vereniging voor Slaap- en Waak Onderzoek (NSWO). Printed by: Offset ...... Besides the lack of stress, rats maintain spontaneous activity throughout, but they do ...
Sleep deprivation in rats: effects on learning and cognitive flexibility
The experiments described in this thesis were performed at the Netherlands institute for neuroscience, Amsterdam, the Netherlands.
Financial support was provided by: the Netherlands Organisation of Scientific Research (NWO), The Hague, program cognition, integrated research project 051-04-010; the Netherlands Institute for Neuroscience; & Achmea Schadeverzekeringen N.V.
Publication of this thesis was financially supported by: Vrije Universiteit Amsterdam, Aurora Borealis, Nederlands Herseninstituut, & Nederlandse vereniging voor Slaap- en Waak Onderzoek (NSWO).
Printed by: Offset Frankie, Leuven, Belgium. Cover design: Offset Frankie, Leuven, Belgium. Photo on cover: Adapted from I. Hattink. Copyright: Cathalijn H.C. Leenaars, 2013.
VRIJE UNIVERSITEIT
Sleep deprivation in rats: effects on learning and cognitive flexibility
ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. L.M. Bouter, in het openbaar te verdedigen ten overstaan van de promotiecommissie van de Faculteit der Aard- en Levenswetenschappen op maandag 1 juli 2013 om 13.45 uur in de aula van de universiteit, De Boelelaan 1105 door Cathalijn Hendrika Cornelia Leenaars geboren te Breda
promotor: copromotor:
prof.dr. E.J.W. van Someren dr. M.G.P. Feenstra
Leden van de leescommissie:
prof.dr. Andries Kalsbeek prof.dr. Gilles van Luijtelaar, prof.dr. Marian Joëls prof.dr. Sabine Spijker dr. Tom de Boer
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TABLE OF CONTENTS PAGE TABLE OF CONTENTS........................................................................................................ 7 LIST OF FIGURES ............................................................................................................ 10 LIST OF TABLES .............................................................................................................. 10 ABBREVIATIONS ............................................................................................................. 11 1. GENERAL INTRODUCTION ....................................................................................... 13 1.1. Behavioural and electroencephalographic aspects of sleep .......................... 16 1.2. Why do we sleep? ........................................................................................ 17 1.3. Sleep and cognition ...................................................................................... 18 1.4. Sleep disturbances ........................................................................................ 21 1.5. Sleep deprivation in rodents ......................................................................... 23 1.6. Rodent equivalents for cognitive tests.......................................................... 24 1.7. Scope & outline of the thesis ........................................................................ 25 1.8. Acknowledgements ....................................................................................... 27 2. A NEW AUTOMATED METHOD FOR RAT SLEEP-DEPRIVATION WITH MINIMAL CONFOUNDING EFFECTS ON CORTICOSTERONE AND LOCOMOTOR ACTIVITY ........................................................................................... 29 3. SWITCH - TASK PERFORMANCE IN RATS IS DISTURBED BY 12H OF SLEEP DEPRIVATION BUT NOT BY 12H OF SLEEP FRAGMENTATION ..................... 31 4. INSTRUMENTAL LEARNING: AN ANIMAL MODEL FOR SLEEP DEPENDENT MEMORY ENHANCEMENT .................................................................... 33 5. TWO-LEVER SPATIAL REVERSAL LEARNING IS ROBUST TO THE EFFECTS OF TOTAL SLEEP DEPRIVATION ............................................................... 35 6. UNALTERED INSTRUMENTAL LEARNING AND ATTENUATED BODYWEIGHT GAIN IN RATS DURING NON-ROTATING SIMULATED SHIFTWORK ............................................................................................................. 37 7. CONCLUDING REMARKS & GENERAL DISCUSSION ................................................. 39 7.1. Summary of the preceding chapters............................................................. 41 7.2. Reflection on methodology ........................................................................... 42 7.2.1. A new automated method for rat sleep-deprivation (chapter 2) .................................................................................... 43 7.2.1.1. Variable forced locomotion protocols........................... 43 7.2.1.2. Confounding by stress and activity .............................. 43 7.2.1.3. Modelling one sleepless night ...................................... 44 7.2.1.4. Motivation after sleep deprivation ............................... 45 7.2.2. Additional results: inactive phase sleep deprivation and recovery sleep ............................................................................... 46 7.2.3. Inversion of the light-dark cycle .................................................... 48
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7.2.3.1.
7.3.
Additional results: Locomotor activity after a light-dark reversal ..................................................... 48 7.2.3.2. Additional results: Sleep-wake patterns after habituation to a light-dark reversal ..................... 51 7.2.3.3. Concluding remarks on the light-dark reversal ........................................................................ 52 7.2.4. General task structure ................................................................... 53 7.2.4.1. Additional results: short and long intertrialintervals in the reversal task ........................................ 54 7.2.4.2. Additional results: Short and long intertrialintervals in the switch-task .......................................... 56 7.2.4.3. Concluding remarks on general task structure ...................................................................... 57 7.2.5. Switch-task parameters ................................................................. 58 7.2.5.1. Additional results: Differences in responding to light and sound stimuli during baseline ................... 59 7.2.5.2. Variability in response latencies ................................... 59 7.2.5.3. Task symmetry ............................................................ 60 7.2.5.4. Task switching with only one stimulus ........................ 60 7.2.5.4.1. Additional results: Light-only task-switching.......................................... 60 7.2.5.4.2. Additional results: Sound-only task-switching.......................................... 61 7.2.5.5. Additional results: High-paced taskswitching...................................................................... 62 7.2.5.6. Additional results: Reversal of the conditional discrimination ............................................ 63 7.2.5.7. Concluding remarks on task switching parameters .................................................................. 64 7.2.6. Spatial reversal learning, additionally measured variables ........................................................................................ 64 7.2.7. Concluding remarks on methodology ............................................ 64 Reflection on sleep and cognition ................................................................. 65 7.3.1. Task switching and sleep disturbance (chapter 3) ........................ 65 7.3.1.1. Task switching and PFC-inactivation............................ 65 7.3.2. Nap prevention disturbs instrumental learning (chapter 4) ................................................................................................... 65 7.3.2.1. Mechanisms underlying sleep-dependent consolidation processes ............................................... 66 7.3.2.2. Concluding remarks on nap-prevention and instrumental learning ................................................... 67 7.3.3. Spatial reversal learning and sleep deprivation (chapter 5) ................................................................................................... 68 7.3.3.1. Inconsistent results between studies........................... 68 7.3.3.2. Sleep deprivation and reversal learning; alternative strategies ................................................... 69 7.3.4. Shiftwork and instrumental learning (chapter 6) .......................... 69 7.3.4.1. Sleep and body weight ................................................ 69 7.3.4.2. Instrumental learning .................................................. 70 7.3.5. Sleep and cognition; other future perspectives ............................. 70 7.3.5.1. Motor sequence learning ............................................. 71 7.3.5.2. Observational learning ................................................. 71
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7.4. 7.5.
7.3.5.3. Mood ............................................................................ 71 7.3.5.4. Neurochemistry ........................................................... 72 Concluding remarks ...................................................................................... 72 Acknowledgements ....................................................................................... 73
8. REFERENCES ............................................................................................................ 75 9. NEDERLANDSTALIGE SAMENVATTING .................................................................... 99 10. RÉSUMÉ FRANÇAIS ................................................................................................ 105 11. DEUTSCHSPRACHIGE ZUSAMMENFASSUNG .......................................................... 111
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LIST OF FIGURES PAGE Figure 1
Recovery sleep after 3h of sleep deprivation and subsequent task exposure, compared to baseline sleep within the same subjects. .......................................................................... 47
Figure 2
locomotor activity (measured by piezo-electric current detection for cages housing 2 rats) under normal and inverted light conditions from arrival onwards ............................. 49
Figure 3
locomotor activity (measured by piezo-electric current detection) for both light regimes during the day 12 period (from Sunday 7:00 to Monday 7:00) .......................................... 50
Figure 4
Sleep & wake times under normal and inverted light conditions ......................................................................... 51
Figure 5
Omissions within blocks of 8 trials after 12h of sleep deprivation and undisturbed control condition with 2 different task structures for a 2-lever reversal of spatial discrimination. ................................................................... 55
Figure 6
Omissions after 12h of sleep deprivation and (movement or undisturbed) control condition with 2 different task structures for a conditional discrimination task offered in blocks (a "switch-task") ......................................................... 57
Figure 7
Baseline switch-task performance on light versus sound trials ............................................................................... 59
Figure 8
Switch-task performance in sessions were one of the stimuli was removed. .................................................................... 61
Figure 9
Performance of a high-paced version of the switch-task ................. 63 LIST OF TABLES PAGE
Table 1
The four experimental conditions in the switch experiment. ............ 58
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ABBREVIATIONS
5-CSRTT
5-Choice Serial Reaction Time Task
AASM
American Academy of Sleep Medicine
ANOVA
Analysis of Variance
Avg
Average
BMI
Body mass Index
cAMP
cyclic Adenosine MonoPhosphate
CCW
Counter ClockWise
CW
ClockWise
EEG
ElectroEncephaloGraphy
EMG
ElectroMyoGraphy
FI40FR3
Fixed Interval 40s Fixed Ratio 3 lever presses task
FIFR
Fixed Interval Fixed Ratio task
FL
Fast Learning
FR
Fixed Ratio
FR3
Fixed Ratio 3 lever presses
hab
Habituation
i.d.
inner diameter
IL
Instrumental Learning
ITI
InterTrial Interval
LD
Light-Dark (cycle / phase)
LP
Lever Press
MC
Movement Control
mPFC
medial PreFrontal Cortex
MWM
Morris Water Maze
NIH
National Institutes of Health (US)
NREMS
Non-REMS
o.d.
outer diameter
OSAS
Obstructive Sleep Apnoea Syndrome
PVT
Psychomotor Vigilance Task
Rec
Recovery
REMS
Rapid Eye Movement Sleep
rms
root mean square
RNA
RiboNucleic Acid
RPM
Rotations Per Minute - 11 -
rPVT
rat PVT
SD
Sleep Deprivation
SEM
Standard Error of the Mean
SF
Sleep Fragmentation
SL
Slow Learning
SPSS
Statistical Package for the Social Sciences
SRA
Stimulus-Response Association
US
United States of America
SWS
Slow Wave Sleep
ZT
ZeitGeber time (in hours from light onset)
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1.
GENERAL INTRODUCTION Cathalijn Leenaars
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General introduction Although all of us have personal experience with sleep, and although many researchers have dedicated their professional lives to investigating this phenomenon since the first official sleep deprivation experiment was performed in 1895 (according to Horne, 2006), no clear definition is available to describe exactly what sleep is (Franken et al., 2009), or why it is important. A lack of sleep does however have very clear consequences for cognition; sleep deprivation induces cognitive impairments. However, the mechanism underlying these impairments remains elusive. The current thesis describes a number of experiments that contribute to the understanding of the mechanism(s) responsible for cognitive impairments after sleep disturbance. Up to the present, research has led to important insights on sleep, and this introduction will provide readers that do not work in this field with a broad (albeit incomplete) overview of relevant available knowledge from literature. Although it is not clear why we need sleep, there are a number of arguments supporting the assumption that sleep is important. First of all, sleep is universally present throughout the animal kingdom. Although sleep has not been investigated in all species, it has been shown to occur in nematodes, fruit-flies, fish, birds, many mammals, and probably even in jellyfish (Hendricks et al., 2000; Shaw et al., 2000; Seymour et al., 2004; Horne, 2006; Vassalli & Dijk, 2009). Circadian patterns in metabolism and activity have even been described in species such as bacteria, sponges, jellyfish and plants (Amano, 1986; Seymour et al., 2004; Horne, 2006). As the state of sleep imposes a substantial risk to an animal, it should not sleep unless sleep has a very important function (Horne, 2006). Second, evolution has lead to adaptations in some species enabling them to sleep in extraordinary circumstances. For example, some species of fish are known to secrete a mucous “sleeping bag” around them, possibly to reduce their scent attracting predators; large animals including horses, sheep and cows can sleep while standing upright without falling over; and seals and dolphins can sleep with half of their brain at the time, the other brain half maintaining continuous swimming in a circle to prevent sinking (Bell, 1972; Marshall, 1972; Dallaire & Rucklebusch, 1974; Siegel, 2005; Lapierre et al., 2007). The third indication that sleep is very important arises from the observation that missed sleep is compensated for at a later time (Vassalli & Dijk, 2009). This phenomenon of recovery sleep would not occur if sleep were not important. Related to this, the fourth indication is that with prolonged wakefulness, the tendency to sleep increases, and the ability to maintain alert and awake decreases (Trachsel et al., 1986; Trachsel et al., 1991; Horne, 2006; Christie et al., 2008). Voluntary wakefulness is only possible up to a point after which sleep will indefinitely follow. The two-process model of sleep suggests that sleep drive - 15 -
- Ch1 is increased by both a circadian and a homeostatic process. First, the circadian process increases sleep drive during the night and decreases sleep drive during the day (for humans at least, the circadian sleep drive is reversed in nocturnal species such as the rat), causing organisms to sleep in the suitable part of the circadian cycle. Second, the homeostatic process increases the sleep drive with prolonged wakefulness; we get more tired when we stay up longer. We usually fall asleep only when both the circadian and the homeostatic process ‘tell us to’, although after prolonged sleep deprivation sleep may occur at unusual circadian times (Horne, 2006). Although we do not know exactly why sleep is so important and what sleep exactly is (Franken et al., 2009), we can describe the phenomenon on both a behavioural and an electrophysiological level. Theories on the function of sleep are described below.
1.1.
Behavioural and electroencephalographic aspects of sleep
Sleep can be defined at both a behavioural and an electroencephalographic level. The behavioural repertoire surrounding sleep starts with preparatory behaviours (Wilcox, 1975); many species have a certain bed-time ritual and find and arrange their dedicated sleeping site to their needs. When going to sleep, most species will adopt a typical body posture (Vassalli & Dijk, 2009), although this posture is not essential when sleep need is high enough; people can sleep while seated (Horne, 2006), and in rats, electroencephalography (EEG, see below) consistent with sleep has even been observed during movement (Leemburg et al. 2010). Usually however, during sleep, organisms move relatively little (Vassalli & Dijk, 2009). Besides, during sleep, organisms are less likely to respond to stimulation; their arousal threshold is increased (Vassalli & Dijk, 2009). When the stimulation is sufficient enough however, the state of sleep can easily be reversed (Vassalli & Dijk, 2009), which is the main difference between sleep and anaesthetic-induced sleep-like states (Horne, 2006). The last aspect of sleep that can be observed behaviourally is the phenomenon of recovery sleep; when species are deprived of sleep, they will compensate this to a certain extent in the future (Vassalli & Dijk, 2009). Most of these behavioural aspects of sleep can be seen in all animal species investigated up to now, and thereby be used to define sleep in organisms in which electrophysiological measurements are difficult. Considered most important in this aspect are the phenomenon of recovery sleep and the increase in arousal threshold, which may distinguish “real sleep” from normal quiescence. EEG is the technique used to register the electrical activity in the brain with electrodes placed on the scalp (Cooper et al., 1969). During sleep, EEG shows a different pattern compared to wakefulness. During wake, the cortical oscillations that are recorded from the electrodes are relatively fast and of low amplitude, while the EEG slows down and amplitudes increase during sleep, resulting in slow oscillations that are characteristic for sleep.
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- Ch1 Based on the EEG, several sleep stages can be discriminated, 5 for humans and usually 2 for rats (Hobson, 1991; Horne, 2006). In humans, sleep usually starts with stage-1 mild sleep, and over time, sleep deepens, first to stage-2 sleep, where sleep spindles (see chapter 4) can be distinguished in the EEG traces, and then to deep or slow-wave sleep (SWS); stages 3 and 4. Recent studies do not always distinguish between stage 3 and 4 and analyse total SWS instead, combined into what is now called stage 3, according to the new guidelines of the American Academy for Sleep Medicine. After deep sleep follows rapid eye movement sleep (REMS), the fifth sleep stage, during which we dream most vividly. During a full night of sleep (approximately 7.5 hours), most people will cycle through these sleep stages a number of times, occasionally waking up between cycles (Dement, 1999; Horne, 2006). In rats, sleep is more fragmented than in humans. Rats habitually sleep for brief periods of 2-20 minutes throughout the day, but also throughout the night during their active phase. Possibly because of the shorter sleep duration in rats it is very difficult to distinguish sleep stages 1-4. Therefore, in rodent sleep research the consensus is to distinguish only between nonREMS (NREMS) and REMS. REMS and NREMS can be distinguished in all birds and mammals investigated so far, with the exception of sea-bound mammals such as dolphins, where no REMS has been observed. The muscle atonia that accompanies REMS would be lethal in species that breathe air but live in the water (Horne, 2006; Roth et al., 2006). In non-mammalian species including fish and fruit flies, electrophysiological correlates of sleep have also been described, and in fish, the brain waves also slow down during sleep (Howard, 1972; Marshall, 1972).
1.2.
Why do we sleep?
After describing sleep, the next question is why this typical behaviour occurs. When the sleep pattern is compared between different mammals, correlations are present between the amount and the nature of sleep and variables such as age, body size & diet, environment and the safety of the sleeping site (Hobson, 1991; Siegel, 2005). Theories on the function of sleep have arisen from cross-species comparisons, assuming that the function of sleep is identical in all these species. Theories on why we sleep are described below. Sleep suppresses behaviour, and as sleep is strongly related to the circadian phase, it can prevent activity at times when this activity is least useful or even dangerous. Sleep has therefore been proposed to occur to provide safety during times when activity would pose a risk (Hobson, 1991; Siegel, 2005). As sleep suppresses movement, it also conserves energy (Siegel, 2005). Energy expenditure is highest in small species and young individuals (due to - 17 -
- Ch1 their higher surface to volume ratio they can lose body heat relatively quickly), and they may benefit most from energy conservation. Correlated with this, they also sleep more (Siegel, 2005). Both the conservation of energy and the provision of safety have therefore been proposed as functions of sleep, but would in fact be served better by consciously being inactive without the decreased arousal threshold that occurs in sleep. Besides, in current human society, with a surplus of available energy (food), and a lack of night-time predators, energy conservation and safety will certainly not be the only reasons for sleep to occur. Another function of sleep that has been widely adopted is that sleep supports the maintenance of normal brain function, at least in higher mammals (Ficca & Salzarulo, 2004; Hobson, 2005). In line with this, there is substantial evidence for cognitive impairments after sleep deprivation (at least in mammals, described below). A number of excellent reviews describe an even greater number of interesting theories on how sleep may affect brain functioning. These reviews suggest that sleep may be essential to memory consolidation, sensory filtering, signal transduction, development and / or restoration via mechanisms comprising neuronal synchronisation, neuronal replay, synaptic plasticity, gene expression, neurotransmitter synthesis and release, and receptor availability and postsynaptic receptor activity (Giuditta et al., 1995; Smith, 1996; Maquet, 2001; Peigneux et al., 2001; Stickgold et al., 2001; Dahl & Lewin, 2002; Hobson & Pace-Schott, 2002; Steriade & Timofeev, 2003; Siegel, 2004; Jones, 2005; Rauchs et al., 2005; Siegel, 2005; Stickgold & Walker, 2005; Walker & Stickgold, 2006; Datta & Maclean, 2007; Meerlo et al., 2008; Franken et al., 2009; Longordo et al., 2009; Rector et al., 2009; Vassalli & Dijk, 2009). As no conclusive evidence is yet available to prove or disregard all of these hypotheses, proper research models are needed. The current thesis describes a number of models that may prove of value in this respect.
1.3.
Sleep and cognition
The lack of sleep is known to impair certain cognitive functions. In real-life situations, this is relatively well-studied in a medical setting, where sleepdeprived doctors are still able to perform routine tasks well, but show less planning, are more hesitant, are less innovative and have poorer social skills (Horne, 2006). Next to these real-life situations, more specific laboratory studies have found a number of cognitive domains to be specifically sensitive to sleep loss. The most salient cognitive consequence of sleep deprivation is attentional impairment. In humans, sustained attention can easily be measured with relatively boring tasks in the laboratory, where subjects have to respond as fast as possible to unpredictable stimuli. These reaction time tasks are particularly sensitive to sleep deprivation protocols. For example, continued sleep deprivation for 88h in healthy subjects leads to increases in psychomotor vigilance task (PVT) reaction times over time compared to - 18 -
- Ch1 controls that are allowed to take a 2 –hour nap every 12 hours (Doran et al., 2001). Daytime performance on the PVT show a dose-response relationship with the amount of sleep during the preceding night; if subjects sleep less, their performance is more severely impaired (Jewett et al., 1999). Individuals differ in their sensitivity to sleep deprivation, as the variability in performance in this study increased over time in the sleep-deprived group (Doran et al., 2001). Also on another attention task, the human 5-choice serial reaction time task (5-CSRTT), performance is impaired after 30h of sleep deprivation (Wilkinson, 1959). Another example of a skill with sensitivity to sleep loss is speech; speech of sleepy people is less articulate, flattened, and longer pauses are interspersed between words and sentences. Besides, sleepy subjects start to mumble (Horne, 2006). Verbal fluency can be tested in humans with different types of tasks where subjects have to freely respond with as many words as possible related to a given word, for example, verbs that can relate to a noun (e.g. “sleep, doze, nap, lean, sit, throw, fight, …” in response to “pillow”). The specific rules to which the responses have to comply are variable, but this type of tasks is very sensitive to sleep loss. The domain of cognitive flexibility can be defined as the flexibility to adapt ongoing behaviour in response to salient changes in the environment or in our current intentions (Kehagia et al., 2010). This also comprises the ability to come up with new ideas and innovative plans, and the ability to switch between performing multiple tasks. Cognitive flexibility is affected by sleep loss (see below). Although at first it may seem difficult to reliably test the ability for innovative planning, a very simple task is available to do just so; the Tower of London task. This task uses 3 differently coloured wooden blocks, stacked onto 3 wooden spindles that can hold 3, 2 or 1 of the blocks. The task is to get the wooden blocks from one configuration to another one, while only moving one block at a time. After sleep deprivation, performing this task is seriously impaired, and subjects are less flexible and perseverate in using previously successful strategies (Horne, 2006). The ability to switch between multiple tasks is often tested with so-called switch-tasks, where subjects have to perform two or more tasks interchangeably, explained in chapter 3. When a switch task was performed every 3 hours during a 40h period of continuous wakefulness in healthy volunteers, task switching was affected by both sleep deprivation and the time of day. Task switching first becomes easier while subjects are learning the task, then it becomes more difficult with prolonged wakefulness, but it becomes a bit easier again during the circadian optimum in the end of the afternoon (Bratzke et al., 2009). For performance of many tasks it is important to retain and / or manipulate sensory input, and neuroscientists use the phrase “working memory” to describe the ability to “keep new information in mind”. Working memory is impaired both after work-related sleep deprivation as for example - 19 -
- Ch1 encountered in medical residents making long days (Gohar et al., 2009), as well as in more controlled laboratory settings (Tucker et al. 2010). An elegant type of tasks to test working memory asks subjects to keep an aspect of a stimulus in mind, and respond to the current stimulus ONLY if it is similar to the stimulus shown n trials before. These so-called n-back tasks become more complicated with increasing n. E.g. it is very easy to compare the current stimulus with the immediately preceding stimulus (n = 1; a 1back task), but when one has to keep the last two (n = 2) or even three (n = 3) stimuli in mind it rapidly becomes more difficult to perform well. A 2back task is already sensitive to the effects of 21h of sleep deprivation (Smith et al., 2002). When new skills or knowledge are acquired, consolidation is required to enable later retrieval from memory. Many different types of information can be distinguished, and many types of learning & memory consolidation appear to be sensitive to sleep deprivation. For example, the skill of visual texture discrimination (between 3 diagonal bars within an array containing 358 horizontal bars) improves after overnight sleep, but is impaired after sleep deprivation (Stickgold et al., 2000; Stickgold et al., 2002). Also explicit learning of word pairs, implicit learning of a list of words, memorising the location of items within a room, remembering the turns in a fictive walk and learning to trace line figures through a mirror are sensitive to sleep deprivation (Plihal & Born, 1997, 1999). Not only nocturnal sleep, but also daytime napping after learning can help consolidation. This has been shown amongst others using a motor memory task consisting of tapping the sequence -4-1-3-2-4- on a numerical keyboard with the non-dominant hand as fast and accurately as possible. Daytime napping leads to significantly enhanced memory consolidation of this sequence tapping (Nishida & Walker, 2007). Also declarative memory, for example the free recall of a previously memorized list of words, improves with a nap during the 60 min post-learning interval (Lahl et al., 2008). Different from free recall, recognition memory may be relatively spared after sleep deprivation. However, memory for the temporal order of previously encountered stimuli seems quite sensitive to a lack of sleep (Horne, 2006). This was tested by showing sets of stimuli, e.g. faces, to subjects at different times, and afterwards asking them first of all if they have seen a specific face before, and if so, when (Horne, 2006; Walker and Stickgold, 2006). Decision making, or the rational judgement in risk taking is also affected by a lack of sleep, which can be tested with games in which subjects gamble with play money (Horne, 2006). In for example the Iowa Gambling Task, subjects choose cards from different decks. Some of these decks are low risk low benefit, while others have higher risks and occasional higher benefits. After 49h of prolonged wakefulness, sleep deprived subjects choose more frequently for cards from the disadvantageous high-risk decks (Killgore et al., 2006). - 20 -
- Ch1 Although sleep deprivation does impair performance on many of these relatively unexciting cognitive tasks, the good news is that performance on more stimulating tasks may be relatively robust to short-term sleep deprivation. For example, one night of total sleep deprivation did not alter performance on a standard intelligence test (Binks et al., 1999). Whereas the cognitive effects of sleep deprivation have received widespread attention over the last decade, some authors have noted that effects on mood are more profound (Pilcher & Huffcutt, 1996). In practice, the relatively small impairments in cognitive performance after sleep deprivation, may be overshadowed by considerable irritability (Pilcher & Huffcutt, 1996; Horne, 2006). The fact remains however, that sleep deprivation does cause cognitive impairments, specifically in attention, verbal fluency, innovative planning, task-switching, working memory, the consolidation of new information and rational decision making. These impairments may decrease the quality of life for people suffering from sleep disorders, but they may also pose a risk when sleep-deprived people are making mistakes while for example driving or working on a job where mistakes and omissions may have severe consequences. If after reading this paragraph you feel like you want to test your cognitive performance in either a normal or a sleep-deprived state, surf to http://cognitivefun.net/ where several versions of most of the described tasks are available.
1.4.
Sleep disturbances
This thesis deals with the cognitive effects of a lack of sleep, which is relevant as many people suffer from disturbed sleep, either because of intrinsic sleep disorders or by extrinsic factors. The American Sleep Disorders Association publishes the International Classification of Sleep Disorders, Diagnostic and Coding manual, which lists nearly 80 recognized sleep disorders, comprising intrinsic and extrinsic sleep disorders and circadian rhythm disorders, parasomnias and sleep disturbances associated with other medical conditions (AASM, 2001). Of special interest because of the relatively high prevalence and potentially serious (cognitive) implications are obstructive sleep apnoea, several types of insomnia, jet-lag syndrome and shift-work sleep disorder. Obstructive Sleep Apnoea Syndrome (OSAS) is characterised by repetitive episodes of upper airway obstruction occurring during sleep, which are usually associated with a reduction in blood oxygen saturation (AASM, 2001). Patients may be aware of the snoring, but are usually unaware of the 20-30s obstructions occurring multiple times each night. The main complaint of OSAS patients is severe daytime sleepiness, due to the repetitive awakenings that re-establish breathing (AASM, 2001). Patients suffering from OSAS also show mild cognitive impairment during daytime, including global intellectual dysfunction and deficits in vigilance, alertness, concentration, short- and long term memory and executive - 21 -
- Ch1 function (Sateia, 2003). However, OSAS comprises both sleep interruptions and repetitive hypoxia, and each factor may individually, or in interaction, contribute to the compromised cognitive performance, inducing more severe impairment than would be expected from just the interrupted sleep. According to the NIH (US National Institutes of Health) consensus statement on insomnia, this disorder may be defined as complaints of disturbed sleep with adverse consequences for daytime functioning, in the presence of adequate opportunity and circumstance for sleep (AASM, 2001). The disturbance consists of one or more of 3 features: 1.) difficulty in initiating sleep, 2.) difficulty in maintaining sleep and / or 3.) waking up too early (NIH, 2005). Insomnia can be either chronic (persisting for at least 30 days) or acute (e.g. the sleeplessness experienced the night before or after an exciting event or stressful situation, NIH, 2005). The cause for insomnia may be intrinsic, in idiopathic insomnia, where the neurologic control of the sleep-wake system is thought to be impaired, and in psychophysiological insomnia, which is caused by somatised tension and learned sleeppreventing associations (AASM, 2001). Extrinsic causes may also be present and include inadequate sleep hygiene, a non-permissive sleep environment (think of noise, temperature, bedpartner, etc.), high altitude (>4000m), stress and anxiety, food allergy, withdrawal of hypnotic substances, use of stimulant drugs and poisoning with certain toxic compounds (AASM, 2001). Insomnia can furthermore be secondary to other diseases, pain and medication (AASM, 2001). Patients suffering from chronic insomnia complain about impairment in day-time cognitive performance, but performance deficits have not conclusively been supported (Altena, 2010). For tips and tricks to improve your sleep, but also if you sleep well and want to contribute to insomnia research by occasionally performing a simple test on your own computer, surf to www.slaapregister.nl. Jet-lag syndrome consists of varying degrees of difficulties in initiating or maintaining sleep, excessive sleepiness, decrements in (subjective) daytime alertness and performance, and even somatic, mainly gastrointestinal symptoms following rapid travel across multiple time zones (AASM, 2001). The severity of these symptoms depends on individual susceptibility, the number of time zones crossed, the direction of travel (east or west), and the timing of takeoff and arrival (AASM, 2001). Although symptoms usually do not last for more than 3 days after a flight, a pattern of alternating good and poor sleep may occur for up to a week (AASM, 2001). Shift-work sleep disorder consists of symptoms of insomnia or excessive sleepiness that occur as transient phenomena in relation to work schedules. Typically, when working night-shifts, people have difficulties in maintaining a normal sleep duration when the major sleep episode starts in the morning (6:00 – 8:00), and wake up “early” and unrefreshed. However, shift-work sleep disorder may also occur with early morning and late evening shifts (AASM, 2001). - 22 -
- Ch1 The body needs 10 days of working at night and sleeping by day to fully adjust to this new situation, which does not occur often in shift-work schemes (Horne, 2006). Daytime sleep following night-shifts is therefore usually lighter and shorter than normal night sleep (Horne, 2006). The main two health issues related to prolonged exposure to shift-work are peptic ulcers and cardiovascular disease (Horne, 2006). The potential cognitive consequences of shift-work are discussed in chapter 6.
1.5.
Sleep deprivation in rodents
Up to the present, it is not understood how and why a lack of sleep affects cognition. Studies in humans may provide important indications for the underlying mechanisms, and e.g. brain scanning studies may indicate the brain regions involved. Still, in order to fully understand the mechanisms involved, more invasive studies are necessary. In order to enable these mechanistic studies it is necessary to use sleep deprivation in combination with cognitive tasks to study brain function in animal models. Therefore, it is essential to optimise methods for sleep disruption. While it is relatively easy to ask humans to stay awake and to provide them with sufficient stimulation to maintain wakefulness, this is more of a challenge in rodents. Conceptually the most elegant method to deprive rodents of sleep is known under the name ‘gentle handling’. This method consists of providing stimulation to rats and mice that will keep them interested and/or mildly aroused and thereby prevent sleep, which is relatively comparable to encouraging human volunteers to stay awake. Stimulation can consist of sound and tactile stimuli, providing “toys”, novel objects or new types of food to the rodents, and even temporary changes in the humidity have been implemented. The type of stimulation is unfortunately not at all standardised (compare for example Sternthal & Webb, 1986; Bodosi et al., 2004; Grassi Zucconi et al., 2006; Deboer et al., 2007; Cai et al., 2009), and the method is very time-consuming for the experimenters. Besides, its effectiveness over periods of sleep deprivation exceeding a few hours is very limited; e.g. during a 6-h sleep deprivation by means of gentle handling rats still manage to sleep 6% of the time (Deboer et al., 2007). Less gentle stimulation has also been implemented to reach sleep deprivation; for example foot-shocks have been used to maintain 24h of wakefulness in rats, but this is not more effective than gentle handling (Sternthal & Webb, 1986). An often used alternative is forced locomotion, where rodents are kept awake by requiring them to move on a rotating disk or drum or a running conveyor belt (Friedman et al., 1979; Gong et al., 2004; Roman et al., 2006). Because methods employing forced locomotion are highly standardised and automated, they are often preferred. During forced locomotion, rodents are kept awake by a combination of tactile stimulation and movement. Tactile stimulation occurs when animals do not move, the urge to move is present because if they do not move they will slide from a disk into surrounding water, slide down from the (often uneven) sides of the drum, or bump into the walls of the boxes. - 23 -
- Ch1 Usually rats manage to deal with the forced locomotion extremely well, and minimize their movement. During prolonged sleep deprivation using forced locomotion, EEG-patterns consistent with sleep have been observed while rats were moving (Leemburg et al. 2010). Therefore, the effectiveness of the previously used protocols appears to have its limits. However, when the protocols are made more variable (and thereby less predictable), this sleep during movement can mostly be prevented (see chapter 2). Besides gentle handling and forced locomotion, the administration of stimulants such as amphetamine has been used to prevent sleep for relatively short periods (Cai et al., 2009). Even more alternatives are available for the selective deprivation of REMS, which can be accomplished by either the administration of anti-depressant compounds or the use of balance methods (e.g. the inverted flowerpot in water and the pendulum), where the muscle atonia that occurs in REMS will lead to instant awakening by falling (Pearlman & Becker, 1974; Van Luijtelaar & Coenen, 1986).
1.6.
Rodent equivalents for cognitive tests
From the preceding section on cognition and sleep it is clear that sleep deprivation causes impairments in specific cognitive domains. Rodent alternatives are available for many of the described human tasks, and may be used in mechanistic studies. A selection of cognitive tasks for rodents will be described here. Rodent alternatives have been developed for the standard attentional tasks that are so sensitive to the effects of sleep deprivation in humans; the 5CSRTT and the PVT. In the rat psychomotor vigilance task (rPVT), rodents have to respond with a quick nose-poke to a light stimulus in order to receive a water reward. Sleep deprivation for 24h increases response latencies and the number of lapses on the rPVT, comparable to human PVT performance after sleep deprivation (Christie et al., 2008). In the rodent version of the 5-CSRTT, rats have to respond to a brief light stimulus in one out of 5 nose-poke holes. Attentional performance of rats is impaired after 4, 7 and 10 hours of total sleep deprivation; the response latency increased, as well as the number of omission errors, while the number of perseverative and premature responses remained unaltered (Cordova et al., 2006). Cognitive flexibility is often tested in rodents with so-called reversal tasks. In a classical reversal task, rats are first trained to perform a certain response, e.g. pressing the left lever, while the alternative response, e.g. pressing the right lever is not rewarded. After acquisition, the responsereward relationship is suddenly reversed; the previously rewarded response becomes unrewarded and vice versa. The effects of sleep deprivation on reversal learning are described in chapter 5. Rodent versions of human switch-tasks, testing another type of cognitive flexibility, were not available before the current studies. A major contribution of this thesis is the design and validation of the first switchtask for rats, which is described in chapter 3. This task may help in starting - 24 -
- Ch1 to understand the brain mechanisms of the typical flexibility deficits after sleep deprivation, but also in other disorders such as Parkinson’s disease. A rodent version of the n-back task for working memory has recently become available. For this means, five spatially separate levers are presented one at the time in certain sequences. Rats can be trained to remember the last or even the one-but-last lever presented (Ko & Evenden, 2009). N-back performance after sleep deprivation has not yet been tested in rodents, but working memory in rodents is impaired after sleep deprivation, as tested using a different task (Beaulieu and Godbout, 2000, Le Marec et al., 2001). Many paradigms are available to investigate learning and consolidation of new information in rodents, but not all of them appear to be sensitive to sleep deprivation. For example learning the position of a submersed platform in a pool (the Morris Water maze, MWM) is not sensitive to deprivation of REMS (Beaulieu and Godbout, 2000, Le Marec et al., 2001). In chapter 4 of this thesis, we describe that a brief period of sleep deprivation does impair learning of a simple response-outcome association between leverpressing and a food reward. Decision making can for example be investigated in rats using a T-maze, where both arms are rewarded. Reward sizes can be varied, as well as the route towards them; rats may have to choose between a high reward after a delay and a small immediate reward, or between a high reward after climbing over a barrier or a small reward without obstacles on route (Denk et al., 2005). For risk-taking, probabilistic tasks are available, where for example pressing one lever will always result in a small reward, while pressing the other lever will occasionally result in a large reward. Preliminary data from our group indicate that choosing between a risky big reward and a safe small reward is not altered by 12h of total sleep deprivation. As described here, rodent versions of many human tasks can be developed. For some others, such as verbal fluency & innovative planning, it is less likely that this will ever be successful. Fortunately, the available tasks will enable future mechanistic research into the cognitive domains of attention, task-switching, flexibility, working memory and risk taking.
1.7.
Scope & outline of the thesis
Eventually, we would like to help people suffering from the cognitive consequences of bad sleep with effective treatments. At the moment, the question remains how a lack of sleep induces cognitive impairments. The work described in this thesis starts to address this question, by assessing the effects of sleep deprivation in rats on learning and cognitive flexibility. It focuses on the (mostly behavioural) prerequisites for future mechanistic studies. This introduction intends to provide non-sleep specialists with sufficient background for interpreting the rest of this thesis. In chapter 2, a new - 25 -
- Ch1 method for inducing sleep deprivation in rats is introduced, based on variable forced locomotion. Although forced locomotion itself may induce a stress response (Roman et al., 2006), our new method does not increase corticosterone, an indicator of the stress response, above normal circadian maximum levels. Also the potential confounding of experimental results by an increase in locomotor activity is limited when this method is used, as activity levels during deprivation do not exceed normal wake levels. When testing behaviour in a sleep-deprived state, other factors than cognitive impairment may affect performance; motivation to “work” for a reward may be decreased, and tiredness may slow motor functioning. These potential problems are assessed using a task where rats show vast levels of leverpressing to receive food rewards, which is highly sensitive to decreases in motivation and motor impairment. Chapter 3 and 4 describe that both very mild (3h during the active-phase, chapter 4 ) and more severe (12h during the inactive-phase, chapter 3 ) sleep deprivation negatively affect cognition in rats. Chapter 3 focuses on the effect of ‘one sleepless night’ and ‘one night of disturbed sleep’ on cognitive flexibility, and a new switch-task for rats is introduced. While 12h of total sleep deprivation during the light (inactive) phase decreases accuracy on switch-task performance, 12h of repetitive sleep disturbance during the inactive phase does not alter task-switching. Chapter 4 describes the impairment in instrumental learning, the acquisition of an association between lever pressing and food reward, after 3h of active phase napprevention. In chapter 5, both 12h of inactive-phase sleep deprivation and 3h of activephase nap prevention are proven not to disturb performance on a different cognitive task, indicating that also in rats, sleep-related cognitive deficits are not generalized but limited to certain cognitive domains. Total sleep deprivation for 12h during the inactive phase does not impair the acquisition of a spatial reversal, and 3h of nap-prevention during the active phase does not impair the consolidation of reversal learning. In chapter 6, rats are exposed to 5 weeks of shift-work, and show no learning deficits on the instrumental learning task in their 5th week on this protocol, which proves that rats can habituate to regular sleep deprivation for 8h per day on 5 days per week (both in the active and in the inactive phase). Furthermore, the undisturbed control groups in this study demonstrate that instrumental learning is similar during the active and the inactive phase. In the discussion (chapter 7), first of all the preceding chapters are summarised. Next, methodological issues are discussed. Additional data are presented in this section to provide more information on shifting the daynight cycle for experimental purposes and on task parameters affecting behavioural results. Although chapter 2 describes that rats can still be motivated to perform a tasks after 12h of inactive phase sleep deprivation, in the discussion it is - 26 -
- Ch1 shown that the task structure also affects motivation; small breaks during the task actually decrease motivation, and in a sleep-deprived state these regular breaks prevent rats finishing the task. The discussion finishes with a reflection on sleep and cognition, including suggestions for future experiments, which should eventually find possible treatments to help people suffering from the cognitive consequences of bad sleep.
1.8.
Acknowledgements
Ruud Joosten, Jennifer Rautamar, Janneke Zant, Matthijs Feenstra and Eus van Someren provided constructive comments on earlier drafts of this chapter. A number of the expressed ideas arose during and following excellent lectures of Jim Horne and Irene Tobler.
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2.
A NEW AUTOMATED METHOD FOR RAT SLEEPDEPRIVATION WITH MINIMAL CONFOUNDING EFFECTS ON CORTICOSTERONE AND LOCOMOTOR ACTIVITY Cathalijn H.C. Leenaars Maurice Dematteis Ruud N.J.M.A. Joosten Leslie Eggels Hans Sandberg Mischa Schirris Matthijs G.P Feenstra Eus J.W. Van Someren J Neurosci Methods 196: 107-117
doi: 10.1016/j.jneumeth.2011.01.014. FREE download from: http://dare.ubvu.vu.nl/bitstream/handle/1871/41482/hoofdstuk_2.pdf?seq uence=5
3.
SWITCH - TASK PERFORMANCE IN RATS IS DISTURBED BY 12H OF SLEEP DEPRIVATION BUT NOT BY 12H OF SLEEP FRAGMENTATION Cathalijn H.C. Leenaars Ruud N.J.M.A. Joosten Allard Zwart Hans Sandberg Emma Ruimschotel Maaike A.J. Hanegraaf Maurice Dematteis Matthijs G.P Feenstra Eus J.W. van Someren Sleep, 35: 211-221.
doi: 10.5665/sleep.1624 Free download from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3250360/ or http://www.journalsleep.org/ViewAbstract.aspx?pid=28410
4.
INSTRUMENTAL LEARNING: AN ANIMAL MODEL FOR SLEEP DEPENDENT MEMORY ENHANCEMENT Cathalijn H.C. Leenaars Carlos E.N.Girardi Ruud N.J.M.A. Joosten Irene Lako Emma Ruimschotel Maaike A.J. Hanegraaf Maurice Dematteis Matthijs G.P Feenstra Eus J.W. Van Someren Submitted
doi: 10.1016/j.jneumeth.2013.04.003 FREE download from: http://dare.ubvu.vu.nl/bitstream/handle/1871/41482/hoofdstuk_4.pdf?seq uence=4
5.
TWO-LEVER SPATIAL REVERSAL LEARNING IS ROBUST TO THE EFFECTS OF TOTAL SLEEP DEPRIVATION Cathalijn H.C. Leenaars Ruud N.J.M.A. Joosten Michiel Kramer Ger Post Leslie Eggels Mark Wuite Maurice Dematteis Matthijs G.P Feenstra Eus J.W. Van Someren Behav Brain Res. 230: 40-47
doi: 10.1016/j.bbr.2012.01.047 FREE download from: http://dare.ubvu.vu.nl/bitstream/handle/1871/41482/hoofdstuk_5.pdf?seq uence=3
6.
UNALTERED INSTRUMENTAL LEARNING AND ATTENUATED BODY-WEIGHT GAIN IN RATS DURING NON-ROTATING SIMULATED SHIFTWORK C.H.C. Leenaars A.Kalsbeek M.A.J. Hanegraaf E. Foppen R.N.J.M.A. Joosten G. Post M. Dematteis M.G.P Feenstra E.J.W. van Someren Chronobiology International, 29: 344-355
doi: 10.3109/07420528.2011.654018. FREE download from: https://www.researchgate.net/publication/221887933_Unaltered_instrume ntal_learning_and_attenuated_body-weight_gain_in_rats_during_nonrotating_simulated_shiftwork?ev=prf_pub
7.
CONCLUDING REMARKS & GENERAL DISCUSSION Cathalijn Leenaars
- Ch7 -
CONCLUDING REMARKS & GENERAL DISCUSSION The work described in this thesis starts to address the question how a lack of sleep may induce cognitive impairments. This discussion will start with a brief summary of the work presented before. It will continue with a reflection on methodology, including data not presented elsewhere. The last section is dedicated to a brief discussion of the findings and their interpretation in light of related previous literature on sleep and cognition. This section includes suggestions for future research.
7.1.
Summary of the preceding chapters
The introduction starts with a resume of the general knowledge and hypotheses on sleep, and its role in cognitive functioning. It includes a description of the behavioural and electroencephalographic characteristics that we can observe during sleep, which are used to define sleep and distinguish it from wake behaviour. This is followed by the general hypotheses on why we sleep, including sleep's contribution to cognitive functioning, and the specific cognitive domains mostly affected by sleep loss. It provides an overview of common human sleep disorders, which can result in cognitive impairment, and finishes with a description of rodent models for both sleep deprivation and cognitive performance. Chapter 2 introduces a new method for inducing sleep deprivation in rats, based on variable forced locomotion. Contrary to some other methods of forced locomotion (e.g. Roman et al., 2006), this method does not induce significant stress, as indicated by the observation that corticosterone levels did not exceed the levels normally seen during the 24-hour day. Moreover, the method did not have the drawback of potential confounding of experimental results by an increase in locomotor activity, as may be the case in some other methods. When our method was applied for 12h of sleep deprivation during the light phase, activity levels did not exceed those normally seen during undisturbed conditions. When testing behaviour in a sleep-deprived state, other possible confounders have to be addressed as well. Notably, the effects of sleep deprivation on a specific cognitive domain may depend on nonspecific cognitive effects that affect performance on the task of interest. For example, the motivation to "work" for a reward may be decreased, and fatigue may slow motor functioning. These potential problems were investigated using a task on which rats show vast levels of lever pressing to receive food rewards, which makes this task highly sensitive to decreases in motivation and motor impairment. Potential decreases in motivation were limited by imposing a food restriction to 12g/rat/day. Chapter 3 describes the modelling of one sleepless night and one night of disturbed sleep in humans, with 12h of inactive-phase sleep deprivation or sleep disruption in rats. It tests the effect of this sleep disruption on cognitive flexibility and introduces a new switch-task. While 12h of total sleep deprivation during the light (inactive) phase decreases accuracy on - 41 -
- Ch7 switch-task performance, 12h of repetitive sleep disturbance during the inactive phase does not alter task-switching. Chapter 4 described the impairment in instrumental learning; the simple association between lever pressing and food reward, after 3h of active phase nap-prevention. EEG was measured before and between task performance. Learning is accompanied by an increase in REM sleep. Baseline sleep parameters do not predict subsequent individual differences in learning abilities. In chapter 5, both 12h of inactive-phase sleep deprivation (as a model for one sleepless night) and 3h of active-phase nap prevention did not disturb performance on a different cognitive task: spatial reversal learning. Total sleep deprivation for 12h during the inactive phase does not impair the acquisition of a spatial reversal, and 3h of nap-prevention during the active phase does not impair the consolidation of reversal learning. This indicates that also in rats, sleep-related cognitive deficits are not generalized but limited to certain cognitive domains. In chapter 6, rats were exposed to 5 weeks of non-rotating shiftwork. They showed no learning deficits on an instrumental learning task (the same task as used in chapter 4) in their 5th week on this protocol, which shows that rats may somehow habituate to regular sleep deprivation for 8h per day on 5 days per week (both in the active and in the inactive phase). Furthermore, the undisturbed control groups in this study demonstrate that instrumental learning is similar during the active and the inactive phase.
7.2.
Reflection on methodology
When investigating the effect of sleep deprivation on cognition in rats, many factors besides sleep deprivation itself can influence the results. The relevant literature has been summarised in chapter 1, while chapter 2 describes the studies which address potential confounding factors with our novel method for sleep deprivation implementing variable forced locomotion. This section will start with a further discussion of the results from chapter 2, and additional results on recovery-sleep after short-term active-phase sleep deprivation, as implemented in chapters 4 and 5. Combining data from different experiments performed during this project can shed some light on the relative effect of two of the potential confounding factors that can influence the results besides sleep deprivation: inversion of the light-dark cycle and behavioural task structure. Inversion of the light-dark cycle; keeping rats in the laboratory with the lights on during our night and with the lights off during our day, is often implemented to allow experimenters to work during normal office hours while performing tests during the dark phase of the animals. As the rats are normally raised under a normal light schedule, this procedure involves a phase-shift of 12 hours, which may induce something like a jet-lag, that could affect subsequent experimental results. - 42 -
- Ch7 Behavioural tasks are designed to answer specific research questions. The tasks themselves will be designed in a certain manner, but not all task parameters are necessarily relevant to answer the research question. Tasks can e.g. consist of a certain number of trials, and trials can be separated by breaks (intertrial intervals) of a certain duration. As tasks implementing different intertrial intervals were used during this project, the effects of the duration of the intertrial interval on performance could be investigated. Additional data are present for the behavioural tests from chapters 3 and 5. These combined and new data are presented in section 7.2.1 to 7.2.6. 7.2.1.
A new automated method for rat sleep-deprivation (chapter 2)
In chapter 2 a novel method for sleep deprivation has been described. Although the upright drum and the type of movement induction are conceptually similar to the disc-over-water-method, our method is more effective in preventing sleep, and does not suffer from the aversive aspects of water. The most likely reason for the increased effectiveness is the variability of the forced locomotion; the movement protocol changes every hour, according to the pre-programmed sequence in Error! Reference source not found., section Error! Reference source not found., thereby providing novelty and minimal arousal throughout. Both the arousal, novelty, and the increasing amount of locomotion over time are thought to counteract increasing sleep pressure during the sleep deprivation. 7.2.1.1.
Variable forced locomotion protocols
Most automated paradigms use standard protocols that are similar throughout deprivation procedures, which may be unnecessary stressful at the onset of sleep deprivation while not being sufficiently arousing at the later stages. Our variable method is sufficiently arousing from the onset to the end, and only parts of the protocol may be minimally stressful as indicated by the temporary mild increase in corticosterone. Because of the high effectiveness and low stressfulness, it can be recommended for sleep deprivation studies to implement protocols with variable forced locomotion; mild movement at the onset of the deprivation procedure and gradually more challenging movement over time. 7.2.1.2.
Confounding by stress and activity
At the end of the 12h sleep deprivation protocol, rats were as easy to handle as when not deprived (casual observations), consistent with the absence of a stress response. However, it would be elegant to confirm the absence of the stress response with additional measurements. Ultrasonic vocalisations in the 22kHz range are thought to relate to a negative state in the rat (e.g. Brudzynski, 2007). This is a non-invasive method that could be implemented to register rat "mood" at multiple time points during the deprivation protocol. Besides the lack of stress, rats maintain spontaneous activity throughout, but they do not have to move faster than they voluntarily would. Although - 43 -
- Ch7 the used infrared-displacement detection is very sensitive, it is difficult to distinguish active movement from passive movement associated with maintaining a fixed position on a rotating of bottom plate. Ideally, movement should therefore be registered on the rat, and not confounded by a passive change of location. Unfortunately, the available actimetric equipment that is so often used in humans (e.g. Gohar et al., 2009) is not yet sensitive enough to detect rat movement. An alternative could be EMG in one of the leg muscles, although this is relatively invasive and therefore not ideal. Current telemetric devices for rats registering location (e.g. Drijfhout et al., 1995) will also be activated by the passive movement of the bottom plate of our deprivation device, and will therefore not provide additional information compared to our infrared displacement detection. Theoretically, it should be possible to deduct actual rat movement by combining measurements of the rat's location (either by infrared or by telemetry) and registration of the movement of the bottom plate. This could well be the most viable movement detection method to aim for. 7.2.1.3.
Modelling one sleepless night
In this thesis, twelve hours of inactive (light) phase sleep deprivation was used to model one sleepless night. This model has some construct validity when investigating the effects of staying awake for one night as it induces 12h of continuous wakefulness during the normally inactive phase. However, important differences occur between experimental sleep deprivation in rodents and human sleep deprivation, limiting construct validity. First of all, while humans may choose to stay awake when they choose to work (or participate in an experiment) during the night, rodent sleep deprivation is never voluntary. Also, the cause of sleep deprivation in our model is external, while insomnia can also be caused by internal factors (see introduction). Sleep has many similarities in rats and humans, warranting the use of rats as model animals in sleep research and enabling careful extrapolation of the results. First of all, in both species sleep follows the behavioural patterns provided in the introduction; sleep is preceded by preparatory behaviours, both humans and rats have a typical sleep posture, sleep occurs regularly within the circadian cycle, arousal threshold is increased during sleep and recovery sleep occurs after sleep deprivation. Second, changes in the EEG show a great degree of overlap. Third, sleep can similarly be affected by pharmacological interventions. However, rat sleep is not exactly the same as human sleep. Sleep is more fragmented in rats than in humans, and in rat sleep EEG only 2 sleep stages can easily be distinguished, compared to 5 in humans (see introduction). Because of the differences in sleep architecture, sleep deprivation of similar duration may differentially affect both species. Besides working with different species, laboratory circumstances are not similar to real life. An important phenomenon which may affect laboratory - 44 -
- Ch7 animal studies, is the lack of need for laboratory animals to be awake. There is no acute danger, and there is no need to hunt for food, which is regularly provided. Zoo animals are known to sleep longer in confinement compared to their wild congeners (Horne, 2006). Assuming that this is also the case for our rats, their surplus sleep may actually make them relatively resistant to the effects of short-term sleep deprivation. Fortunately, our method for sleep deprivation in rats does reproduce cognitive impairments after sleep deprivation (chapter 3). This proves that the method does have decent face validity, and can be used for future mechanistic studies. 7.2.1.4.
Motivation after sleep deprivation
The effects of sleep deprivation on a specific cognitive domain may depend on nonspecific cognitive effects such as the motivation to "work" for a reward. Motivation can be investigated with fixed-interval (FI) fixed-ratio (FR) tasks. Chapter 2 describes performance of sleep-deprived rats on an FI40FR3 task. On a 16g/rat/day diet, sleep deprivation decreases the response rate on this task. In the following part of this discussion, it will be shown that motivation is not only affected by internal state (which is altered by amongst others sleep deprivation and food restriction), but that task structure also substantially affects motivation. The continuous presence of a lever in the skinner-box stimulates continuous responding, thereby inhibiting the initiation of recovery sleep between trials. It is generally thought that variable ratio and interval schedules lead to the highest rate of responding (Feldman et al., 1997; Mazur, 1983). It would therefore be of interest to see if a VI40VR3 task would be relatively robust to the effects of sleep deprivation compared to the FI40FR3 task used here. In the current studies, when motivated performance was affected by sleep deprivation, rats usually still performed relatively normally on the first trials of these tasks and only ceased responding later on (e.g. Figure 5; comparable data for the other tasks not shown). This may indicate that motivation after sleep deprivation decreases with prolonged time-on-task. Not many compounds are available that can truly alleviate all the symptoms of sleep deprivation. However, in humans, the relatively novel stimulant Modafinil can restore multiple executive functions, as well as alertness, after sleep deprivation to normal baseline levels (Killgore et al., 2009; Wesensten, 2006). In a non-sleep deprived state Modafinil can increase motivation in rats (Young & Geyer, 2010). As rats are less motivated to perform certain instrumental tasks after sleep deprivation, it would be of interest to test if the administration of Modafinil can restore normal motivation in a sleep-deprived state. If so, the decreased performance on the FI40FR3 task described in chapter 2 could be used as a model for decreased motivation after sleep deprivation.
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- Ch7 7.2.2.
Additional results: inactive phase sleep deprivation and recovery sleep
Chapters 4 and 5 describe the cognitive effects of short-term (3h) activephase sleep deprivation, using the same method as described above. Rats normally nap substantially during their active phase. The amount of sleep that is prevented by sleep deprivation in the active phase, and subsequent recovery sleep, were quantified by EEG measurements during the experiments in chapter 4. Rats were exposed to 2 daily sessions of instrumental learning. EEG was measured before, between and after these sessions. Sleep deprivation was scheduled during the 3 hours between the first and the second session. EEG could not be measured during learning, but recovery sleep was observed during the 6 30-minute intervals following the second session of instrumental learning. Methods are described in detail in chapter 4. Sleep deprivation effectively prevented sleep; during the 3 hour sleep deprivation protocol, SWS decreased from 39.4% to 0.8% of the time and REMS from 1.4% to 0.0% compared to the same time on the preceding day. Recovery sleep, or increases in sleep following sleep deprivation compared to baseline sleep, were tested for the half-hourly intervals subsequent to the second session of instrumental learning using ANOVAs with time (six 30minute intervals) and day (baseline or recovery) as within-subject factors. Planned simple contrasts were used to locate potential time effects and interactions. Results are shown in Figure 1. For SWS duration, a generalized effect over the 3h period was observed (F1.0,15.0 = 13.9; p = 0.002), indicating a generalized increase in SWS. For REMS duration, the interaction between time and day was significant (F5.75 = 2.8; p = 0.024), but the simple contrasts for this effect were not significant. The significant day-by-time interaction for REMS duration could not be located with the simple contrasts, therefore individual time points were compared post-hoc using paired Student's t-tests. These post-hoc ttests indicated that the increase in REMS was only significant during the fourth half hour (t16 = -2.1; p = 0.048). Recovery effects were also observed when sleep deprived rats were compared to undisturbed control rats (e.g. p = 0.001 for SWS and p = 0.008 for REMS, data not shown). For cortical spindle duration, both the day-effect (F1.0,9.0 = 11.0; p = 0.009) and the day-time interaction (F5,45 = 3.2; p = 0.015) were significant. Spindle duration was decreased during the 1st - 3rd and 5th half hour of recovery (p = 0.038). For cortical spindle number, the day * time interaction was significant (F5,50 = 3.6; p = 0.007). Cortical spindle numbers were increased during the 1st half hour of recovery sleep (p = 0.002). For parietal spindle duration, the day-effect was significant (F1.0,9.0 = 9.2; p = 0.014), spindle duration was decreased throughout recovery sleep. For parietal spindle number, the day * time interaction was significant (F5,50 = 3.6; p = 0.007). The number of spindles was increased during the first half hour of recovery sleep (p = 0.021). - 46 -
- Ch7 No significant recovery effects were present for spindle amplitude (data not shown). Figure 1
Recovery sleep after 3h of sleep deprivation and subsequent task exposure, compared to baseline sleep within the same subjects.
A.)SWS duration; b.) REMS duration; c.) spindle duration measured from the cortical electrode; d.) spindle number measured from the cortical electrode; e.) spindle duration measured from the parietal electrode; f.) spindle number measured from the parietal electrode.
To conclude, even a brief 3h period of sleep deprivation during the active phase results in significant recovery sleep.
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- Ch7 7.2.3.
Inversion of the light-dark cycle
As the current thesis focuses on rat studies, and rats are preferably nocturnal animals, their light-dark cycle is often inverted to enable experiments during their active, dark phase, without causing substantial sleep deprivation and night shifts for the experimenters. Unfortunately, few studies have been performed to identify the minimal time required for a complete behavioural and physiological adaptation to the inverted lightdark cycle (Bertoglio and Carobrez, 2002). Inversion of the light-dark cycle is not necessary for all experiments. In the current thesis, if continuous measurements were made for prolonged periods, rats were housed under normal light conditions. In chapter 6 we present an experiment with equal numbers of rats on a normal and an inverted light cycle, for which locomotor activity measurements were performed. Also, baseline electroencephalographic (EEG) registrations, and thereby sleep architecture data, are available for normal light-dark conditions (all EEG-experiments in chapter 2 and the EEG-experiment in chapter 5, in total n = 20), as well as for inverted light conditions (all EEGexperiments in chapter 3, n = 43). Comparisons of spontaneous locomotor activity and time spent sleeping under normal light conditions and after inversion of the light dark cycle are described in section 7.2.3.1 and 7.2.3.2 respectively. 7.2.3.1.
Additional results: Locomotor activity after a light-dark reversal
The experiment described in chapter 6 includes both a group on regular light cycle (n = 16) and a group on an inverted light cycle (n = 16). For these rats, activity was measured from arrival from the animal supplier (Harlan, Horst, the Netherlands) onwards, by means of piezo-electric elements responding to vibration, connected to metal plates below the home cages, as previously described (Cailotto et al., 2005). These data allow for a study of the adaptation of the locomotor activity pattern to an inversion of the light-dark cycle. At our animal supplier (Harlan), rats are housed under a regular 12:12h light-dark cycle. Rats are transported in dark boxes, and housed in the requested light-dark cycle upon arrival. In Figure 2 the activity of these rats is plotted in hourly intervals, for the first 11 days after arrival. Separate lines represent activity of the group maintained on the regular light cycle and the group that were exposed to the inverted light cycle from arrival onwards. After arrival, a clear circadian pattern can be observed in the locomotor activity, consistent with the normal light cycle at the supplier. The noninverted group shows high locomotor activity during the dark phase (19:007:00) while their activity is low during the light phase (7:00-19:00). The group that underwent a 12h phase-shift upon arrival slowly adapts to the inverted light-dark-cycle. After 11 days, they show high locomotor activity during the dark phase (7:00-19:00) and low activity during the light phase (19:00-7:00). - 48 -
- Ch7 Figure 2
locomotor activity (measured by piezo-electric current detection for cages housing 2 rats) under normal and inverted light conditions from arrival onwards
Data are presented as mean ± SEM.
Total locomotor activity throughout the light and the dark period on day 12 after inversion (from Sunday 7:00 to Monday 7:00) was compared with an ANOVA with circadian phase (light or dark) as within subject factor and circadian phase inversion as a between-subject factor. Activity at preceding intervals was not tested as data for half of the cages was not available due to a technical failure. A Greenhouse-Geisser correction was applied as data violated the assumption of sphericity In these data (Figure 3), a clear effect of circadian period was observed (light versus dark, F1.0,14.0 = 398.0; p = 0.000), locomotor activity is higher during dark than during light. However, no between group effect of light regime (p = 0.37) was present, and the interaction between light regime and light-dark activity was not significant either (p = 0.31). From these results we can conclude that 12 days after phase-shifting, the activity of the inverted light group had fully adapted to the inverted light regime. In future experiments, a 12 day adaptation period to inverted light conditions is therefore sufficient as far as spontaneous locomotor activity patterns are concerned.
- 49 -
- Ch7 Figure 3
locomotor activity (measured by piezo-electric current detection) for both light regimes during the day 12 period (from Sunday 7:00 to Monday 7:00)
Data are presented as mean ± SEM. *: p right lever Light left lever; sound -> right lever Light right lever; sound -> left lever Light right lever; sound -> left lever
First SRA learned Light left lever Sound right lever Light right lever Sound left lever
SRA = Stimulus Response Association
As only four rats were trained on each condition, the data sets are too small to reliably investigate potential effects of the order of learning the stimulus response associations or of the stimulus response associations themselves. In chapter 3, the data from these four conditions were therefore always pooled; average latencies and percentages correct were calculated for all switch-trials and all 5th repetition trials over whole sessions for each rat. However, additional analyses can be performed to investigate if performance on light and sound trials is different. - 58 -
- Ch7 7.2.5.1.
Additional results: Differences in responding to light and sound stimuli during baseline
Differences may occur in responding to sound and light trials. Therefore, data from a baseline experimental session after the last interventions from chapter 3 were analysed without pooling the data for sound and light trials. Differences between light and sound trials were tested with an ANOVA including trial type (light versus sound) and trial (first versus fifth) as within-subject factors. For latencies (Figure 7), no main effect of stimulus type was observed (p = 0.2). Significant latency switch-costs occurred (F1,12 = 15.9; p = 0.002), and the interaction of trial and stimulus was significant (F1,12 = 12.0; p = 0.005). Post-hoc paired-samples t-tests for both trial types indicated that the latency-switch-effect was only significant for light blocks (T12 = 0.001), but not for sound blocks (p = 0.7). Figure 7
Baseline switch-task performance on light versus sound trials
Significant accuracy switch-costs were also observed when both stimulus types were separately analysed (F1,12 = 5.3; p = 0.040, Figure 7). Stimulus type and the interaction of stimulus-type with trial were not significant (p = 0.08 for both the main effect and the interaction), indicating that the switch-effect on accuracy was similar for both stimuli. 7.2.5.2.
Variability in response latencies
The absence of significant latency switch-costs in the sound blocks described above, and in some of the experiments described in chapter 3, may be due to the relatively high variation in latencies on this task. A potential manner to improve the latency data in future studies is by adjusting the task to prevent the occasionally present long response times - 59 -
- Ch7 affecting the data. In the current task, rats can respond to the stimulus for up to 30s. Usually, they respond within a second or 2, but latencies for occasional slow trials were included in the analyses. The task could be adjusted to prevent slow trials by decreasing the maximal response time to e.g. 2s. Alternatively, data analysis might be adjusted to ignore these slow trials. A pilot with the data from the infusion experiment in chapter 3 shows that this procedure reduces the SEM for the latency data with 40-79%. When reanalysing the data this way, the latency switch-costs became significant (F1,10 = 14.4; p = 0.004). Treatment (saline versus the muscimol-baclofen mixture) did still not affect response latencies (p = 0.2 for the treatment and p = 0.4 for the treatment-by-trial interaction). 7.2.5.3.
Task symmetry
A manner to tackle the difference between responses to sound and light stimuli is by developing a more symmetric task. Two distinct conditional discriminations using compound stimuli could be offered, and the discrimination of choice made dependent on the presence or absence of another stimulus. This type of task could be developed e.g. with nose-poke units with multiple coloured stimulus-lights. For example, when a sound is present rats can be made to respond to a red light, and when the sound is absent, to a green light, ignoring irrelevant other lights. The reason that the current studies used a single conditional discrimination is the relative simplicity. On a more complex switch-task, latencies are expected to increase and accuracy is expected to decrease. The performance may thereby become less comparable with human switch-task performance, which is highly accurate and fast (see chapter 3). 7.2.5.4.
Task switching with only one stimulus
In theory, rats can perform the type of conditional discrimination task used in the current studies by observing only one out of the two stimuli, e.g. with a light, press the right lever, without a light, press the left lever, ignoring the presence of a sound stimulus on the no-light trials. To verify that both the light and the sound stimulus were used in task performance, after finishing the chapter 3 experiments, rats were exposed to a new experiment. Versions of the task containing only one stimulus were used, alternating e.g. blocks of normal light trials with blocks without stimulus, which were rewarded when the lever usually rewarded on sound-trials was pressed. Both light-only and sound-only sessions were tested. Performance was compared with the preceding baseline days. Beforehand, we verified that no day-of-week effect was present for the respective days in normal training weeks (chapter 3), using an ANOVA with both day (baseline versus altered task) and trial (first versus fifth) as within-subject factors. 7.2.5.4.1.
Additional results: Light-only task-switching
To investigate if the sound-stimuli were essential for switch-task performance, rats were exposed to a task switching between light-trials and - 60 -
- Ch7 no-stimulus trials, where the no-stimulus trials were rewarded when the sound-matched lever was pressed. When rats had to switch between blocks of light trials and blocks of no-tone trials (rewarded as tone trials), latency (Figure 8) was not altered compared to the preceding baseline day (p = 0.07). The interaction between trial and day was not significant either (p = 0.4). Latency switch-costs were observed as a significant effect of trial (1st vs. 5th, F1,12 = 14.7; p = 0.002). To conclude, latencies are unaltered by removing the sound stimulus. For accuracy (Figure 8), the effect of trial (1st versus 5th, F1,12 = 118.3; p = 0.000), a main impairing effect of task alteration (F1,12 = 51.2; p = 0.000) and the interaction between these factors (F1,12 = 40.2; p = 0.000) were significant. Post-hoc paired-samples t-tests indicated that switch-costs were significant in both baseline (t12 = -2.3; p = 0.04) and light-only (t12 = -13.8; p = 0.000) conditions. To conclude, removing the sound stimuli impairs accuracy on the switch-task, mainly on the switch-trials. After the first 2 blocks and the first 2 switches, accuracy and latency stayed constant throughout the rest of the session. Figure 8
7.2.5.4.2.
Switch-task performance in sessions were one of the stimuli was removed.
Additional results: Sound-only task-switching
To investigate if the light-stimuli were essential for switch-task performance, rats were exposed to a task switching between sound-trials and no-stimulus trials, where the no-stimulus trials were rewarded when the - 61 -
- Ch7 light-paired lever was pressed. For one of the rats in this condition, latency data were deleted from the analysis as too many errors were present. When rats had to switch between blocks of tone trials and blocks of no-light trials (rewarded as light trials), latency (Figure 8) was not altered compared to the preceding baseline day (p = 0.1). The interaction between day and trial was not significant either (p = 0.3). The effect of trial (1st vs. 5th) was significant (F1,11 = 8.8; p = 0.013), indicating latency switch-costs. To conclude, latencies are unaltered by removing the light stimulus. For accuracy in sound-only sessions (Figure 8), the effect of trial (1st versus 5th, F1,12 = 103.9; p = 0.000), a main impairing effect of task alteration (F1,12 = 221.4; p = 0.000) and the interaction between these factors (F1,12 = 82.1; p = 0.000) were significant. Post-hoc paired-samples t-tests indicated that switch-costs were significant in sound-only (t12 = -12.1; p = 0.000) conditions, but not on the preceding baseline day (p = 0.1). In conclusion, removing the light stimuli impairs accuracy on the switch-task, mainly on the switch-trials. As with light-only switching, after the first 2 blocks and the first 2 switches, accuracy and latency stayed constant throughout the rest of the session. Overall, it can be concluded that both sound and light stimuli are used in performing the switch-task, as deleting either of these stimuli impairs performance. 7.2.5.5.
Additional results: High-paced task-switching
To test if rats could also perform a switch-task while switching between the 2 stimuli at a higher pace, rats were exposed to a task where they had to switch between stimulus-response associations after 1 or 2 trials of one type. To compare switch and repetition trials, only the 2nd trials of a block (when present) could be chosen as repetition trials. Performance on the fast version of the switch-task was compared with first and second trials on the preceding baseline day (Figure 9).
- 62 -
- Ch7 Figure 9
Performance of a high-paced version of the switch-task
Switch-costs were observed for both latency (F1,12 = 36.4; p = 0.000) and for accuracy (F1,12 = 12.0; p = 0.005); second trials were performed faster and more accurate than first trials. Latencies were increased (F1,12 = 6.7; p = 0.024) and accuracy was decreased (F1,12 = 7.8; p = 0.02) on the fast switch task compared to the preceding baseline day. No interaction between tasktype and trial was observed (p = 1.0 for latencies and p = 0.2 for accuracy), indicating that the effects were generalised and not switch- or repetition specific. Concluding, rats can perform a switch-task at a higher pace. Using a higher pace of switching, a switch-task of a certain length can comprise more switch- and repetition trials. A faster switch-rate could therefore be advantageous, as the average of the switch- and repetition trials is then based on a higher number of observations, and thereby more reliable. However, this alteration is not preferable as latencies are increased and accuracy is decreased on the faster version of the task. The performance will become less comparable with human switch-task performance, which is highly accurate and fast (see chapter 3). 7.2.5.6.
Additional results: Reversal of the conditional discrimination
Using the switch-task, a more complex form of cognitive flexibility could be tested by reversing the stimulus-response associations. In a pilot experiment, two rats, after extensive (18 weeks) experience with taskswitching, were exposed to reversed stimulus-response associations for a single session. Performance remained below chance levels (15% correct and 33% correct respectively) and did not improve over this session; correct - 63 -
- Ch7 responses were occasionally made throughout the session. This indicated that after long training, the type of cognitive flexibility needed to reverse the stimulus-response associations is hardly present within a single session. Reversing stimulus-response associations in this switch-task is therefore not likely to be a useful short-term procedure. 7.2.5.7.
Concluding remarks on task switching parameters
The switch-task from chapter 3 is a useful paradigm for future studies. The additional results describe that differences occur in responding to light and sound stimuli. For future experiments, these stimuli could be analysed separately, but it is most important to implement strict counterbalancing for the various stimulus response associations (SRAs). Performance of the switch task remains fairly good when one of the stimuli is no longer presented, and also when the task is offered at a higher-paced switch-rate. Performance is severely impaired in a single-session exposure to a reversal of the conditional discrimination. As also mentioned in the discussion within chapter 3, the switch-task can be altered in many ways to address specific experimental needs. 7.2.6.
Spatial reversal learning, additionally measured variables
Chapter 5 describes the number of correct responses after a spatial reversal. Other parameters were registered during these tasks, further confirming the absence of an effect of sleep deprivation on spatial reversal learning. No clear differences between sleep-deprived and well-rested groups were observed on: The number of omissions (generally none or one), the number of nosepokes made, the latency to press the levers and the activity during the task (as measured by infrared displacement detectors, similar to those used in the sleep deprivation devices). The moment of the first reversed response (on the newly rewarded lever) in the first reversal session was also compared between the sleep-deprived and well-rested groups, as an indication for perseveration. Again, no clear differences were present (data not shown). 7.2.7.
Concluding remarks on methodology
The current thesis describes a novel automated method for sleep deprivation, with several advantages over other available methods: It is automated and highly efficacious, while potential confounding by stress and locomotor activity are limited. The advantages are thought to be related to the variability of the forced movement protocol. Inversion of the light-dark cycle, allowing sufficient time (12 days) for habituation, is preferable when performing sleep-related experiments. The inverted light-dark cycle may minimise sleep disturbance, while experiments can easily be performed during the rats' active phase.
- 64 -
- Ch7 Concerning task structure, long ITI's appear to decrease the motivation to perform. It would be advisable to prevent unnecessary breaks and to keep ITI's as short as possible when developing new tasks, at least when interested in cognition instead of motivation. The switch-task from chapter 3 is a useful paradigm for future studies, but as differences occur in responding to light and sound stimuli, it is most important to implement strict counterbalancing for the various SRAs. The switch-task can be altered in many ways to address specific experimental needs.
7.3.
Reflection on sleep and cognition
7.3.1.
Task switching and sleep disturbance (chapter 3)
From the results in chapter 3 we can conclude that task switching accuracy is impaired after 12h of sleep deprivation. Milder sleep deprivation (occurring in the control conditions and the sleep fragmentation condition) did not have such effects. A dose-response relationship between the amount of sleep deprivation and the resulting cognitive impairments could be postulated. Alternatively, a threshold amount of sleep is needed to be able to perform certain cognitive tasks, and sleep deprivation that does not bring the amount of sleep below this threshold will not affect cognitive performance. To distinguish between these two relationships between sleep and cognition, switch-task performance should be compared after sleep deprivation of various durations. 7.3.1.1.
Task switching and PFC-inactivation
The studies in chapter 3 show that the rat mPFC is involved in performing the switch task. However, while sleep deprivation induces switch-specific accuracy impairments, PFC-inactivation has a more generalised effect on both switch and repetition trials. This implies that sleep deprivation probably has a more complex effect than simply inhibiting the mPFC. The finding of impaired task switching accuracy after 12h of sleep deprivation can be used to investigate the mechanisms underlying cognitive impairments after sleep deprivation. It would be of interest to see first if stimulant compounds that decrease sleepiness can reverse this effect. By testing several compounds from different pharmacological classes, such as caffeine, amphetamine, modafinil and nicotine, the involved receptor system(s) may be revealed. Next, it would be of interest to perform neurochemical measurements during task-switching in a sleep-deprived and a control state. Using microdialysis, for example mPFC dopamine, noradrenalin and serotonin release could be measured. If neurotransmitter release is not altered by sleep deprivation, post-synaptic alterations should be further investigated. 7.3.2.
Nap prevention disturbs instrumental learning (chapter 4)
Chapter 4 shows that three hours of total sleep deprivation during the active phase (nap prevention) following initial task-exposure disturbs subsequent - 65 -
- Ch7 instrumental learning. Instrumental learning occurs within discrete sessions, and learning may occur both within and between sessions. Lever pressing during the first session of instrumental learning did not appear to be altered by experimental interventions in this study. Sleep deprivation was limited to the 3h after the first session of instrumental learning. This one-time brief period of sleep deprivation may alter memory processing within the period of sleep deprivation, but it may also affect subsequent behaviour. Sleep deprivation either affects processing of previously acquired information, or the learning process within the next session(s), or both. In line with the hypothesis that sleep-deprivation affects processing of previously acquired information, alterations in sleep can be observed in this period in non-deprived control rats; exposure to an instrumental learning paradigm affects spontaneous subsequent sleep in the 3h after initial taskexposure. The main effect observed was an increase in REM duration within this interval. The increase in REM-sleep occurs after learning, but not after a subsequent non-learning session. Furthermore, the increase in REM-sleep also occurs after late learning in slow-learning rats. The results of both sleep deprivation and EEG measurements are in line with the hypotheses that sleep is beneficial for memory consolidation (e.g. Rudoy et al., 2009) and that some form of memory processing takes place within a 3h post-task exposure time window (e.g. Sara et al., 1999). The possibility that previous sleep deprivation also affects the learning process within the next session(s) cannot however be rejected. 7.3.2.1.
Mechanisms underlying sleep-dependent consolidation processes
In the current studies, only cortical EEG was measured. Alterations in cell firing as indicated by the surface EEG are present after learning, but potential alterations in deeper brain regions that do not clearly affect the EEG may also occur. For example Eschenko & Sara have shown that during SWS 2h after odour discrimination learning, neuronal firing in the locus coeruleus is transiently increased without affecting the cortical EEG (Eschenko & Sara, 2008). The anatomical location of sleep-related learning was not determined in the current studies. Instrumental learning was previously reported to depend on neural circuits comprising dopaminergic and glutamatergic transmission between the nucleus accumbens, prefrontal cortex and amygdala (Kelley et al., 2003), providing an indication of the mechanistic whereabouts. Using virtually the same paradigm as in chapter 4, during task performance, dopamine was found to increase in the nucleus accumbens (Cheng & Feenstra, 2006). During the first session, this increase was more pronounced in rats that learned the task than in rats that did not learn the task within two sessions, indicating relevance of this dopamine release for learning. As our 3h nap-prevention protocol between the first and the second session drastically disturbs instrumental learning, one possible mechanism is that it affects dopamine transmission. - 66 -
- Ch7 After release, dopamine can bind to presynaptic autoreceptors or to postsynaptic receptors of the D1 (stimulatory) or the D2 (inhibitory) type. Dopamine receptors can influence cellular function through mechanisms comprising stimulation or inhibition of adenylate cyclase, thereby increasing or decreasing the second messenger cyclic adenosine monophosphate (cAMP) and modulation of inositol phosphate production, but also by direct effects on potassium and calcium channels (Feldman et al., 1997, Cooper et al., 2003). These reactions or alterations that occur further downstream, like the stimulation or inhibition of RNA synthesis or neurogenesis, may somehow be altered by sleep deprivation. Recent work shows that sleep deprivation reduces the normal increase of the transcription factor phosphorylated cAMP response element binding protein, which may underlie the simultaneously observed impaired memory formation (Hagewoud et al., 2010a). Recently, a number of studies has focussed on the effect of sleep deprivation on hippocampal neurogenesis in relation to memory formation, which might be another possible mechanism in disturbed instrumental learning after nap prevention. Neurogenesis can be affected by sleep, circadian time and activity (Meerlo et al., 2008a). Prolonged sleep deprivation does reduce hippocampal neurogenesis independent of adrenal stress hormones (Meerlo et al., 2008a). Although these findings are interesting for learning over the course of a few days, it is unlikely that they are very relevant to the findings in chapter 4. First of all, as neurogenesis itself is unlikely to be severely affected by our brief period of nap prevention. Neurogenesis is impaired by 4 or 7 days of sleep fragmentation, but not by a single day (Guzman-Marin et al., 2007). Differentiation, proliferation and integration of newly formed neurons may be affected by shorter periods of sleep deprivation (Meerlo et al., 2008a), but these processes usually take longer than the few hours in which many rats learn this task. Last, although occasional neurogenesis may occur in regions throughout the cortex, neurogenesis is generally limited to a few restricted areas within the brain: mainly the hippocampal dentate gyrus and the subventricular zone (Cheung et al., 2007, Hagg, 2009), areas that are generally not thought to be involved in instrumental learning. 7.3.2.2.
Concluding remarks on nap-prevention and instrumental learning
Overall, spontaneous napping after a session of instrumental learning is important for the consolidation of this learning, although it is not essential to all subjects (chapter 4). Spontaneous napping is also not essential to all types of memory consolidation; consolidation of a previously learned spatial discrimination is not altered by a similar post-task period of nap prevention (chapter 5). The negative effect of post-task exposure sleep disturbance can furthermore be prevented by substantial habituation to sleep deprivation, as indicated by the results described in chapter 6. The relationship between sleep and learning is extremely complex.
- 67 -
- Ch7 7.3.3.
Spatial reversal learning and sleep deprivation (chapter 5)
The data in chapter 5 show that twelve hours of total sleep deprivation during the light phase, as a model for one sleepless night, did not alter PFCdependent reversal learning. Also, three hours of total sleep deprivation during the active phase subsequent to reversal learning, preventing spontaneous napping, did not affect consolidation of reversal learning. These conclusions were based on the number of correct responses throughout the sessions of reversal learning and are strengthened by additionally measured variables described in section 7.2.6. 7.3.3.1.
Inconsistent results between studies
The results from chapter 5 clearly indicate that spatial reversal learning is relatively robust to the effects of sleep deprivation. Other authors have shown that 24 hours of sleep interruption did not impair reversal learning of familiar odour and texture discriminations in rats either (McCoy et al., 2007). However, spatial reversal learning on a Y-maze is sensitive to the effects of sleep deprivation in mice, although performance was not different from control conditions either when sleep deprivation only commenced after the first session of Y-maze reversal learning (Hagewoud et al., 2010). The Y-maze task is different from ours; the number of trials per day is lower (6 per day) than in spatial reversal learning in a skinnerbox (2*64 trials per day), and trials last a bit longer, as mice have to walk over the T-maze. This however is unlikely to explain the differences in the results. The trial duration is longer and the number of trials needed to learn a reversal are also lower in the McCoy et al. (2007) study, where rats have to walk to a bowl and dig out their reward (instead of quickly pressing a close by lever). The sleep-deprivation induced impairment in the Hagewoud (2010) study, in contrast to the other two, can be explained either by a species-effect; mice are perhaps more sensitive to the effect of sleep deprivation on reversal learning than rats, or by a critical effect of the amount of sleep deprivation; In the Y-maze study, mice were exposed to 5h of daily sleep deprivation immediately after training. During the first reversal session, mice had been exposed to 6 days of previous discrimination sessions, and sleep deprivation at that stage therefore totalled 30h (Hagewoud et al., 2010). It would be of interest to test if longer sleep deprivation (e.g. 23h or longer) does impair spatial reversal learning in our set-up, and if repeated 5h posttraining sleep deprivation in rats does impair reversal learning on a comparable Y-maze paradigm. My hypothesis is that more severe sleep disruption would induce stronger cognitive impairments (although also decreased motivation after more severe sleep disruption could affect the results). After long-duration total sleep deprivation, spatial reversal learning could be impaired.
- 68 -
- Ch7 7.3.3.2.
Sleep deprivation and reversal learning; alternative strategies
Interestingly, while training on a similar spatial discrimination on T-maze, mice in the sleep-deprived condition were shown to use a different learning strategy, and sleep deprived mice had increases in the transcription factor pCREB, a critical element in memory formation, in different brain regions (Hagewoud et al., 2010). Although reversal learning is robust to the effects of 12h of sleep deprivation, this does not mean that the PFC is performing normally. It would be of interest to test if reversal learning activates the same brain regions in a sleep-deprived and a control state. 7.3.4.
Shiftwork and instrumental learning (chapter 6)
The data in chapter 6 show that when rats were trained on an instrumental learning paradigm during the fifth week of shiftwork, instrumental learning was not affected by (shift)work or by circadian phase. Furthermore, shiftwork decreased the normal weight gain. This result contrasts with previous studies. The difference probably arose because our shiftwork rats do not decrease their home-cage activity between the periods of shiftwork, as they did in the previous studies (Salgado-Delgado et al., 2008, 2010). The difference in home-cage activity could be related to experimental factors, and it would be of interest to investigate in our paradigm if single housing would alter the results. Also, it would be of interest to see if the shiftwork procedure itself (the sleep deprivation devices with a variable movement protocol versus the Lafayette activity wheels with a continuous protocol) is of effect. 7.3.4.1.
Sleep and body weight
With respect to the effect of sleep deprivation on body weight gain, there are no studies proving that chronic short sleep can cause a substantial weight gain. The main two health issues related to prolonged exposure to shiftwork are peptic ulcers and cardiovascular disease (Horne, 2006). Regular short sleepers are usually not overweight and overweight people are usually not short sleepers (Horne, 2008). When people are (at a risk to become) overweight, it would therefore be advisable for them to take more exercise instead of sleeping more (Horne, 2008). However, the general notion remains that short sleep may, in a subpopulation of humans, contribute to obesity (Spiegel et al., 2009). A main methodological problem arises when investigating this phenomenon in model animals. Whereas humans truly fast during their sleep, releasing some of its energy stores during sleep to prevent waking up hungry in the middle of the night, this phenomenon hardly occurs in animals. Bigger herbivores and carnivores are digesting their meals during their sleep, thereby supplying their bodies with a continuous nutrient influx (Horne, 2006). Small rodents that are generally used in laboratory studies however do not have consolidated sleep and wake up regularly to nibble on some lab chow (or to look for food in the wild, Horne, 2006). As this truly is a substantial difference in physiology, it may be very difficult to reliably - 69 -
- Ch7 investigate the relationship between body weight gain and sleep in model animals. However, important information may still arise from studies in model animals. An important finding from the current study in combination with results described in literature (Salgado-Delgado et al., 2008, 2010), is, that reversing the rhythm may increase body weight gain, while shiftwork without reversing the spontaneous activity pattern prevents this increase. In line with this theory, non-rotating night-shift workers had similar BMIs compared to day-time shifts, even though sleeping less (de Assis et al., 2003). Controlled studies in humans could test if not fully reversing the activity pattern is also preferable during human shiftwork. 7.3.4.2.
Instrumental learning
Instrumental learning was not altered by regular work or shiftwork. It would be of interest to see if instrumental learning is altered by shiftwork protocols where between-work home-cage activity is decreased compared to non-working rats. Reversing the circadian activity pattern during shift work appears to be disadvantageous regarding increases in body weight gain. What happens to cognition during shiftwork when the circadian activity pattern is reversed is presently not known. Although instrumental learning is robust to the effects of (shift)work, in parallel to the discussion on chapter 5, this does not necessarily mean that the brain is functioning completely normally. It would be of interest to test if instrumental learning during undisturbed control conditions and during work and shiftwork activates the same brain regions. In the current study, rats were trained on an instrumental learning paradigm during the 5th week of (shift)work. Habituation to the daily period of work may prevent negative effects on cognition. It would therefore be of interest to investigate when habituation starts to occur, and to elucidate underlying brain mechanisms. Instrumental learning should be tested during the first week of (shift)work. As a 3h period of sleep deprivation after first task exposure was sufficient to disturb instrumental learning (chapter 4), impaired instrumental learning would be expected up to some point at least in the 1st week of (shift)work. EEG measurements could show if sleep efficiency during sleep is increased. Increased sleep efficiency during sleep deprivation, thereby maintaining sleep homeostasis, has been described previously (Leemburg et al. 2010). Up to the present, there is no support for the idea that regular exposure to sleep deprivation can lead to tolerance or immunity to the effects (Horne, 2006), so this finding could be of great importance. 7.3.5.
Sleep and cognition; other future perspectives
Besides the studies suggested in the preceding paragraphs, other interesting research questions came up during the current work. A few of these will be described here. - 70 -
- Ch7 First of all, a number of rodent cognitive tasks have been described in the introduction, and some of these could very well be implemented in sleep research. Of particular potential is the 5-CSRTT (see introduction). Attentional performance on this task is negatively affected by sleep deprivation (Cordova et al., 2006), but the task itself has many more possibilities because it uses five differential responses. 7.3.5.1.
Motor sequence learning
In humans, motor sequence learning is sensitive to sleep, both on a conscious task consisting of tapping a numerical sequence on the numeric keys on a standard keyboard, and on a subconscious task when a standard sequence is continuously offered on a CSRTT without telling the subjects (Walker et al., 2002, Cajochen et al., 2004, Cohen et al., 2005, Fischer et al., 2006). In mice, the 5-CSRTT has already been used to model sequence learning (Christie and Hersch, 2004), but the effect of sleep on sequence learning in rodents has not yet been described. 7.3.5.2.
Observational learning
Sleep has also been shown to benefit observational sequence learning; when sleep followed the viewing of a video of a finger-tapping hand, performance was improved if the viewed sequence was congruent with the tested sequence (Van Der Werf et al., 2009). Observational learning also occurs in rats; observation of instrumental responding enhances instrumental learning in naive rats (Zentall & Levine, 1972). Perhaps observational learning also occurs for sequence learning on the 5-CSRTT, and the effect of sleep on observational learning may then be further investigated in rats. 7.3.5.3.
Mood
Besides the cognitive effects, sleep deprivation has an even more pronounced effect on mood (Pilcher & Huffcutt, 1996, Horne, 2006). Sleep deprivation usually impairs mood, but it can actually improve mood in patients suffering from major depressive disorder (Meerlo et al., 2008b). Although mood is not an important topic in this thesis, during the experiments from chapter 3 and in other (not reported) experiments, an interesting observation was made. Rats that were exposed to 3 or 12h of total sleep deprivation were somewhat less active afterwards, but responded normally to handling. However, rats that were exposed to 12h of sleep fragmentation had an altered behavioural response to handling; they were moving more than usual and more escape-prone, consistent with increased ‘grumpiness’. From this observation the theory arises that sleep fragmentation may decrease mood more strongly than total sleep deprivation. If sleep-interrupted rats do show depression-like symptoms in, for example, the Porsolt forced swim test (learned helplessness) or in a sucrose preference test (anhedonia), sleep fragmentation might be used to model a depression-like state in rodents.
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- Ch7 7.3.5.4.
Neurochemistry
Eventually, this line of work intends to resolve the mechanisms underlying sleep-deprivation induced cognitive impairments. In chapter 3, I first mentioned the hypothesis that after sleep deprivation, neurotransmission is altered in certain brain regions. Therefore, neurochemical studies should be performed before, during and after sleep deprivation, and during task performance in a normal and a sleep-deprived state. Neurotransmission can be altered on either the presynaptic or the post-synaptic side. Presynaptic alterations should be observable on neurotransmitter release, which can be measured in freely-moving animals with for example microdialysis, voltammetry and biosensors. On the post-synaptic side, for example receptor density can be measured with immunohistochemistry or receptor autoradiography (Feldman et al., 1997). Receptor density does not necessarily correlate with the induction of second messengers, but second messengers such as cAMP and IP3 can also be measured with microdialysis (e.g. Cadogan et al., 1994, Gur et al., 1996).
7.4.
Concluding remarks
In the introduction, I described that we still do not know exactly what sleep is and why we need it (Franken et al., 2009). A lack of sleep does however have very clear consequences for cognition, as described in the introduction and in chapters 3 and 4, but the mechanisms underlying cognitive impairments after sleep deprivation are currently not known. To elucidate this mechanism, rodent models are needed, and the current thesis describes a number of new and in this respect very useful models. In this discussion, additional data were presented to provide supplementary information on these models. In humans, simple, dull and monotonous tasks are very sensitive to the effects of sleep deprivation, but cognitively demanding, relatively interesting or exciting tasks are relatively robust to the effects of shortterm sleep deprivation (Horne, 2006). Naturally, there are limits to the amount of sleep deprivation up to which people are willing, motivated and still able to perform a task. Also in rats, some cognitive functions are relatively robust to a lack of sleep (e.g. chapter 5). The lack of an observable effect of sleep deprivation on task performance does however not mean that brain function is unaltered; on relatively interesting tasks, the phenomenon of reactive reinforcement (see chapter 3) may prevent observable deficits. Besides, as indicated by studies in mice, other strategies may be used to perform a task in a sleepdeprived state, and other brain regions may be activated (Hagewoud et al., 2010a).Substantial experimental work is still needed to elucidate how cognition benefits from sleep. Suggestions for future studies were made throughout this discussion. Although personal health and cognition can clearly be affected by a lack of sleep, the main risk of sleep deprivation consists of sleepiness-related accidents (Dement, 1999). Sleepiness -related crashes induce more - 72 -
- Ch7 mortality than alcohol-related crashes, irrespective of traffic density (Dement, 1999, Horne, 2006). A problem in this respect is the inability of human beings to accurately monitor their own degree of sleepiness, their tendency to fall asleep and their moments of sleep (Dement, 1999, Horne, 2006). Furthermore, a lack of sleep may impair decision making and increase risk-taking (see introduction), which will make sleepy drivers more inclined to keep driving and less inclined to pull over and take a nap. When sleep-deprived, we should not try to drive or operate dangerous equipment. On the other side, mild sleep deprivation might even be beneficial in certain circumstances (e.g. shiftwork, chapter 6). Thus, overestimating the importance of sleep for other health factors than sleepiness-related accidents may lead to unfounded concerns about a mild lack of sleep, and may thereby increase "medical consumption", stress and the use of over the counter sleep aids (Horne, 2006). Although the effects of sleep deprivation on cognition and weight are regularly overrated in both scientific and popular literature, sleep is important, and further research is essential to find methods that can help people suffering from the consequences of bad sleep.
7.5.
Acknowledgements
This discussion includes data that are not presented elsewhere in this thesis. Ewout Foppen, Maaike Hanegraaf, Ruud Joosten, Carlos Girardi, Leslie Eggels, Mark Wuite, Irene Lako, Michiel Kramer, Hans Sandberg, Allard Zwart, Emma Ruimschotel and Ger Post (roughly in the order of data appearance) made essential contributions to performing these experiments. Ruud Joosten, Matthijs Feenstra, Eus van Someren and Victoria Foster reviewed preceding draft(s) of this chapter.
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9.
NEDERLANDSTALIGE SAMENVATTING Cathalijn Leenaars
Nederlandstalige Samenvatting Slaapdeprivatie in ratten: effecten op leren en cognitieve flexibiliteit Dit proefschrift beschrijft een reeks experimenten rond de centrale vraag hoe cognitie (de mentale activiteit die leren, waarnemen, herinneren, denken, interpreteren, geloven en het oplossen van problemen omvat) verstoord wordt door slaaptekort. De introductie vat de bestaande literatuur rond dit onderwerp kort samen, en begint met een beschrijving van het slaapgedrag. Slaapgedrag omvat bijvoorbeeld een voorkeurs-slaaphouding aannemen, weinig bewegen en een verminderde reactie op licht en geluid. Verder wordt gemiste slaap later bijna altijd ingehaald. Electroencephalografie (EEG) is een techniek om de electrische activiteit van de hersenen te meten. Het EEG is gedurende slaap anders, gedurende gewone slaap vertragen de hersengolven en worden ze groter. Gedurende REM-slaap (Rapid Eye Movement; snelle oogbewegingen) bewegen de ogen en is het EEG sneller. Dit slaapgedrag en deze hersengolven worden gebruikt om slaap te definieren en te onderscheiden van wakker zijn. Er zijn verschillende ideeën over waarom we slapen. Een van deze ideeën is dat slaap nodig is voor het goed werken van de hersenen, ofwel cognitie. Een aantal cognitieve domeinen zijn specifiek gevoelig voor slaaptekort: aandacht, spraak, flexibiliteit (het wisselen tussen verschillende taken), werkgeheugen, lange termijn geheugen, beslissingen nemen en stemming. Slaap kan worden verstoord door een onrustige slaapomgeving, maar ook door een aantal veelvoorkomende slaapstoornissen zoals slaapapneu en insomnie, en door jet-lag of wisseldienst. Mensen die hier last van hebben rapporteren vaak cognitieve problemen naast de slaapproblemen. Ook als gezonde vrijwilligers wakker worden gehouden op een slaaplaboratorium kunnen we zien dat ze het slechter doen op bepaalde cognitietesten. Om onderzoek te doen naar wat er bij deze cognitieproblemen in de hersenen gebeurt , willen we graag in de hersenen kunnen meten. Hiervoor hebben we een diermodel nodig, maar de bestaande diermodellen zijn niet optimaal. Het tweede hoofdstuk van dit proefschrift beschrijft de ontwikkeling van een nieuwe methode om ratten wakker te houden. We gebruiken hiervoor een rechtopstaande draaiende ton met een stilstaande middenwand , die op een onvoorspelbare steeds wisselende manier 2 kanten op draait (een foto van de ton is te vinden in figuur 1 in sectie Error! Reference source not found., tijdens de proeven ligt er zaagsel op de bodem). In deze ton kunnen de ratten gewoon eten en drinken, en door gaten in de middenwand kunnen ze contact met elkaar houden. In hoofdstuk 2 hebben we met EEG-metingen laten zien dat de ratten met deze methode gedurende 12h goed wakker blijven. Door het stresshormoon corticosteron te meten gedurende het wakker blijven konden we laten zien - 101 -
dat onze methode niet erg stressvol is. Verder laten activiteitsmetingen zien dat de ratten gedurende het wakker blijven meer bewegen dan nodig zou zijn om het draaien van de box bij te houden, maar dat de pieken in activiteit niet hoger zijn dan op een onverstoorde dag. Als we willen kijken naar het effect van slaaptekort op leren en geheugen, is het belangrijk om te weten of onze ratten na 12h verplicht wakker blijven nog wel een leertaak kunnen en willen doen. In hoofdstuk 2 beschrijven we een test waarbij ratten voor slaapverstoring al geleerd hadden om op een pedaal te drukken voor een voedsel-beloning. Op een licht dieet (15g/rat/dag) werkten ratten na slaapverstoring minder hard om de beloning te verdienen, maar op een iets strenger (12g/rat/dag) dieet (waarop de ratten nog steeds groeien) deden ze het net zo goed als wanneer ze vooraf hadden geslapen. Om het wisselen tussen taken (flexibiliteit) in ratten te kunnen onderzoeken, hebben we een nieuwe wisseltaak voor ratten ontworpen, waarbij in de aanwezigheid van lampjes de ene taak moest worden uitgevoerd, in de aanwezigheid van geluid de andere. Deze wisseltaak is beschreven in hoofdstuk 3. In dat hoofdstuk laten we zien dat ratten na een 12h uur durenden continue verstoring van de slaap slechter kunnen wisselen tussen 2 bekende taken. Als de verstoring van de slaap werd afgewisseld met onverstoorde rust-perioden (door de ton steeds aan en uit te zetten) deden ze het even goed als wanneer ze vooraf hadden geslapen. De prefrontale cortex, een hersengebied wat bij mensen actief is gedurende wisseltaken, wordt ook door ratten gebruikt bij het uitvoeren van deze wisseltaak; als we dit hersengebied tijdelijk uit zetten (met locale anesthesie) dan maken de ratten meer fouten. Hoofdstuk 4 beschrijft hoe slaap verandert na het leren dat het drukken op een pedaal een voedselbeloning geeft. Er zijn geen verschillen in de normale slaap vooraf tussen de ratten die dit snel leren en de ratten die dit langzaam leren. Na het leren zien we dat de ratten meer droomslaap hebben. Als we de ratten 3h wakker houden na hun eerste leersessie, dan leren ze minder snel, dus deze droomslaap kan heel belangrijk zijn voor het leren. In hoofdstuk 5 laten we met een omkeertaak zien dat niet alle soorten van leren verslechteren na slaapverstoring. Ratten hadden eerst geleerd dat alleen de rechterpedaal beloond werd, bij het indrukken van de linkerpedaal kregen ze niets (voor de helft van de ratten was dit omgekeerd en werd eerst de linkerpedaal beloond). Als dit wordt omgedraaid, moeten de ratten ineens leren op de andere pedaal te drukken. Als de ratten 12h moesten wakker blijven voordat we het beloonde pedaal wisselen, deden ze dit even goed als wanneer ze vooraf hadden geslapen. Ook als ze 3h wakker moesten blijven na de eerste keer dat we het pedaal gewisseld hadden deden ze het daarna even goed. In hoofdstuk 6 hebben we onze tonnen gebruikt om ratten een soort van ploegendienst te laten doen, ze moesten 8 uur per etmaal ‘werken’ (lopen - 102 -
in de rechtopstaande draaiende ton), 5 dagen per week, ofwel in hun actieve periode (vergelijkbaar met onze gewone werkweek) ofwel in hun slaapperiode (vergelijkbaar met continue nachtdienst). Gedurende de ‘nachtdienst’-periode gingen de ratten in totaal minder slapen. In de 5de werkweek leerden we deze ratten op een pedaal drukken (net als in hoofdstuk 4). De werkende en shiftwerkende ratten leerden dit net zo snel als ratten die niet hadden gewerkt. Het lijkt alsof de ratten in deze 5de werkweek gewend waren aan het lopen in de ton. Dit proefschrift laat zien dat slaaptekort bij ratten, net als bij de mens, de prestatie op wisseltaken en het aanleren van iets nieuws verstoort, maar dat sommige soorten van leren ook na slaaptekort nog goed gaan. In de discussie worden de mogelijkheden voor verder onderzoek beschreven. Uiteindelijk moet verder onderzoek een oplossing vinden voor cognitieve problemen na slaapverstoring.
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10.
RÉSUMÉ FRANÇAIS Cathalijn Leenaars Traduit par: Claire Verbelen
10. RÉSUMÉ FRANÇAIS La privation de sommeil chez les rats: les effets sur l'apprentissage et la flexibilité cognitive Cette thèse décrit une série d'expériences autour d'une question centrale: comment la cognition (l'activité mentale comprenant l'apprentissage, la perception, la mémoire, la pensée, l'interprétation, la croyance et la résolution de problèmes) est-elle perturbée par le manque de sommeil? L'introduction résume brièvement la littérature existante à ce sujet et commence par une description du comportement pendant le sommeil. Le comportement pendant le sommeil comprend, par exemple, le fait de se mettre dans une position de sommeil préférée, de peu remuer et une réaction réduite à la lumière ou au son. Le manque de sommeil est presque toujours récupéré par après. L'électroencéphalographie (EEG) est une technique qui permet de mesurer l'activité électrique du cerveau. L'EEG varie pendant le sommeil ; pendant le sommeil normal, les ondes cérébrales ralentissent et deviennent plus grandes alors que pendant le sommeil REM (rapid eye movement, mouvements oculaires rapides), les yeux bougent et l'EEG s'accélère. Ce comportement pendant le sommeil et ces variations des ondes cérébrales sont utilisés pour définir le sommeil et le distinguer de l'éveil. Il y a différentes hypothèses qui tentent d’expliquer pourquoi nous dormons. L’une d’entre elles est que le sommeil est nécessaire au bon fonctionnement du cerveau, ou la cognition. Un certain nombre de domaines cognitifs sont particulièrement sensibles à la privation de sommeil: l'attention, la parole, la flexibilité (passer d'une tâche à l'autre), la mémoire active (de travail), la mémoire à long terme, la prise de décision et l'humeur. Le sommeil peut être dégradé par un environnement de sommeil perturbé, mais aussi par un certain nombre de troubles du sommeil fréquents, tels que l'apnée du sommeil ou les insomnies, et par les décalages horaires ou le travail en pauses. Les gens qui en souffrent font souvent état de problèmes cognitifs en plus des troubles du sommeil. Même lorsque des volontaires sains sont maintenus éveillés dans un laboratoire du sommeil, nous pouvons remarquer qu'ils obtiennent de moins bons résultats à certains tests cognitifs. Pour analyser ce qui se passe dans le cerveau lors de ces troubles cognitifs, nous voudrions pouvoir prendre des mesures dans le cerveau. Pour cela, nous avons besoin d'un modèle animal, mais les modèles animaux existants ne sont pas optimaux. Le deuxième chapitre de cette thèse décrit le développement d'une nouvelle méthode pour maintenir des rats éveillés. Pour cela, nous utilisons un tonneau vertical, rotatif, contenant une paroi intérieure centrale fixe, qui tourne dans deux directions de façon imprévisible et irrégulière (une photo du tonneau est visible à la figure 1 de la section 2.3.3; au cours des - 107 -
essais, il y a de la sciure sur le sol). Dans ce tonneau, les rats peuvent manger et boire normalement, et par des trous dans la paroi centrale, ils peuvent rester en contact les uns avec les autres. Dans le chapitre 2, nous avons démontré grâce à des mesures EEG que les rats restaient bien éveillés pendant 12h avec cette méthode. En mesurant l'hormone de stress corticostérone pendant l'éveil, nous avons pu démontrer que notre méthode n'est pas très stressante. Ensuite, la mesure du degré d'activité montre que les rats bougent plus que ce qui est nécessaire pour compenser la rotation du tonneau pendant la période d'éveil, mais que les pics d'activité ne sont pas supérieurs à ceux d'une journée tranquille. Si nous voulons étudier l'effet de la privation de sommeil sur l'apprentissage et la mémoire, il est important de savoir si nos rats après avoir été tenus éveillés pendant 12h sont encore capables et ont encore envie d'effectuer une tâche d'apprentissage. Dans le chapitre 2, nous décrivons un test dans lequel les rats avaient déjà appris à appuyer sur une pédale pour recevoir une récompense alimentaire avant que l'on perturbe leur sommeil. Avec un régime alimentaire léger (15g/rat/jour) et après les perturbations du sommeil, les rats travaillaient moins bien pour recevoir la récompense, alors qu'avec un régime légèrement plus sévère (12g/rat/jour; permettant encore la croissance des rats), ils travaillaient aussi bien que s’ils avaient dormis auparavant. Afin d'étudier la capacité des rats de passer d'une tâche à l'autre (flexibilité), nous avons développé une nouvelle épreuve changeante; les rats devaient accomplir une certaine tâche en présence de lumière et une autre tâche en présence de bruit. Cette épreuve changeante est décrite au chapitre 3. Dans ce chapitre, nous démontrons que les rats ont plus de difficultés à passer d'une tâche familière à l'autre après une perturbation continue de sommeil de 12h. Si la perturbation du sommeil est entrecoupée de périodes de repos non perturbé (en allumant et éteignant continuellement le tonneau), ils passaient aussi bien d’une tâche à l’autre que s’ils avaient dormi auparavant. Le cortex préfrontal, une région du cerveau active chez l'homme lors de l'exécution de tâches variables, est également utilisé par les rats lors des changements de tâche; lorsque nous rendons cette région du cerveau temporairement inactive (par une anesthésie locale), les rats font plus d'erreurs. Le chapitre 4 décrit les modifications du sommeil après que les rats aient appris à appuyer sur une pédale pour recevoir une récompense alimentaire. Avant l'expérience, le sommeil normal n'est pas différent chez les rats qui apprennent vite et chez les rats qui apprennent lentement. Après l'apprentissage, nous voyons que les rats font plus de sommeil REM. Lorsque nous tenons les rats éveillés pendant 3h après leur première séance de formation, ils apprennent moins vite, ce qui suggère que ce sommeil REM peut être très important pour l'apprentissage.
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Dans le chapitre 5, nous démontrons grâce à une tâche réversible que tous les types d'apprentissage ne s'altèrent pas après les perturbations du sommeil. Dans cette expérience, les rats avaient d'abord appris à n’utiliser que la pédale de droite afin de recevoir une récompense et ils ne recevaient rien en appuyant sur la pédale de gauche (chez la moitié des rats, l'expérience inverse avait été réalisée et seule la pédale de gauche leur permettait de recevoir une récompense). Lorsque l'expérience est inversée, les rats doivent soudainement apprendre à appuyer sur l'autre pédale. Les rats, qui avaient été maintenus éveillés pendant 12h avant le changement de pédale de récompense, le faisaient aussi bien que ceux qui avaient dormi auparavant. De plus, les rats qui avaient été maintenus éveillés pendant 3h après le premier changement de pédale, l’ont aussi bien fait par après. Dans le chapitre 6, nous avons utilisé nos tonneaux afin que les rats effectuent une sorte de travail en pauses, ils devaient « travailler » 8 heures par jour (marcher dans un tonneau vertical en rotation), 5 jours par semaine, soit dans leur période active (semblable à notre semaine de travail standard) soit dans leur période de sommeil (semblable aux équipes de nuit en continu). Pendant les périodes de « service de nuit », les rats dormaient moins au total. Au cours de la 5ème semaine, nous avons appris aux rats à appuyer sur une pédale (comme dans le chapitre 4). Les rats qui travaillaient avec des horaires de travail standards et des horaires en pauses apprenaient cela aussi rapidement que les rats qui n'avaient pas travaillé. Il semble qu’au cours de la cinquième semaine, les rats avaient pris l'habitude de marcher dans le tonneau. Cette thèse montre que le manque de sommeil chez les rats, comme chez les humains, influence certaines prestations, telles que la capacité de passer d'une tâche à l'autre et l'apprentissage de choses nouvelles, mais que d'autres types d'apprentissage se passent toujours bien même après un manque de sommeil. Dans la discussion, les perspectives de recherche futures sont décrites plus en détail. Ces recherches futures devraient permettre de trouver une solution aux problèmes cognitifs après les troubles du sommeil.
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11.
DEUTSCHSPRACHIGE ZUSAMMENFASSUNG Cathalijn Leenaars Uebersetzt durch: Nikola Buelow
Deutschsprachige Zusammenfassung Schlafentzug bei Ratten: Auswirkungen auf Lernen und kongnitive Flexibilitaet Diese Doktorarbeit beschreibt eine Reihe von Experimenten, um die zentrale Frage wie Kognition (die mentale Aktivitaet, die Lernen, Wahrnehmen, Erinnern, Denken; Interpretieren; Glauben und das Loesen von Problemen umfasst) durch Schlafmangel gestoert wird. Die Einleitung fasst die bestehende Literatur dieses Themas kurz zusammen und beginnt mit der Beschreibung des Schlafverhaltens. Das Schlafverhalten beinhaltet z. B. eine Vorzugsschlafhaltung Einnehmen, wenig Bewegen und eine verminderte Reaktion auf Licht und Geraeusche. Verpasster Schlaf wird fast immer nachgeholt. Elektroenzephalografie (EEG) ist eine Technik, die elektrische Aktivitaet des Gehirns zu messen. Das EEG ist waehrend des Schlafs anders. Waehrend des normalen Schlafs verzoegern sich die Gehirnwellen und werden groesser. Waehrend des REM-Schlafs (Rapid Eye Movement; schnellle Augenbewegungen) bewegen sich die Augen und das EEG ist schneller. Das Schlafverhalten und die Gehirnwellen werden benutzt, um Schlaf zu definieren und vom Wachzustand zu unterscheiden. Es gibt verschiedene Theorien, warum wir schlafen: Eine von diesen Theorien ist, dass Schlafen noetig ist, damit das Gehirn gut arbeitet, d.h. Kognition. Eine Anzahl kongnitiver Anwendungsbereiche sind besonders anfaellig fuer Schlafmangel: Aufmerksamkeit, Sprache, Flexibilitaet (das Wechseln zwischen verschiedenen Aufgaben), Kurzzeitgedaechtnis, Langzeitgedaechtnis, Entscheidungsfaehigkeit und Stimmung. Schlaf kann durch eine unruhige Schlafumgebung gestoert werden, aber auch durch eine Anzahl haeufig vorkommender Schlafstoerungen wie Schlafapnoe und Isomnie und durch Jet-lag oder Schichtdienst. Menschen, die hierunter leiden, berichten haeufig von kognitiven Problemen neben Schlafproblemen. Auch wenn gesunde Freiwillige in einem Schlaflaboratorium wach gehalten werden, koennen wir sehen, dass sie bei bestimmten Kognitionstests schlechter abschneiden. Um zu untersuchen, was bei diesen Kognitionsproblemen im Gehirn passiert, moechten wir gern im Gehirn messen koennen. Hierfuer benoetigen wir ein Tiermodel, jedoch die existierenden Tiermodelle sind nicht optimal. Das zweite Kapitel der Doktorarbeit beschreibt die Entwicklung einer neuen Methode, Ratten wach zu halten. Wir benutzen hierfuer eine aufrechtstehende, sich drehende Tonne mit einer still stehenden Mittelwand, die sich auf eine unvorhersehbare, stets wechselnde Art hin und her dreht (ein Foto dieser Tonne ist im Bild 1 im Kapitel 2.3.3 zu finden; waehrend der Versuche liegt Saegespaene auf dem Boden). In dieser Tonne koennen die Ratten normal essen und trinken; und durch Loecher in der Mittelwand koennen sie miteinander Kontakt halten.
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Im Kapitel 2 haben wir mit EEG-Messungen gezeigt, dass Ratten mit dieser Methode 12 Stunden lang gut wach bleiben. Durch Messung des Stresshormons Kortikosteron waehrend des Wachbleibens konnten wir zeigen, dass unsere Methode nicht viel Stress hervorruft. Weiter zeigen Aktivitaetsmessungen, dass sich die Ratten waehrend des Wachbleibens mehr als noetig bewegen, um mit dem Drehen der Box mithalten zu koennen, aber dass die Aktivitaetsmaxima nicht hoeher sind als an einem ungestoerten Tag. Wenn wir die Auswirkung von Schlafmangel auf das Lernen und Gehirn betrachten wollen, ist es wichtig zu wissen, ob unsere Ratten nach 12 Stunden gezwungenem Wachseins noch eine Aufgabe erlernen koennen und wollen. Im Kapitel 2 beschreiben wir einen Test, bei dem Ratten vor der Schlafstoerung schon gelernt hatten, ein Pedal zu druecken, um Nahrung als Belohnung zu bekommen. Mit einer leichten Diaet (14g/Ratte/Tag) arbeiteten die Ratten nach der Schlafstoerung weniger hart, um sich die Belohnung zu verdienen, aber mit einer strengeren Diaet (12g/Ratte/Tag; womit die Ratten noch immer wuchsen) arbeiteten sie genauso gut, als haetten sie vorher geschlafen. Um das Wechseln zwischen Aufgaben (Flexibilitaet) bei Ratten untersuchen zu koennen, haben wir eine neue, wechselnde Aufgabe fuer Ratten entworfen, wobei in Anwesenheit von Lampen die eine Aufgabe ausgefuehrt werden musste, in Anwesenheit von Geraeuschen die andere. Diese wechselnde Aufgabe ist in Kapitel 3 beschrieben. In diesem Kapitel zeigen wir, das Ratten nach einer 12-stuendigen kontinuierlichen Schlafstoerung schlechter zwischen zwei bekannten Aufgaben wechseln koennen. Wenn die Schlafstoerung mit ungestoerten Ruheperioden abwechselt (durch An- und Ausschalten der Tonne,) machten sie es genauso gut, als wenn sie vorher geschlafen haetten. Der praefrontale Cortex, ein Hirngebiet, das bei Menschen waehrend wechselnder Aufgaben aktiv ist, wird auch von Ratten beim Ausfuehren von wechselnden Aufgaben gebraucht; wenn wir dieses Hirngebiet voruebergehend ausschalten (mit einer lokalen Betaeubung), machen die Ratten mehr Fehler. Kapitel 4 beschreibt, wie sich der Schlaf nach dem Erlernen, dass das Druecken auf ein Pedal mit Nahrung belohnt wird, veraendert. Es gibt vorher keine Unterschiede im normalen Schlaf zwischen den Ratten, die schnell lernen, und den Ratten, die langsam lernen. Nach dem Lernen sehen wir, dass die Ratten mehr Traumschlaf haben. Wenn wir die Ratten nach ihrer ersten Lerneinheit drei Stunden wach halten, lernen sie langsamer. Demnach kann dieser Traumschlaf fuer das Lernen sehr wichtig sein. Im Kapitel 5 zeigen wir mit einer Umkehraufgabe, dass sich nicht alle Lernarten nach einer Schlafstoerung verschlechtern. Ratten hatten erst gelernt, dass nur das rechte Pedal belohnt wird; beim Druecken des linken Pedals bekamen sie nichts (fuer die Haelfte der Ratten war das umgekehrt, bei diesen wurde erst das linke Pedal belohnt). Wenn dies umgedreht wird, - 114 -
muessen die Ratten auf einmal lernen, auf das andere Pedal zu druecken. Wenn die Ratten 12 Stunden wach bleiben mussten, bevor wir das belohnende Pedal wechselten, taten sie das genauso gut, als ob sie vorher geschlafen haetten. Auch wenn sie drei Stunden wach bleiben mussten nachdem wir das erste Mal das Pedal gewechselt hatten, taten sie es danach genauso gut. In Kapitel 6 haben wir unsere Tonnen benutzt, um die Ratten eine Art von Gruppenaufgabe machen zu lassen. Sie mussten acht Stunden pro Essmahlzeit ‘arbeiten’ (laufen in der aufrechtstehenden, sich drehenden Tonne), fuenf Tage pro Woche, entweder in ihrer aktiven Periode (vergleichbar mit unserer gewoehnlichen Arbeitswoche) oder in Ihrer Schlafperiode (vergleichbar mit kontinuierlichem Nachtdienst). Waehren der ‘Nachtdienst’-Perioden schliefen die Ratten insgesamt weniger. In der fuenften Arbeitswoche brachten wir diesen Ratten bei, auf ein Pedal zu druecken (wie im Kapitel 4). Die arbeitenden und Schicht-arbeitenden Ratten lernten dies genauso schnell wie die Ratten, die nicht gearbeitet hatten. Es scheint, als ob die Ratten in dieser fuenften Arbeitswoche an das Laufen in der Tonne gewoehnt waren. Diese Doktorarbeit zeigt, dass Schlafmangel bei Ratten genauso wie beim Menschen die Leistung bei wechselnden Aufgaben und das Erlernen von etwas Neuem stoert, aber das einige Arten von Lernen auch nach Schlafmangel noch gut funktionieren. In der Diskussion werden Moeglichkeiten weiterer Untersuchungen beschrieben. Letztendlich muss eine weitere Untersuchung eine Loesung fuer kognitive Probleme bei Schlafstoerungen finden.
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