Altered serotonergic and corticotropin releasing hormone regulation of the hypothalamo-pituitary-adrenal stress system in

chronic partial sleep deprivation

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

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Earlier studies have indicated that chronically restricted sleep leads to changes in hypothalamo-pituitary-adrenal (HPA) axis regulation and altered stress reactivity. The present study aimed to establish whether changed serotonergic and corticotropin releasing hormone (CRH) signalling may play a role in the sleep loss-induced alteration in neuroendocrine stress reactivity. In order to measure stress hormone levels under undisturbed conditions, we provided rats with permanent jugular vein cannulas. The serotonergic and corticotropin modulation of the HPA-axis was investigated by intravenous injection of a serotonergic agonist or CRH, and then measure plasma levels of the adenocorticotropin hormone (ACTH) and corticosterone (CORT). The results show that chronic partial sleep deprivation decreases ACTH release in response to both the serotonergic agonist and CRH. This indicates that reduced serotonin-1A and/or CRH receptor sensitivity may be an underlying mechanism for sleep loss-induced alterations of stress reactivity.

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The body’s stress systems play a critical role in adapting to a continuously changing and challenging environment (Johnson et al., 1992). One of the main neuroendocrine stress systems involved in the response to stressors is the HPA-axis. Whereas a normally functioning HPA-axis is required for environmental adaptation, a dysfunction of the HPA-axis may be associated with the development of psychopathologies (Holsboer, 2000; Meerlo et al., 2001b). One factor that could be involved in altered HPA-axis function and increased vulnerability to disease is frequently disrupted and restricted sleep. Indeed, various studies have shown that sleep deprivation can be accompanied by an increase in the activity of the HPA-axis both in humans (Leproult et al., 1997;

Vgontzas and Chrousos, 2002; Voderholzer et al., 2004) and rodents (Meerlo et al., 2002;

Suchecki et al., 2002; Andersen et al., 2005). Although, in some animal studies this activation may partly be related to stress involved in the given sleep deprivation procedure, part of the HPA-axis activation seems to be inherently related to sleep loss alone (Meerlo et al., 2002). More importantly, whether or not attempts are made to experimentally separate sleep loss effects from stress effects, in real life sleep loss and stress often go hand in hand. Therefore, the complex interaction of sleep loss and HPA-axis activity is a relevant issue in human society.

While a number of studies suggest that sleep loss may be associated with a mild activation of the HPA-axis, very little is known about how this system under conditions of restricted sleep responds to novel stressors (Meerlo et al., 2002). This is a particularly important gap in our knowledge on sleep and stress because sleep restricted people have to deal with challenges of real life. Perhaps sleep loss gradually changes the HPA-axis and eventually its response to real stressors. Indeed, earlier studies have shown that sleep deprivation alters HPA response to a new stressor: particularly a reduced pituitary ACTH response to restraint stress (Meerlo et al., 2002;

Sgoifo et al., 2006).

Importantly, disrupted sleep and an altered HPA-axis activity are both hallmarks of human depression (Adrien, 2002). Among other physiological changes, CRH levels in the cerebrospinal fluid are increased in depressed subjects, which is accompanied by a blunted release of ACTH in response to a CRH challenge (Nemeroff et al., 1984; Dinan, 1996a). This blunted ACTH response in depression may be explained by excessive ligand-induced receptor down-regulation (Ferguson and Caron, 1998; Aguilera et al., 2004).

Given the complex interaction of sleep and HPA-axis function, the aim of the present study was to investigate how restricted sleep affects the regulation and activity of the HPA-axis.

Particularly, we were interested in the underlying mechanisms of sleep loss-related changes in stress-reactivity reported earlier (Meerlo et al., 2002; Sgoifo et al., 2006).

A main candidate for mediating sleep loss-induced alterations in stress-reactivity is CRH.

The functioning of the HPA-axis is under the control of CRH produced in the hypothalamic paraventricular nucleus (Johnson et al., 1992). It has been suggested that sleep loss may increase the mRNA of CRH in the paraventricular nucleus and it may also increase the amount of the

Fujihara et al., 2003; Koban et al., 2006). In the long run, such a sleep loss-induced increase in CRH production might change the sensitivity of CRH receptors. Therefore, the first aim of the present study was to establish, whether chronic partial sleep loss alters the sensitivity of CRH receptors as measured by the ACTH and CORT response to an injection of CRH.

A second candidate that might be responsible for sleep restriction-induced changes in stress reactivity is serotonin, which is an important modulator of HPA-axis function at various levels including the hypothalamus, pituitary and adrenal cortex (Fuller, 1992; Dinan, 1996b; Matheson et al., 1997; Mikkelsen et al., 2004; Porter et al., 2004). This serotonergic modulation of the HPA-axis is mediated in particular by the serotonin-1A receptor, which has a more dominant role than other receptor subtypes (Dinan, 1996b). One of our earlier studies has shown that the overall serotonin-1A receptor sensitivity is decreased in sleep restricted rats (Roman et al., 2005). Therefore, our second aim was to examine whether chronic partial sleep loss desensitizes those particular serotonin-1A receptor populations that are involved in the modulation of the HPA-axis.

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Animals and housing

In the present experiment, we used adult male Wistar rats (Harlan, Horst, The Netherlands) weighing approximately 350 g at the start of the experiments. Animals were housed under a 12h light/12h dark cycle, with lights on from 09.00 h to 21.00 h. Temperature in the room was maintained at 21 ± 1^oC. Rats were provided with food and water ad libitum in all experiments. Experiments were approved by the Ethical Committee of Animal Experiments of the University of Groningen.

Sleep restriction and forced activity

Rats were subjected to a protocol of repeated partial sleep deprivation for 8 days allowing them 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 forced activity during the last 10h of the dark phase, their circadian activity phase (Fig. 1). Before starting the experiments, rats were habituated to the experimental apparatus by placing them in the wheels for 1h on 3 successive days.

Permanent heart cannulations

To achieve stress-free blood sampling, permanent heart catheters were used that permit frequent blood sampling in unrestrained and freely moving animals (Steffens, 1969). Rats were provided with a polyethylene catheter in the right atrium of the heart under isoflurane/N2O/O2 inhalation anaesthesia. The catheter was inserted through the right jugular vein and externalized on the top of the skull according to techniques described earlier (Steffens, 1969). After surgery, rats were allowed at least 10 days of recovery before the start of experiments. During this period, animals were habituated to handling and blood sampling procedures.

Serotonergic challenge

In order to examine the effect of sleep loss on serotonergic modulation of HPA-axis function, sleep restricted and control rats received an intravenous (i.v.) injection of the serotonergic 1A receptor agonist (±)-8-hydroxy-2-(di-n-propyl-amino) tetralin hydrobromide (8-OH-DPAT; Sigma, St. Louis, MO, USA). After 7 days of sleep restriction, during the 4h rest period in the home cage, rats were connected to sampling tubes. After 1½ h, when any handling-induced changes would have disappeared, rats received an i.v. injection of 8-OH-DPAT dissolved in saline through the jugular vein catheter (0.1 mg/kg body weight). The concentration of 8-OH-DPAT was based on earlier studies and was chosen to cause intermediate hormone responses (Korte et al., 1995). To measure plasma levels of ACTH and CORT in response to serotonin-1A receptor activation, blood samples were taken shortly before as well as 5, 15, and 60 min after the 8-OH-DPAT injection. Blood was

collected in pre-chilled plastic tubes containing EDTA as anti-coagulant. Samples were centrifuged at 2600 G and stored at -80^oC until radioimmunoassay analysis of hormones (ICN Biomedicals, Costa Mesa, CA, USA).

After the last blood sampling, sampling tubes were removed and rats were placed back in the rotating wheels to continue the sleep restriction regime.

Figure 1. Experimental set-up of sleep restriction protocol and forced activity control. Top bar: rats were sleep restricted by forced locomotion (FA) for 20h each day (grey section of the bar) and were allowed 4h of rest (R) in their home cage (first 4 hours of the light phase). Middle bar: rats were subjected to a protocol of forced activity at double speed (FA2) for half the time.

Animals were subjected to the 10h of forced activity in one block (dark grey section of the bar) which coincided with the last 10 h of the dark phase of the light-dark cycle. Lower bar depicts the 24h light-dark cycle. (*) Serotonin 1A agonist and corticotropin releasing hormone injections on day 7 and 8 took place between the third and fourth hour of the light phase.

CRH challenge

In order to investigate whether sleep loss alters the regulation of the HPA-axis by corticotropin releasing hormone (CRH), sleep restricted and control rats received an i.v. injection of ovine CRH (oCRH; American Peptide Company, Sunnyvale, CA, USA). One day after the challenge with 8-OH-DPAT, thus after 8 days of sleep restriction, rats were again connected to the sampling tubes, while in their home cage during the daily 4h resting phase. Animals received an iv injection of CRH dissolved in saline (0.5 μg/kg body weight) through the jugular vein catheter. The concentration of oCRH was based on earlier studies and was known to induce intermediate ACTH and CORT responses (Buwalda et al., 1999). Blood samples were taken to assess the sensitivity of the pituitary gland to CRH. Blood sampling and hormone measurements for ACTH and CORT were carried out as after the serotonergic challenges.

Data analysis and statistics

To test for effects of sleep restriction on the hormone responses to the 8-OH-DPAT and CRH injection, hormone data were subjected to analysis of variance (ANOVA) with repeated measures. When appropriate, post hoc Tukey test was applied to establish at which time points after the pharmacological challenges the experimental and control groups differed.

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Serotonergic challenges

The injection of 8-OH-DPAT induced a clear HPA-axis response in all animals. On average, the ACTH response of the sleep restricted rats was lower than that of the home cage control animals, which in turn was lower than that of the forced activity control rats. Repeated measures ANOVA revealed a significant treatment effect (treatment x time interaction: F(6,75)=4.318; p=0.001; overall effect: F(2,25)=6.16; p=0.007), and the post hoc Tukey test indicated that the response of the sleep restricted animals was significantly lower than that of the forced activity group at t=15 min (p=0.006) and t=60 min (p=0.016) (Fig. 2A). In contrast, the CORT response to 8-OH-DPAT was not significantly different between sleep restricted and control rats (Fig. 2B).

CRH challenges

Injection of CRH resulted in a clear ACTH and CORT response. However, the magnitude of the ACTH response differed between the groups (repeated measures ANOVA treatment x time interaction: F =4.029; p=0.001; overall effect: F =3.543; p=0.044). Post hoc Tukey test

the forced activity controls at t=5 min (p=0.021), and lower than that of the home cage controls at t=15 min (p=0.041) (Fig. 3A). Yet, despite the lower ACTH response, CORT levels were not significantly different between the treatment groups (Fig. 3B).

Figure 2. ACTH and CORT responses to a pharmacological challenge with the sero-tonin-1A receptor agonist 8-OH-DPAT in sleep restricted (n=10), forced activity (n=10) and home cage controls (n=8). A, Plasma levels of ACTH were significantly different between sleep restricted rats and animals of the forced activity group at t=15 and 60 min (b), with home cage controls showing intermediate hormone levels. ACTH levels peaked around t=15 min at values of approximately 800 pg/ml. B, Plasma levels of CORT were not altered by the experimental treatment.

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In line with the earlier finding of a sleep loss-induced blunted pituitary ACTH response to novel stress, our present results show that rats subjected to chronic partial sleep deprivation have reduced ACTH response to pharmacological stimulation with CRH and the serotonin-1A agonist 8-OH-DPAT. At the same time, the release of CORT in response to an injection with the serotonergic and corticotropic agents remained unaltered. Together these results confirm that losing too much sleep alters neuroendocrine stress reactivity and suggest that these changes are partially mediated by decreased CRH and serotonin-1A receptor sensitivity in the pituitary and/or in other central structures.

The present data show that the ACTH response to CRH in sleep restricted animals was lower than that of the home cage controls, whereas in forced activity controls it was higher than in home cage animals. In other words, restricted sleep and forced activity seem to have opposite effects. This increase in ACTH response in the forced activity control group may be the result of stress experienced by those animals (Roman et al., 2005). Indeed, a similar increase in ACTH response to CRH was found in a model of social stress (Buwalda et al., 1999). It may thus be that the mild physical exercise involved in our sleep restriction procedure partly counteracted the effects

of sleep loss per se. Thus, sleep loss without any activity might have resulted in an even stronger attenuation of the pituitary ACTH response.

Figure 3. ACTH and CORT responses to a pharmacological challenge with corticotropin releasing hormone in sleep restricted (n=9), forced activity (n=10) and home cage controls (n=9). A, Plasma levels of ACTH were significantly different between sleep restricted rats and home cage controls at t=15 min (a)and between sleep restricted animals and forced activity controls at t=5 min (b). ACTH levels peaked around t=5 min at values of approximately 400 pg/ml. B, Plasma levels of corticosterone were not altered by the experimental treatment.

The blunted ACTH response to an injection with CRH in sleep restricted rats suggests a desensitization of CRH receptors in the pituitary gland. This desensitization may be due to overstimulation of CRH receptors by their own ligand (Fadda and Fratta, 1997; Fujihara et al., 2003;

Koban et al., 2006). Although information is limited, a number of animal studies suggest that CRH expression and release may be elevated during sleep deprivation. The desensitization of CRH receptors can be due to adaptational mechanisms such as receptor phosphorylation-induced G-protein uncoupling, receptor internalization and down-regulation by increased G-protein degradation or reduced receptor mRNA and protein synthesis (Ferguson and Caron, 1998; Aguilera et al., 2004). Interestingly, human depression is also characterized by higher amounts of CRH in the cerebrospinal fluid and by a blunted ACTH response to a challenge with CRH (Nemeroff et al., 1984; Von Bardeleben and Holsboer, 1988; Dinan, 1996a).

The second main finding of the present study is a blunted pituitary ACTH release in response to serotonin-1A receptor stimulation in sleep restricted animals. Similar to the results of the CRH challenge, the sleep restricted animals and forced activity controls showed opposite changes, suggesting that effects of sleep loss per se may have been partly counteracted by activity involved in the sleep deprivation procedure.

The attenuated ACTH response to serotonin-1A stimulation in sleep restricted animals may be due to changes at the level of the HPA-axis itself or due to changes in other brain areas that provide input to the HPA-axis. Part of the attenuated response may be an indirect effect that is

stimulates not only ACTH production but CRH release too (Dinan, 1996b). Thus, the decreased ACTH response after the serotonergic challenge can be a result of decreased serotonin-1A and CRH receptor sensitivity. On the other hand, the attenuated response to 8-OH-DPAT may also be a direct consequence of a reduced sensitivity of the serotonin-1A receptors alone, both at the level of the pituitary or in other brain areas that provide input to the HPA-axis (Calogero et al., 1989;

Calogero et al., 1990; Dinan, 1996b). Along these lines, in a study, bilateral lesions of the central amygdaloid nucleus, a structure that innervates the HPA-axis, lead to a marked decrease in the ACTH release in response to stress (Beaulieu et al., 1986; Herman et al., 2005).

The hypothesis that altered HPA-axis regulation in sleep restricted rats is not only related to altered CRH sensitivity but also associated with an attenuated serotonin-1A sensitivity is supported by our earlier studies showing that another 1A receptor-mediated response is also attenuated in sleep loss. In particular, we demonstrated by in vivo measurements of the hypothermic response to an injection with 8-OH-DPAT that chronic partial sleep deprivation significantly desensitized the serotonin-1A receptor system (Roman et al., 2005). The present study supports that too little sleep may indeed lead to a decrease in serotonin-1A receptor sensitivity including serotonin-1A receptor populations which are involved in the regulation of neuroendocrine function (Calogero et al., 1989; Calogero et al., 1990; Dinan, 1996b).

Importantly, desensitized serotonin-1A responses and altered regulation of the HPA-axis are present in depression (Porter et al., 2004). Also, disrupted sleep is a hallmark of depression and has been suggested as a causal factor for this disorder (Riemann and Voderholzer, 2003). In other words, disrupted and restricted sleep may lead to alterations in neurobiological systems (e.g.

serotonin-1A receptor system) and altered regulation of stress systems (e.g. HPA-axis), which eventually may sensitize individuals to stress-related disorders such as depression.

Our results show that both in response to the serotonergic and CRH challenge, despite the attenuated ACTH response, sleep restricted animals had a CORT response that was not different from that of the controls. An unchanged CORT response, in the face of blunted ACTH levels, can be explained by increased ACTH sensitivity in the adrenal cortex which implies that sleep restriction alters regulation of the HPA-axis at multiple levels.

In conclusion, the present study shows that losing too much sleep alters the sensitivity of central serotonin-1A and CRH receptors and thereby it attenuates the release of ACTH from the pituitary gland. Importantly, disrupted sleep and HPA-axis dysregulation are both hallmarks of human depression and have been suggested as causal factors in the development of mood disorders (Adrien, 2002; Porter et al., 2004). Our data support the notion that disrupted sleep and altered HPA-axis regulation may be interrelated and, in fact, changes in sleep may be a causal factor in the altered HPA-axis.

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The authors thank Jan Bruggink for his assistance with the surgeries, blood samplings and hormone analysis.

Losing too much sleep alters serotonin signalling in the