Viktor Román, Roelina Hagewoud, Paul G. M. Luiten, Peter Meerlo
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BSTRACTSeveral studies indicate that chronically disrupted sleep may be involved in the development of mood disorders. In addition, animal experiments support the notion that sleep loss alters emotionality and stress reactivity. The first aim of the present study was to establish whether sleep loss alters behavioural stress reactivity in a contextual fear conditioning paradigm. To this end, we exposed sleep restricted and control rats to the stress of electric shocks in a box and 1 day later measured the amount of immobility upon re-exposure to the fearful environment, this time without shocks. The results show that a week of restricted sleep gradually diminishes the immobility response during re-exposure to the shockbox. Our second aim was to establish how sleep restriction alters the activation of brain areas involved in contextual fear conditioning; particularly, the amygdala and the dentate gyrus of the hippocampus. Immunohistochemical analysis of the brains showed that the expression of the neuronal activity marker c-Fos was not changed in the basolateral complex and central nucleus of the amygdala. However, c-Fos expression was significantly increased in the dentate gyrus. In conclusion, chronic partial sleep deprivation alters stress-related behaviour and neuronal activation patterns in the brain. The diminished fear response was not accompanied by altered neuronal activation of the amygdala but it was associated with an increased activation in the hippocampus. This suggests that the sleep loss-associated reduction in conditioned fear response might depend on alterations in brain areas where the amygdala projects to and not on changes in the amygdala itself.
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NTRODUCTIONA number of longitudinal studies have indicated that chronic partial sleep loss may be involved in the development of mood disorders in humans (Breslau et al., 1996; Chang et al., 1997; for review see Riemann and Voderholzer, 2003; Gregory et al., 2005). Various animal studies support the notion that sleep loss alters emotionality and stress reactivity (Meerlo et al., 2002; Sgoifo et al., 2006). In particular, it has been suggested that the loss of sleep may affect amygdala-dependent aspects of emotionality and behavioural stress reactivity such as fear or anxiety (Hicks and Moore, 1977; Moore et al., 1979; Mogilnicka et al., 1985; Graves et al., 2003). However, there are no controlled animal studies concerning behavioural stress reactivity and fear-related behaviour under conditions of chronic partial sleep deprivation as it often occurs in modern society. Accordingly, the first aim of the present study was to assess in rats whether chronic partial sleep deprivation alters emotionality and behavioural stress reactivity. To this end, we tested the behaviour of chronically sleep restricted and control rats in a fear-conditioning paradigm, and measured the freezing behaviour evoked by re-exposure to the box where they had received shocks the day before (Davis, 1992; Le Doux, 2000; McGaugh, 2004). As our second aim, we wanted to establish whether changes in behavioural stress reactivity are associated with altered neuronal activation. It has been shown that stress-related behaviours such as the expression of fear result in the activation of transcription factors including c-Fos (Schafe et al., 2001; Stork and Pape, 2002).
Based on this, in the present study we measured the number of cells expressing c-Fos in the amygdala and the hippocampus, both being structures that are linked to emotionality and stress reactivity (Davis, 1992; Feldman et al., 2000; LeDoux, 2000).
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ATERIALS AND METHODSAnimals and housing
In the present study, we used male Wistar rats (n=108) weighing ± 250 g at the start of the experiment (purchased from Harlan, Horst, The Netherlands). Animals were housed under a 12h light/12h dark cycle, with lights on from 09.00 h to 21.00 h. The average temperature of the animal room was 21 ± 1oC. Rats were provided with food and water ad libitum throughout the experiments. The experiment was approved by the Animal Experimentation Committee of the University of Groningen.
Sleep restriction and forced activity
The sleep restriction protocol allowed the rats to sleep 4h per day at the beginning of the light phase (09.00-13.00 h) in their home cage (Meerlo et al., 2002; Roman et al., 2005). The remainder of the time, animals were kept awake by placing them in slowly rotating wheels (40 cm in diameter) driven by an engine at constant speed (0.4 m/min). Since the sleep deprivation procedure includes mild forced locomotion, we used forced activity controls to test whether effects of sleep restriction might be due to forced activity rather then sleep loss per se. Animals of the forced activity group were placed in the same plastic drums as the ones that were used for sleep restriction. However, these wheels rotated at double speed (0.8 m/min) for half the time (10h). With this protocol, rats walked the same distance as sleep restricted ones, but had sufficient time for sleep (14h). Animals were subjected to the 10h of forced activity in one block which coincided with the last 10 h of the dark phase of the light-dark cycle. Before starting the experiments, rats were habituated to the experimental apparatus by placing them in the wheels for 1 h over 3 days.
Contextual fear conditioning
In order to assess the effect of sleep loss on behavioural stress reactivity and fear, rats were subjected to a fear conditioning paradigm after 1 or 7 days of partial sleep deprivation. On these days, rats returned to their home cage after their daily sleep deprivation session, and had 2h recovery sleep before being exposed to a fearful environment. Rats were placed in a shock-box (25 x 25 x 15 cm) and after 3 min habituation they received a 2-sec electric shock of 0.5 mA. Then, after 1 min intervals two more identical shocks were delivered. After the third shock, rats stayed in the box for another 30 sec before returning to their home cage.
A noise generator was used to mask any external disturbing sounds. Between successive animals, the shock box was thoroughly cleaned with ethanol and then dried. At 13.00 h, rats were placed in the wheels to continue the sleep restriction protocol for one more period of 20h sleep deprivation. The next day at 09.00 h, rats returned to their home cage after the daily sleep deprivation session and 2h later were re-exposed to the shock box for a 15 min period, this time without shocks, in order to measure the conditioned fear response.
During the first shock session and the re-exposure, the behaviour of the rats was recorded on videotape for 6.5 and 15 min, respectively. The behaviour of the rats was scored according to a number of categories by an experimenter unaware of the group assignment of the animals. Behavioral categories included exploration (walking and sniffing around in the box), rearing (standing on the hindlegs), grooming (cleaning face and fur) and immobility (complete cessation of movements with eyes open). For behavioural scoring, the program ELINE was used (Frans Maes, University of Groningen).
Collection and cutting of brains
Two hours after the re-exposure to the shock box, rats were deeply anesthetized with pentobarbital and, subsequently, transcardially perfused with physiological saline followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed from the skull and rinsed overnight in 0.01 M phosphate buffered saline (PBS, pH 7.4). After dehydration in 30% buffered sucrose solution, brains were frozen with liquid nitrogen and stored at -80oC. With a cryostatic microtome, 30μm sections were cut between bregma -1.8 and -3.8 (Paxinos and Watson, 1986). Brain sections were stored in 0.01 M PBS containing 0.1% sodium azide until further processing.
Immunohistochemical stainings
In order to examine the effect of sleep loss on neuronal activation in the brain, sections were immunostained for the transcription factor c-Fos. First, sections were pretreated with 0.3% H2O2 for 30 min then rinsed in phosphate buffered saline (0.01 M, pH 7.4, PBS). Non-specific binding of immunoreagents was blocked with 5% normal goat serum in PBS (Zymed, San Fransisco, CA, USA). Subsequently, sections were incubated overnight at 4oC with rabbit-anti-c-Fos primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA;
1:1000 in 5% normal goat serum in PBS). After rinsing in PBS and a second blocking step with 5% normal goat serum in PBS, the secondary antibody was added for 2h (biotinylated goat-anti-mouse; Jackson, West Grove, PA, USA; 1:1000 in 5% normal goat serum in PBS). This was followed by a thorough rinsing step in PBS and then by incubation with Avidin-Biotin-Complex for 2 h (1:300, ABC Elite kit, Vector Laboratories, Burlingame, CA, USA). Labelled cells were visualized with 0.2 mg/ml diaminobenzidine, 30 mg/ml nickel-sulphate, 16 mg/ml sodium-acetate, and 1% H2O2. Stained sections were mounted on gelatinized glass slides.
Quantification of the immunohistochemistry
The number of c-Fos positive cells was determined in the central and basolateral nucleus of the amygdala and in the granular cell layer of the dentate gyrus of the dorsal hippocampus bilaterally in 4 sections per animal.
Areas of interest were delineated with a computerized system (Leica Qwin, Rijswijk, The Netherlands). Within the demarcated areas, the number of cells was measured with a custom-written macro.
Data analysis and statistics
Behavioural and immunohistochemical data were statistically analysed by applying one-way ANOVA followed by the post hoc Tukey test if appropriate. The level of significance was set to p=0.05. Data are expressed as group averages ± SEM.
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ESULTSBehavioral stress reactivity
On the first day of fear conditioning paradigm, when the animals received a series of shocks, there was no difference between sleep restricted and control rats in any aspect of behaviour during the 6.5 min of exposure, neither after 1 or 7 days of sleep restriction (Table 1). On the second day of
the fear conditioning paradigm, when rats were re-exposed to the shock box to measure their conditioned fear response, the behaviour of the rats that had been sleep restricted for two days did not significantly differ from that of forced activity controls or home cage controls (Fig. 1A). However, after 8 days of partial sleep deprivation, the conditioned freezing response was significantly affected by the treatment (F(2,21)=4.65; p=0.023). This was caused by a significant reduction of context-induced immobility in sleep restricted animals compared to home cage controls (p=0.021) (Fig. 1B).
c-Fos immunopositive cells
To estimate general neuronal activation in the amygdala and hippocampus, two areas involved in contextual fear conditioning, brain sections were immunostained against the immediate early gene c-Fos (Fig. 2). Two or 8 days of sleep restriction in combination with fear conditioning did not result in significantly changed c-Fos-positive cell counts in the basolateral and central nuclei of the amygdala (Table 2). One-way ANOVA revealed that 2 days of treatment significantly increased the number of c-Fos-positive cells in the dentate gyrus of the hippocampus (F(2,15)=4.16; p=0.036) (Fig.
3A). The post hoc test showed that sleep restricted rats significantly differed from home cage rats (p=0.031). Also, 8 days of treatment resulted in significantly increased c-Fos-positive cell counts in the dentate gyrus (F(2, 20)=9.46; p=0.001) (Fig. 3B). The Tukey test showed that sleep restricted animals significantly differed from home cage (p=0.001) and forced activity controls (p=0.017).
1 DAY 7 DAYS
Behaviour HC FA SR HC FA SR
Exploration 54.2 ± 2.3 59.0 ± 2.1 55.0 ± 2.8 68.0 ± 2.7 64.9 ± 1.9 71.1 ± 1.4 Rearing 20.5 ± 3.0 25.6 ± 2.6 25.9 ± 3.2 19.1 ± 3.0 20.4 ± 2.2 20.3 ± 1.8 Grooming 2.1 ± 0.6 4.0 ± 0.8 3.3 ± 1.0 0.0 ± 0.0 3.6 ± 0.7 0.4 ± 0.3 Immobility 23.6 ± 4.0 12.8 ± 2.6 16.8 ± 2.9 14.4 ± 3.3 13.1 ± 2.0 9.3 ± 1.5
Table 1. Baseline behaviour during the first exposure to a fearful environment (schedule: 3 min habituation, 3 electric shocks of a duration of 2 sec with 1 min intervals, 30 sec rest). Behaviour in a shock-box was scored according to 4 categories: exploration, rearing, self-grooming and immobility. Neither after 1 nor after 7 days of partial sleep deprivation did sleep restricted rats significantly differ in their behaviour in the shock-box from control rats (group size: 7-8). Abbreviations: FA, forced activity; HC, home cage control; SR, sleep restriction.
2 DAYS 8 DAYS
HC FA SR HC FA SR
BLA 167.3 ± 15.7 137.2 ± 6.1 131.5 ± 24.6 141.6 ± 9.3 150.7 ± 11.3 174.1 ± 9.9 CeA 191.7 ± 15.7 163.3 ± 21.0 168.6 ± 52.6 175.0 ± 22.1 135.0 ± 14.7 199.6 ± 29.1
Table 2. Number of c-Fos-positive cells in the basolateral (BLA) and the central nucleus (CeA) of the amygdala. Neither 2 nor 8 days of partial sleep deprivation did significantly change the number of c-Fos-immunopositive cells (group size: 5-8). Abbreviations: FA, forced activity; HC, home cage control; SR, sleep restriction.
Figure 1. Immobility during re-exposure to the fearful context. During re-exposure, rats were placed again in the same shock-box for 15 min and their behaviour was scored. For clarity, here we show only immobility which is an accepted read-out of amygdala-related behavioural stress reactivity.
Immobility was defined as complete cessation of movements, with eyes open. [A] After 2 days of treatment, sleep restricted rats (n=7) did not show significantly different amounts of immobility in comparison with forced activity (n=7) and home cage controls (n=8). [B] Rats that were sleep restricted for 8 days (n=8) showed a significantly diminished fear response in comparison with home cage controls (n=8). Forced activity controls (n=6) were not significantly different from either groups.
Figure 2. Representative photomicrographs of brain sections containing the amygdala [A] and the hippocampus [B] after immunohistochemical staining against the transcription factor and neuronal activation marker c-Fos. [A] The number of c-Fos-positive cells was determined in two regions of the amygdala; the basolateral complex (BLA) and the central nucleus (CeA). For anatomical orientation, the optic tract is indicated (OT). [B] C-Fos-positive cells were counted in the dentate gyrus of the dorsal hippocampus (DG).
Two further hippocampal subfields are indicated; the CA1 and CA3 regions. The scale bar is 200 μm.
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ISCUSSIONThe results of the present study show that partial sleep deprivation for 8 days alters behavioural stress reactivity in a contextual fear conditioning paradigm. This manifested in the present study as reduced freezing response in a fearful environment. We further examined whether this change in behavioural stress reactivity was associated with altered activation within brain areas mediating contextual fear behaviour. In order to test this hypothesis, we counted cells expressing the immediate early gene c-fos in the amygdala and the dentate gyrus of hippocampus. Contrary to our expectations, the number of c-Fos-positive cell in the amygdala did not change in sleep restricted rats. Instead, there was a sleep deprivation-related increase in c-Fos-positive cell counts in the dentate gyrus.
Based on the altered fear response reported here and our earlier findings of an altered serotonergic signalling in the amygdala (Chapter 5), we expected changes in neuronal activation and c-Fos expression in this particular region of the brain. The amygdala controls several physiological functions including emotionality as well as neuroendocrine and behavioural stress
reactivity (Beaulieu et al., 1986; Sanford et al., 1995; Feldman et al., 2000); functions that are partly under control of the serotonergic system by means of a rich serotonergic innervation originating from the dorsal raphe nucleus (Schreibner and De Vry, 1993; Hensler, 2006). The serotonin released in the amygdala is important in maintaining the balance between excitation and inhibition by modulating GABAergic interneurons (Rainnie, 1999). Our earlier results indicated that chronic partial sleep loss increased the number of serotonin-1A receptor-associated inhibitory G-proteins in the amygdala. The serotonin-1A receptor-linked G-proteins are known to inhibit molecular pathways that lead to c-fos expression (Kovacs, 1998; Raymond et al., 1999). Since in one of our earlier studies we found increased amounts of inhibitory G-proteins, we expected to find an increased inhibition of c-fos expression. Thus, our hypothesis was that a diminished fear response should be accompanied by a decreased expression of c-fos in the amygdala (Schafe et al., 2001;
Stork and Pape, 2002). However, we did not find significant changes in the numbers of cells expressing the transcription factor c-Fos both in the central nucleus and the basolateral complex of the amygdala. However, the variation in c-Fos expression between individual animals was large, which may be partly related to a cumulative variation in response to sleep loss and variation in stress responsivity in the fear conditioning paradigm. With this variation in mind, our conclusions have to be drawn carefully. We suggest that the reduction of fear-related behaviour may be caused by altered neuronal activation in other brain areas where the amygdala projects to (Davis, 1992).
A number of studies have shown that rapid eye movement or total sleep deprivation results in altered emotionality and behavioural stress reactivity. In a study by Hicks and Moore (1977), rats spent more time in the central area of an open field indicating reduced fear due to rapid eye movement (REM) sleep deprivation. Other studies reported diminished neophobia to a novel object or to a Y-maze in rats deprived of their REM sleep (Moore et al., 1979; Moglinicka et al., 1985).
Recently, Graves et al. (2003) and Ruskin et al. (2004) showed that total sleep deprivation in rodents also interferes with the consolidation of contextual fear memories. Likewise, the present study shows that partial sleep deprivation for 8 days resulted in a significantly reduced fear response during exposure to a fearful environment.
In our earlier studies, based on a reduced serotonergic receptor sensitivity, we described chronic partial sleep deprivation as a condition that makes the brain more vulnerable to mood disorders (Roman et al., 2005; 2006). Alterations in stress-related behaviour have been found in the present study, however difficult to interpret. Firstly, these results may indicate a decreased fear response, which may be due to a sleep loss-induced hyperaroused or hyperactivated condition.
Secondly, this reduced fear response may be linked to reduced general responsiveness or compromised emotionality. In order to draw more straightforward conclusions, additional experiments investigating other aspects of emotionality such as anxiety, motivation, anhedonia have to be done.
The results of the present study show that sleep loss increased c-Fos-positive cell counts in the dentate gyrus of the dorsal hippocampus. Such an increase in c-fos expression is known to be induced by increased hippocampal activity that is associated with contextual fear conditioning
expression is associated with hippocampal activity and ultimately the formation of fear memories: a number of other transcription factors such as zif-268, JunB and Arc may play a role as well (Hall et al., 2001; Strekalova et al., 2003; Huff et al., 2006). Consequently, we can draw only limited conclusions based on our Fos cells counts. Even so, the increased number of hippocampal c-Fos-positive cells may indicate an increased hippocampal activity at least on the input side, i.e. in the dentate gyrus. Taking the increased number of c-Fos-positive hippocampal cells and the diminished fear into account, this suggests that the diminished fear response is not a result of a reduced hippocampal activity, rather, it might be a result of general hyperactivity or hyperarousal due to sleep restriction (Gessa et al., 1995).
Figure 3. Number of c-Fos-positive cells in the dentate gyrus of the hippo-campus. Immunpositive cells were counted in the granular cell layer. [A]
Two days of sleep restriction signi-ficantly increased the number of c-Fos expressing cells in sleep restricted rats (n=6) in comparison with home cage controls (n=6). Two days of forced activity (n=6) was not significantly different from the other two experimental groups. [B] Partial sleep deprivation for 8 days significantly increased c-Fos-positive cell counts in the dentate gyrus of sleep restricted rats (n=7) compared to both forced activity (n=8) and home cage controls (n=8).
conclusion, chronic partial sleep deprivation diminished fear-related behaviour, which as no
NOWLEDGEMENTS
he authors thank Jan Keijser for his help with the quantification of immunostainings.
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w t accompanied by a reduction in neuronal activation within the amygdala. Instead, sleep restriction increased neuronal activation in the hippocampus. These results on neuronal activation patterns indicate that (1) the reduced fear response might depend on alterations in brain areas were the amygdala projects to and not in the amygdala itself and that (2) the sleep loss-induced diminished fear may not be a result of reduced hippocampal activity but, rather, an altered activity.
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