Viktor Román, Jan N. Keijser, Paul G. M. Luiten, Peter Meerlo
A number of studies in humans indicate that chronic partial sleep deprivation adversely changes mood, and eventually may lead to psychopathologies associated with an altered serotonergic signalling. In earlier pharmacological challenge studies we found that chronic sleep loss in rats gradually and persistently desensitized the serotonin-1A receptor system. The first aim of the present study was to perform serotonin-1A receptor autoradiography in order to measure possible changes at the receptor level. The results show that the number of serotonin-1A receptors was not significantly altered by sleep loss in the brain areas examined. As a next step, we autoradiographically examined the serotonin-1A receptor-associated inhibitory G-proteins as downstream elements of the serotonin-1A receptor signal transduction cascade. Our results show that chronic partial sleep deprivation for 8 days selectively and significantly increased the number of 1A receptor-linked G-proteins in the amygdala. Thus, chronic partial sleep deprivation alters serotonin signalling in the amygdala, a brain structure important in the regulation of stress and emotionality.
Chronic partial sleep deprivation is frequent in modern society and on the long term it may increase the individual’s vulnerability to mood disorders such as depression (Chang et al., 1997). Alterations in the brain’s serotonergic system have long been implicated in the pathogenesis of this disorder (Cryan and Leonard, 2000; Sobczak et al., 2002). Yet, it has not been investigated whether chronic sleep loss would alter serotonergic neurotransmission as it is seen in depression (Adrien, 2002). In our earlier studies in rats we have shown that chronic partial sleep deprivation indeed changes the serotonin-1A receptor system similarly to mood disorders: while 2 days of restricted sleep had little effect, a week of repeated partial sleep deprivation gradually and persistently desensitized the postsynaptic serotonin-1A receptor system (Roman et al., 2005; 2006). This desensitization was represented by blunted physiological responses to pharmacological challenges with the serotonin-1A receptor agonist 8-OH-DPAT. Importantly, similar reduced physiological responses to injections with serotonin-1A agonists have been described in depressed subjects (Meltzer and Maes, 1995;
Shapira et al., 2000; Sobczak et al., 2002).
While pharmacological challenge studies give a good indication of the functional sensitivity of a receptor system, they provide little information on the exact localization of possible changes in the receptors. Therefore, the first aim of the present study was to measure the amount of serotonin-1A receptors after chronic partial sleep deprivation in brain areas involved in the regulation of mood and stress-reactivity by applying receptor autoradiography. Although receptor autoradiographical techniques are widely used to localize and assess changes of serotonin-1A receptor binding in the brain (Nyakas et al., 1997; Knapp et al., 1998; Meerlo et al., 2001b), no autoradiographical investigation has been carried out after chronic partial sleep deprivation so far.
To our knowledge, only one such study has been done on the serotonergic system in sleep loss but that one investigated the effects of rapid eye movement sleep deprivation on the serotonin transporter (Hipolide et al., 2005).
As a second aim, we set out to further explore the serotonin-1A receptor signalling cascade by performing G-protein autoradiography in sleep restricted and control rats (Raymond et al., 1999). A number of studies have indicated that changes in functional sensitivity of the serotonin-1A receptor system may depend on downstream elements of the signal transduction pathway rather than on the 1A receptors themselves (Li et al., 1997b; Hensler, 2002; Castro et al., 2003). However, similar to the serotonin-1A receptors, sleep loss-induced changes in G-proteins also constitute an uncharted territory within the sleep research field. One autoradiographical study has shown that the activity of adenosine A1 receptor-associated inhibitory G-proteins increased in the frontal and cingular cortex after a few hours of sleep deprivation in rats (Alanko et al., 2004), but no studies have specifically examined G-proteins associated with serotonin receptor activation.
MATERIALS AND METHODS
Animals, housing and experimental set-up
In the present study, we used young male Wistar rats (n=60) weighing ± 250 g at the start of the experiments (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 temperature of the animal room was approximately 21 ± 1oC. Rats were provided with food and water ad libitum throughout the experiments. The experiments were 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 control rats 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. The two treatments, sleep restriction and forced activity, are similar in the sense that both involve forced locomotion, however, they differ in the time available for sleep and the amount of stress (Roman et al., 2005; 2006). In other words, the sleep restriction protocol is characterized by major sleep loss accompanied by little stress, and the forced activity schedule by little sleep loss but more stress.
Collection and cutting of brains
After 2 and 8 days of treatment, sleep restricted and control rats were anaesthetized on dry ice and decapitated. Brains were removed from the skull as quick as possible, frozen with liquid nitrogen and stored at -80 oC. In a cryostate, 20-μm-sections were cut from the prefrontal cortex (bregma 3.70 – 3.46 mm), the medial septum (bregma 1.20 – 0.96 mm), the hypothalamus (bregma -0.26 – 0.50 mm), the hippocampus/amygdala (bregma -2.12 – 2.36 mm) (Paxinos and Watson, 1986). Slides were mounted on SuperFrost®Plus glass slides (Menzel-Gläser; Menzel GmbH & Co KG, Braunschweig, Germany) and stored at -80 oC.
Autoradiography of serotonin-1A receptors
The in vitro autoradiography of serotonin-1A receptors was performed according to published methods (Nyakas et al., 1997, Meerlo et al., 2001b). Brain sections were preincubated in Tris/HCl buffer (0.17 M, pH 7.6; Merck, Darmstadt, Germany) containing 4 mM CaCl2 (Merck) and 0.01% L-ascorbic acid (Sigma, St.
Louis, MO, USA) for 30 min. Preincubation was followed by a drying step in cool air stream. The incubation buffer was made by adding 10 μM N-metyl-N-2-propynyl-benzylamine (pargyline, Sigma) and 1.5 nM [3 H]8-hydroxy-2-(di-n-propylamino)tetraline ([3H]-8-OH-DPAT; specific activity: 217 μCi/nM; Amersham, Roosendaal, The Netherlands) to the Tris/HCl buffer. Sections were incubated in the incubation solution for 1 h. The incubation was followed by rinsing 3 times for 90 sec in ice cold Tris/HCl buffer and once for 5 sec in ice cold destilled water. Rinsing was followed by a drying step in cool air stream. Then, slides were desiccated with silica gel (Merck) overnight. On the next day, sections were placed in cassettes with standard autoradiographic [3H] micro-scales (Amersham) and Hyperfilm-3H film (Amersham) was placed on them. After an exposition time of 8 weeks at room temperature, films were developed according to standard photographical procedures (Kodak D-19 developer containing paramethylaminophenol sulphate, sodium sulphite, hydroquinone, sodium carbonate, citric acid and potassium metasulphate in destilled water, and fixative containing 30% w/v sodium thiosulphate in destilled water). Autoradiograms were analysed with a computer-assisted image analysis system (Quantimet 500; Leica, Cambridge, England) by an experimenter who was unaware of the group assignment of animals. After background correction, the optical density of [3H]8-OH-DPAT binding was determined and calculated into nCi of radioligand bound per mg of brain tissue according to calibration curves that were generated with the aforementioned autoradiographic scales.
Autoradiography of serotonin-1A receptor-associated G-proteins
The in vitro autoradiography of serotonin-1A receptor-activated G-proteins was performed according to published methods with slight modifications (Sim et al., 1997; Waeber and Moskowitz, 1997). Brain sections were preincubated in the assay buffer containing 3 mM MgCl2 (Merck), 100 mM NaCl (Sigma), and 0.2 mM EGTA (Merck) in Tris/HCl (50 mM, pH 7.4, Merck) for 15 min. Then, sections were incubated in the assay buffer containing 2 mM GDP (Sigma) and 9.5 mU/ml adenosine-deaminase (EC 188.8.131.52, type VI from calf intestinal mucosa; Sigma) for 30 min. After this, sections were incubated in the assay buffer, to which 2 mM GDP, 9.5 mU/ml adenosine-deaminase, 40 pM [35S]GTPγS (specific activity: 1187μCi/nM; Amersham), and 1 μM 8-OH-DPAT (Sigma) was added for 90 min. In order to assess basal binding, 8-OH-DPAT was omitted from the incubation buffer on some of the slides. After the 90-min incubation, slides were rinsed 3 times for 1.5
min in Tris/HCl buffer (50 mM, pH 7.4) and once for 5 sec in ice cold destilled water. Rinsing was followed by a drying step in cool air stream. Then, slides were desiccated with silica gel (Merck) overnight. On the next day, sections were placed in cassettes with standard autoradiographic [14C] micro-scales (Amersham) and Kodak Bio Max films (Amersham) were placed on them. After an exposition time of 96 h at room temperature, films were developed according to standard photographical procedures (Kodak D-19 developer and fixative).
Autoradiograms were analysed with a computer-assisted image analysis system (Quantimet 500; Leica, Cambridge, England) by an experimenter who was unaware of the group assignment of animals. After background correction, the optical density of [35S]GTPγS binding was determined and calculated into nCi of radioligand bound per mg of brain tissue according to calibration curves that were generated with the aforementioned autoradiographic scales.
Data analysis and statistics
Binding was measured in forebrain areas including prefrontal cortex, septum, hypothalamus, stratum oriens and radiatum of the CA1, 3, 4 subregions of the hippocampus, inner and outer granule cell layers of the dentate gyrus of the hippocampus and amygdala. Binding values obtained from the autoradiograms were calculated into percentages of the average level of the home cage control group. These data were statistically analysed by applying one-way ANOVA. The level of significance was set to p=0.05. Data are expressed as group averages ± SEM.
Serotonin-1A receptor autoradiography
To assess effects of chronic sleep restriction on the serotonergic signalling system in the brain, we performed serotonin-1A receptor autoradiography after 2 or 8 days of repeated partial sleep deprivation. Figure 1A shows a representative photomicrograph of a brain section after serotonin-1A receptor autoradiography. Two days of sleep restriction and forced activity did not significantly change the overall binding of serotonin-1A receptors (Table 1A). Also, 8 days of sleep restriction and forced activity had no significant effect on 1A receptor numbers in any of the brain areas examined (Table 1B).
Figure 1. Representative autoradiograms of brain sections labelled with [3H]8-OH-DPAT for serotonin-1A receptors [A] and with [35S]GTPγS for serotonin-1A receptor linked inhibitory G-proteins [B]. Scale bar: 4 mm.
Serotonin-1A receptor-associated G-protein autoradiography
In order to further explore serotonin-1A receptor signalling, we performed 1A receptor-associated inhibitory G-protein autoradiography after 2 or 8 days of partial sleep deprivation and forced activity. Figure 1B shows a representative photomicrograph of a brain section after G-protein autoradiography. Two days of sleep restriction did not significantly change the number of serotonin-1A receptor-activated G-proteins (Table 2A). But, 8 days of partial sleep deprivation resulted in
[A] Sleep restriction Forced activity
Brain area 2 d SR CTRL 2 d FA CTRL
Prefrontal cortex 105.5 ± 7.8 100.0 ± 3.8 112.6 ± 9.4 100.0 ± 7.8
Septum 98.5 ± 4.6 100.0 ± 5.9 103.2 ± 6.9 100.0 ± 8.5
Hypothalamus 92.1 ± 13.1 100.0 ± 12.9 78.8 ± 8.3 100.0 ± 9.7
CA1 oriens 83.9 ± 4.5 100.0 ± 2.3 99.4 ± 6.1 100.0 ± 8.3
CA1 radiatum 95.7 ± 6.8 100.0 ± 2.9 96.3 ± 6.1 100.0 ± 10.2 CA3 oriens 99.9 ± 12.8 100.0 ± 11.9 99.9 ± 28.8 100.0 ± 12.7 CA3 radiatum 86.4 ± 10.7 100.0 ± 8.2 81.1 ± 19.9 100.0 ± 13.2 CA4 oriens 109.8 ± 12.1 100.0 ± 11.9 101.4 ± 19.9 100.0 ± 8.9 CA4 radiatum 94.6 ± 11.4 100.0 ± 10.2 91.5 ± 16.6 100.0 ± 10.6
DG inner 93.7 ± 4.9 100.0 ± 6.9 92.3 ± 7.9 100.0 ± 9.1
DG outer 95.9 ± 4.1 100.0 ± 7.4 91.5 ± 5.9 100.0 ± 8.6
Amygdala 116.46 ± 10.6 100.0 ± 6.2 99.5 ± 10.2 100.0 ± 16.4
[B] Sleep restriction Forced activity
Brain area 8 d SR CTRL 8 d FA CTRL
Prefrontal cortex 93.6 ± 6.3 100.0 ± 6.5 81.1 ± 5.4 100.0 ± 10.7
Septum 90.4 ± 4.8 100.0 ± 8.1 84.9 ± 6.7 100.0 ± 8.5
Hypothalamus 73.1 ± 6.7 100.0 ± 21.5 64.9 ± 7.9 100.0 ± 22.2
CA1 oriens 105.2 ± 6.5 100.0 ± 5.6 87.6 ± 17.9 100.0 ± 8.5
CA1 radiatum 106.2 ± 5.9 100.0 ± 6.6 89.9 ± 12.5 100.0 ± 6.6 CA3 oriens 126.6 ± 26.9 100.0 ± 15.4 68.4 ± 15.9 100.0 ± 19.1 CA3 radiatum 123.4 ± 29.9 100.0 ± 17.3 69.0 ± 11.5 100.0 ± 21.6 CA4 oriens 107.7 ± 18.2 100.0 ± 16.3 79.8 ± 11.8 100.0 ± 16.8 CA4 radiatum 104.6 ± 13.3 100.0 ± 13.7 77.9 ± 13.3 100.0 ± 17.3
DG inner 103.6 ± 6.2 100.0 ± 3.8 89.8 ± 10.0 100.0 ± 7.5
DG outer 97.5 ± 3.4 100.0 ± 4.6 89.2 ± 6.7 100.0 ± 5.9
Amygdala 114.6 ± 14.3 100.0 ± 13.9 90.6 ± 10.3 100.0 ± 9.7
Table 1. Serotonin-1A receptor autoradiography after 2 [A] and 8 days [B] of partial sleep deprivation in sleep restricted and control rats. Data are expressed as percentage of values measured in home cage controls which were housed under undisturbed conditions. None of the brain areas examined showed significantly different serotonin-1A receptor binding. Data are represented as group averages ± SEM. Abbreviations: CA, cornu Ammonis; CTRL, control; DG, dentate gyrus; FA, forced activity; SR, sleep restriction.
[A] Sleep restriction Forced activity
[B] Sleep restriction Forced activity
Brain area 8 d SR CTRL 8 d FA CTRL
Table 2. Serotonin-1A receptor-associated G-protein autoradiography after 2 [A] and 8 days [B] of partial sleep deprivation in sleep restricted and control rats. Data are expressed as percentage of values measured in home cage controls which were housed under undisturbed conditions. The amygdala in partially sleep deprived rats shows significantly elevated levels (*). Data are represented as group averages ± SEM.
Abbreviations: CA, cornu Ammonis; CTRL, control; DG, dentate gyrus; FA, forced activity; SR, sleep restriction.
Figure 2. Autoradiographical labellings for the serotonin-1A receptor and for the inhibitory G-proteins that are linked to the 1A receptors in the amygdala. Serotonin-1A receptors were labelled with [3H]8-OH-DPAT and G-proteins were labelled with [35S]
GTPγS. [A] Partial sleep deprivation for 8 days significantly increased the amount of serotonin-1A receptor-associated inhibitory G-proteins (*), while the number of 1A receptors were not changed by the same treatment. [B] Forced activity for 8 days did not change the amount of receptors and that of the G-proteins. Data are expressed as percentage of values measured in home cage controls which were housed under undisturbed conditions. Data are represented as group averages ± SEM.
The results of the present study show that neither 2 nor 8 days of partial sleep deprivation caused a significant change in the number of postsynaptic serotonin-1A receptors in the rat brain.
However, 8 days of partial sleep deprivation significantly increased the amount of serotonin-1A receptor-associated G-proteins in the amygdala. Forced activity for 2 or 8 days, which involved strenuous physical exercise and stress, was not associated with any change in receptors or the G-proteins coupled to them, indicating that the alterations in G-G-proteins were due to sleep loss per se.
In our earlier studies, chronically sleep restricted rats showed significantly blunted serotonin-1A receptor-mediated physiological responses to the injection of the 1A receptor agonist 8-OH-DPAT (Roman et al., 2005; 2006). These decreased physiological responses suggested desensitization of the postsynaptic receptor system (Bill et al., 1991). However, in the present study the reduced functional sensitivity of the receptor system was not matched by significant alterations in the number of postsynaptic serotonin-1A receptors.
There are various explanations for the lack of significant alterations in the number of postsynaptic serotonin-1A receptors in the face of a significantly reduced functional sensitivity of this receptor system. Firstly, it may be that changes in serotonin-1A receptor-mediated temperature and neuroendocrine responses are related to changes in 1A receptor numbers in specific areas that are small and may not be distinguishable in autoradiograms; for instance, certain hypothalamic subregions. Secondly, it may be that the changes in functional responses are related to serotonin-1A populations in brain areas that were not included in the present study, for example, the brain stem. Finally, functional alterations may not be associated with alterations in receptor numbers but, rather, with changes in the phosphorylation state of the receptor (Raymond, 1991; Albert and Tiberi, 2001; Wurch et al., 2003) or with changes in more downstream elements of the 1A signalling cascade (Li et al., 1997a; Hensler, 2002; Castro et al., 2003; O’Connor et al., 2005).
Indeed, the literature shows that changes in functional 1A receptor-mediated responses are not necessarily associated with changes in receptor numbers. For example, in agreement with our findings, rat lines that were bred for different sensitivity to 8-OH-DPAT differ greatly in their hypothermic response to the agonist, however the expression of 1A receptors is the same (Knapp et al., 1998; 2000). Also, various other studies have shown experimentally induced changes in 1A receptor-mediated responses without changing receptor numbers. In a study by Van Riel et al.
(2003), chronic unpredictable stress led to significantly reduced serotonin-1A receptor-mediated hyperpolarization in CA1 pyramidal neurons nevertheless, receptor mRNA levels were unchanged.
The authors suggested that changes in the posttranslational modification of the receptor proteins might have caused the decreased serotonin-1A receptor-mediated response. Similarly, Tokarski et al. (1996) found that chronic imipramine treatment increased postsynaptic serotonin-1A receptor-mediated responses in CA1 pyramidal cells however, failed to change 1A receptor numbers. In accordance with this finding, numerous studies have shown changes in protein coupling or G-protein expression after chronic antidepressant treatment which was not accompanied by changes at the receptor level (Li et al., 1997a; Hensler, 2002; Castro et al., 2003; O’Connor et al., 2005).
Thus indeed, sensitization or desensitization of receptors in the brain may happen by means of alterations in the signal transduction pathways rather than at the receptor level.
The present study also shows that various brain regions we measured did not show changes in terms of the amount of inhibitory G-proteins after partial sleep deprivation for 8 days.
However, one area showed significant changes; sleep restriction for 8 days significantly increased the amount of serotonin-1A receptor-associated inhibitory G-proteins in the amygdala. The lack of significant alterations in other brain areas may be partly explained by the fact that the method is just not sensitive enough to detect minor changes (Alanko et al., 2004). Furthermore, autoradiographical GTPγS assays label all inhibitory G-protein subtypes which are coupled to the stimulated receptors. However, there still might be a differential effect of sleep loss on Gi1, Gi2, Gi3 and Go proteins, but changes in the amount of one of these proteins remains masked by the unaltered and invariably high expression of the others (Li et al., 1997b). Finally, the desensitized physiological response to the 1A agonist may be a result of changes due to changes at other elements of the signal transduction pathway such as the regulators of G-protein signalling or RGS proteins which thus remain undetected in a G-protein assay (Ni et al., 1999; Carrasco et al., 2004).
An increase of inhibitory G-proteins, thus a presumably enhanced serotonin-1A signalling in the amygdala seems paradoxical, since our earlier studies showed reduced 1A receptor-dependent functional responses (Roman et al., 2005; 2006). However, it may be, that sleep loss affects the serotonergic system of the brain in a region specific, localized manner. Indeed, a number of studies have indicated that experimental treatments other than sleep deprivation can act in a region specific manner. Region specificity is an important factor for example in the effects of antidepressants on G-proteins: the selective serotonin reuptake inhibitor fluoxetine has different effects at pre and postsynaptic locations in terms of amount of G-proteins (Castro et al., 2003). On top of this, marked regional differences within the postsynaptic compartment of the serotonin-1A receptor system have also been reported in other studies with antidepressants (Li et al., 1997a;
Shen et al., 2002).
Taken together, the results of the present study indicate that chronic partial sleep deprivation alters serotonergic signalling in the amygdala. It is known that the amygdala receives serotonergic innervation (Hensler et al., 2006) and that this serotonergic modulation influences other brain functions such as stress reactivity, anxiety, arousal and rapid eye movement sleep (Beaulieu et al., 1986; Schreiber and De Vry, 1993; Sanford et al., 1995; Feldman et al., 2000). It seems that serotonin exerts this influence by balancing excitatory and inhibitory activity within the basolateral amygdala (Rainnie, 1999). It might be that this sleep loss-specific alteration of serotonergic signal transduction in the amygdala found in the present study has significant functional consequences on the aforementioned amygdala-related functions.