2003). Several studies suggest that frequently disrupted and restricted sleep may in the long run contribute to the development of mood disorders such as depression (Chang et al., 1997; Riemann and Voderholzer, 2003). Depression is one of the most prevalent psychopathologies and among its proposed pathophysiological mechanisms is an abnormal interaction between the monoaminergic and cholinergic neurotransmitter systems (Dilsaver, 1986a). This abnormal interaction leads to an imbalanced neurochemical state where a reduced serotonergic neuro-transmission is accompanied by an enhanced cholinergic signalling.

Indeed, several lines of evidence indicate that the serotonergic system is impaired in depression. Pharmacological challenge studies have shown that physiological responses linked to the activation of the serotonin-1A receptor system are blunted in depressed patients. These attenuated physiological responses include reduced hypothermia and decreased release of cortisol and prolactin in response to an injection of serotonin-1A agonists (Lesch, 1991; Meltzer and Maes, 1995; Shapira et al., 2000). Positron emission tomography studies have also shown a reduced serotonin-1A receptor signalling in some brain areas of depressed subjects (Drevets et al., 1999;

Sargent et al., 2000).

In contrast, opposite changes have been described for cholinergic signalling in depression, in particular a hypersensitive muscarinic receptor system (Meyerson et al., 1982; Dilsaver, 1986b).

Evidence for a hypersensitization of the muscarinic cholinergic receptor system in depression comes from pharmacological challenge studies and rapid eye movement (REM) sleep induction tests with muscarinic ligands, which have shown increased cortisol and REM sleep responses in depressed patients (Krieg and Berger, 1987; Gillin et al., 1991; Lauriello et al., 1993; Riemann et al., 1994; Perlis et al., 2002). Also, chronic stress, which may contribute to the pathogenesis of depression, has been shown to increase the sensitivity of the muscarinic receptor system in rodents (Finkelstein et al., 1985; Dilsaver et al., 1986; Takayama et al., 1987; Gonzalez and Pazos, 1992; Orsini et al., 2001).

In line with the serotonergic-cholinergic imbalance hypothesis of depression is the fact that many classical antidepressants have both pro-serotonergic and anti-muscarinic properties (Dilsaver, 1986b; Markou et al., 1998).

Given the notion that disturbed and restricted sleep may sensitize individuals to depression, it may do so by altering serotonergic or cholinergic receptor sensitivity. In an earlier study, we experimentally tested the hypothesis that chronic partial sleep deprivation gradually alters the sensitivity of the serotonin-1A receptor system in a direction that is similar to what is seen in depression. Indeed, chronic sleep restriction in rats gradually caused a blunted physiological response to a serotonin-1A agonist which persisted for many days even after subsequent recovery sleep (Roman et al., 2005).

The first aim of the present study was to investigate whether this blunted receptor