Chronic stress during paradoxical sleep deprivation increases ...

Psychoneuroendocrinology (2008) 33, 1211—1224

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n

Chronic stress during paradoxical sleep deprivation increases paradoxical sleep rebound: Association with prolactin plasma levels and brain serotonin content Ricardo Borges Machado, Sergio Tufik, Deborah Suchecki * õ Paulo, Sa õ Paulo, Brazil Department of Psychobiology, Universidade Federal de Sa Received 26 December 2007; received in revised form 6 June 2008; accepted 19 June 2008

KEYWORDS Paradoxical sleep deprivation; Footshock stress; Sleep homeostasis; Heart rate; Prolactin; Corticosterone; Dopamine; Serotonin

Summary Previous studies suggest that stress associated to sleep deprivation methods can affect the expression of sleep rebound. In order to examine this association and possible mechanisms, rats were exposed to footshock stress during or immediately after a 96-h period of paradoxical sleep deprivation (PSD) and their sleep and heart rate were recorded. Control rats (maintained in individual home cages) and paradoxical sleep-deprived (PS-deprived) rats were distributed in three conditions (1) no footshock — NF; (2) single footshock — SFS: one single footshock session at the end of the PSD period (6—8 shocks per minute; 100 ms; 2 mA; for 40 min); and (3) multiple footshock — MFS: footshock sessions with the same characteristics as described above, twice a day throughout PSD (at 7:00 h and 19:00 h) and one extra session before the recovery period. After PSD, animals were allowed to sleep freely for 72 h. Additional groups were sacrificed at the end of the sleep deprivation period for blood sampling (ACTH, corticosterone, prolactin and catecholamine levels) and brain harvesting (monoamines and metabolites). Neither SFS nor MFS produced significant alterations in the sleep patterns of control rats. All PS-deprived groups exhibited increased heart rate which could be explained by increased dopaminergic activity in the medulla. As expected, PS deprivation induced rebound of paradoxical sleep in the first day of recovery; however, PSD + MFS group showed the highest rebound (327.3% above the baseline). This group also showed intermediate levels of corticosterone and the highest levels of prolactin, which were positively correlated with the length of PS episodes. Moreover, paradoxical sleep deprivation resulted in elevation of the serotonergic turnover in the hypothalamus, which partly explained the hormonal results, and in the hippocampus, which appears to be related to adaptive responses to stress. The data are discussed in the realm of a prospective importance of paradoxical sleep for processing of traumatic events. # 2008 Elsevier Ltd. All rights reserved.

1. Introduction * Corresponding author at: Department of Psychobiology — UNIFESP, Rua Napolea õ de Barros 925, Sa õ Paulo — SP 04024-002, Brazil. Tel.: +55 11 2149 0159; fax: +55 11 5572 5092. E-mail address: [email protected] (D. Suchecki).

Numerous studies have pointed out to a sleep reboundinducing effect of acute stress (Cespuglio et al., 1994; Rampin et al., 1991). This effect follows a temporal pat-

0306-4530/$ — see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2008.06.007

1212 tern insofar as both slow wave sleep (SWS) and paradoxical sleep (PS) are reduced during stress exposure (Marinesco et al., 1999) and remain so inhibited for some hours after the end of the stimulus (Tiba et al., 2004); 2—3 h after the end of the stimulus, however, sleep begins to rise, and reaches the highest levels during the dark phase of the cycle (Dewasmes et al., 2004; Koehl et al., 2002; Tiba et al., 2003, 2004). Sleep rebound, and especially, increased slow wave activity during SWS, is believed to play a recovery function that helps restore internal balance after a traumatic event (Meerlo et al., 1997, 2001a). Nevertheless, long periods of immobilization stress (4 h) have been reported to induce less sleep rebound than shorter periods (1 h or 2 h), suggesting that prolonged secretion of corticosterone could be involved in the suppression of sleep rebound (Marinesco et al., 1999). Sleep deprivation using instrumental methods, but not stimulation of the midbrain reticular formation, results in sleep rebound (Kovalzon and Tsibulsky, 1984). Due to this dissociation, some authors have claimed that sleep rebound is a consequence of the stress inherent to these methods (Coenen and Van Luijtelaar, 1985; Jouvet, 1994; Rechtschaffen et al., 1989). However, when two related methods of PS deprivation (PSD) were compared, it became clear that both completely suppressed PS and reduced the amount of SWS to the same extent; nonetheless, there was a difference in the resulting sleep rebound between the methods. Thus, rats submitted to PSD by the modified multiple platform method (MMPM) displayed a lager amount of PS rebound than rats PSdeprived by the single platform method (SPM) (Machado et al., 2004). Interestingly, we have shown previously that the immediate activation of the hypothalamic—pituitary— adrenal (HPA) axis after 96 h of PSD is similar for both methods (Suchecki et al., 2002a), but the behavioral and physiological responses of the rats to a psychological stressor (the elevated plus maze) is smaller in rats deprived by the MMPM than in rats deprived by the SPM (Suchecki et al., 2002b). Based on this evidence we sought to examine whether a high intensity stressor would be capable to prevent the sleep rebound that follows a prolonged period of PSD. We hypothesized that acute application of stress would impair the sleep rebound, whereas chronically it would not interfere with PS rebound that typically follows a prolonged period of sleep deprivation in the SPM, because animals would have habituated to the stressor. To test this hypothesis, we chose a stressor that had been shown in previous studies to impair sleep. From the literature data, acute footshock appears to be the most detrimental to sleep, increasing sleep latency and reducing the time spent in PS (Palma et al., 2000; Pawlyk et al., 2005; Sanford et al., 2003a; Va ´zquez-Palacios and Velazquez-Moctezuma, 2000). Therefore, in the present study we assessed the effects of footshock stress on the expression of sleep rebound and heart rate of PS-deprived rats. The secretion of hormones of the HPA, of prolactin and of peripheral catecholamines and central monoamines and their metabolites were determined in discrete areas of the brain to unravel the mechanisms responsible for the observed changes.

R.B. Machado et al.

2. Methods 2.1. Subjects Groups containing 8—10 adult Wistar rats (300—400 g) from our own colony were used after prior approval from the Ethics Research Committee of Universidade Federal de Sa õ Paulo was issued in accordance with international guidelines for care in animal research. Rats were allowed free access to water and food during all experiments. A constant 12 h light to 12 h dark cycle was maintained in the experimental rooms with fluorescent white lamps (lights on at 7:00 h) during the entire study. Temperature in the experimental rooms was kept at 20—22 8C.

2.2. Electrophysiological procedures After anaesthesia with ketamine-diazepam (140.0—5.5 mg/ kg, i.p.), the rats were fitted with electrodes to monitor sleep. Two bipolar electrodes placed ipsilaterally with stainless-steel micro-screws (1 0.9 mm) were used for EEG monitoring: one pair on the right lateral parieto-parietal (for minimum theta activity EEG) and the other on the left medial fronto-parietal (for maximum theta activity EEG) areas (Rosenberg et al., 1976; Timo-Iaria et al., 1970). One pair of insulated nickel— chromium flexible fine wire electrodes was implanted in the dorsal neck muscle for EMG recording and other additional pair of the same material was positioned on either side of the rib cage, bilaterally, to record heart rate (EKG). After the surgical procedure, antibiotics (Pentabio ´tico1 Fort-Dodge) and sodium diclofenac were administered and the animal was allowed to recover from surgery for 15 days. Electrophysiological signals were recorded on a digital polygraph (Neurofax QP 223 A #Nihon Kohden Co., Tokyo, Japan) and recordings were displayed on 30 s epochs and submitted to visual scoring routine, as described previously (Machado et al., 2005). Behavioral states were classified as wakefulness (W); low amplitude slow wave sleep (LSWS), high amplitude slow wave sleep (HSWS) and paradoxical sleep. LSWS is classified by the presence of a voltage in the EEG of 20.0—30.0 mV (usually 200—400 mV, peak to peak) and low frequency activity (delta waves) in less than 50% of the epoch. HSWS was characterized by average voltage of 30.0 mV (usually 250—500 mV, peak to peak) and presence of delta waves in 50% or more of the epoch. Additionally, transition-type sleep (spindles and fast high voltage activity) was considered as HSWS. The sleep—wake states were analyzed off-line in 3 h blocks. The following parameters were compared within each recording period throughout the study: 1. Sleep time (latency, total time of sleep represented as percentage of recording time). 2. Slow wave sleep (time of low [LSWS] and high [HSWS] amplitude fractions, and total time of slow wave sleep, presented as percentage of sleep time). 3. Paradoxical sleep (latency, total time, represented as percentage of sleep time, number and duration of episodes). Control and paradoxical sleep-deprived rats submitted to the single footshock session were not recorded during the sleep deprivation period, because they were exactly the

Chronic stress during paradoxical sleep deprivation increases rebound of paradoxical sleep same as their non-stressed counterparts, which provided sleep data during the deprivation procedure. Six seconds of electrocardiograms recordings with normal QRS complex morphology and cycle lengths were selected for each sleep state (i.e. LSWS, HSWS and PS, when present) at every 3 h and were included in the analysis of sleep heart rate index. Acquisition of the heart rate during wakefulness was avoided, because of the presence of interference and artifacts on EKG signal, especially during sleep deprivation. Animals were habituated to the cables and to the recording environment for 3 days before baseline recording. Baseline sleep was recorded on two consecutive days (2 24-h) and the parameters are represented by the average of these 2 days. After the baseline recording, in the period that preceded paradoxical sleep deprivation, animals were adapted to the sleep deprivation chambers for 30 min per day for three consecutive days.

2.3. Sleep deprivation procedure It was accomplished by the single platform method, in which each animal was placed onto a narrow cylindrical platform, 6.5 cm in diameter surrounded by water about 1 cm below the platform surface. Temperature of the water inside the water tanks was similar to the environmental temperature (22 8C). Food and water were provided ad libitum throughout the whole study. Hanging food containers were placed inside the water chambers and filled whenever necessary. Water bottles were fixed on the grid that covers the water chamber, cleaned and refilled daily. In addition, the water in the tanks was changed everyday. After 4 days under this protocol, rats were allowed to sleep freely in their individual home cages (recovery period) for 3 days (R1—R3), being continuously monitored.

2.4. Footshock stress The footshock stress was administered by placing each animal in an individual chamber (30 cm 20 cm 30 cm) with electrified floor that delivered unavoidable shocks (6—8 shocks per minute; 100 ms; 2 mA) for 40 min (Palma et al., 2000). Control and PS-deprived groups were either submitted to: (1) single footshock stress (SFS), applied immediately after the end of PSD or at the corresponding time for control rats. These groups were not recorded during the PSD period; (2) multiple footshock stress (MFS), applied twice a day, immediately before light shift (at 7:00 h and 19:00 h) to prevent the introduction of a possible additional zeitgeber. Footshock sessions were delivered during the 4 days of PSD and one extra session was given immediately before the recovery period, summing up nine footshock sessions; (3) no footshock stress (NFS): rats were placed inside the chamber onto the electrified grid also for 40 min, but no footshock was delivered; this group served as a control for the stress procedure. Therefore, this study was composed by six groups: (1) control no footshock stress (CTL + NFS); (2) CTL single footshock stress (CTL + SFS); (3) CTL multiple footshock stress (CTL + MFS); (4) paradoxical sleep deprivation NFS (PSD + NFS); (5) PSD + SFS; (6) PSD + MFS.

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2.5. Blood analyses Trunk blood was obtained approximately 2 h after the end of stress procedures from matched groups, run simultaneously with the sleep study. To prevent animals from sleeping during these 2 h, and to avoid the effects of sleep rebound on these measurements, rats were placed back in their previous environments until decapitation was performed. Blood was collected in chilled 7.5% EDTA-containing vials, and was centrifuged at 2300 rpm at 4 8C for 20 min; plasma was collected and separated for subsequent determination of prolactin, ACTH and corticosterone (CORT) levels. All assays were performed in duplicate. Prolactin concentrations were determined by a specific immunenzimatic commercial kit, based on AChE competitive substrate (SPI Bio — Bertin Group, Montigny-le-Bretonneux, France). Plasma ACTH was determined by sequential immunometric assay (DPC Immulite, Los Angeles, CA); when necessary, samples were diluted with appropriate solution to avoid high-dose hook effect. Corticosterone levels were evaluated by specific radioimmunoassay (INC Biomedicals, Costa Mesa, CA).

2.6. Monoamine concentrations in discrete brain areas After 96 h of sleep deprivation and/or stress procedures, animals were sacrificed by decapitation, their brains were removed and the frontal and parietal cortex, striatum, hippocampus, hypothalamus, medulla and pons regions, separately, were dissected on a cold surface. The tissue samples were weighed individually and homogenized by sonication in 500 mL of extraction solution (0.1 M perchloric acid containing 0.4 mM sodium metabissulfite and 0.2 mM EDTA). The homogenates were centrifuged at 20,000 g for 10 min, then filtered through 0.22 mm membrane and stoked at 80 8C for further analysis. Precipitates were dissolved in 0.1N NaOH and assayed to protein estimation (Bicinchoninic acid method, Pierce Chemical, Rockford, IL). Supernatants were submitted to fast isocratic separation through a C18 HPLC reversed-phase column system (Spheri-5, C18, ODS, 5 mm, 25 cm 4.6 mm column; linked to a NewGuard Cartridge Column, RP-18, 7 mm pre-column; PerkinElmer Brownlee Columns, Shelton, CT) and electrochemically detected using an amperometric detector (L-ECD-6A, Shimadzu, Japan), by oxidation on glass carbon electrode at +850 mV in relation to an Ag—AgCl reference electrode. The mobile phase consisted of 0.163 M citric acid, 0.06 M sodium phosphate dibasic anhydrous, 0.69 mM octyl sodium sulfate, 12 mM EDTA, acetonitrile 4%, tetrahydrofuran 1.7% and orthophosphoric acid sufficient to bring the pH to 2.85, diluted in double distilled water. The mobile phase was filtered through a 0.2 mm filter membrane, degassed under helium and delivered at a flow rate of 1.4 mL/min. Each sample was analyzed in duplicate for concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and their non-conjugated metabolites 3,4-dihydroxyphenilacetic acid (DOPAC), homovanilic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA). The recovery of the analytes was determined by adding a fixed concentration of internal standard DHBA (dihydroxybenzylamine) before tissue homogenization. All standards and salts were purchased from Sigma (St. Louis,

The values are expressed in beats per minute (b.p.m.) S.E.M. of 8—10 rats/group. CLT, home-cage controls; PSD, paradoxical sleep-deprived; NFS, non-footshock stress; SFS, single footshock stress; MFS, multiple footshock stress; D, sleep deprivation period; R, recovery period; NR, non-recorded. a Different from baseline levels. b Different from respective CTL group. c Different from respective NFS group.

343.38 10.6 331.04 4.2 318.80 3.8 401.69 12.0a, b NR 410.23 5.9a, b 365.75 6.0a, b NR 372.19 5.3a, b 296.63 7.8 294.53 4.9 315.63 5.0

448.75 9.4a, b NR 441.56 7.4a, b

467.56 9.1a, b NR 462.58 6.0a, b NFS SFS MFS PSD

378.54 2.7a, b 357.40 3.1a, b 335.99 4.8a,b, c

286.38 4.6 306.30 9.2 281.04 4.0 289.17 3.0 NR 287.08 4.8 293.25 2.9 NR 294.38 4.2 304.42 5.9 296.38 7.2 291.82 4.71

295.58 4.2 NR 287.76 3.5

290.83 5.0 NR 291.93 6.2 NFS SFS MFS CLT

R1 D2 D1 Baseline

D3

D4 Stress

A three-way interaction during sleep deprivation was shown F (4,128) = 2.76; p 0.05. Regardless of the stress regimen, PSdeprived rats exhibited higher heart rate throughout the entire sleep deprivation period when compared to their respective CTL counterparts ( p 0.005) and to their baseline heart rate ( p 0.001). No differences were observed in CTL rats. During the recovery period, heart rate was influenced by an interaction of all factors F (6,138) = 6.59; p 0.0001. Analysis of this interaction showed that all PS-deprived rats exhibited higher heart rate on R1 than on baseline ( p 0.05). Moreover, a difference due to stress regimen was also observed, in which the heart rate of PSD + MFS rats was lower than that of PSD + NFS rats on R1 ( p 0.01). Finally, PS-deprived rats showed higher heart rate than their respective CTL counterparts on R1 ( p 0.05).

Group

3.1. Heart rate during sleep (Table 1)

Heart rate during sleep in PS-deprived and CTL rats, submitted or not to single or multiple footshock stress

3. Results

Table 1

EEG data were analyzed by a two-way ANOVA for repeated measures, with main factors Group (Control [CTL], Paradoxical sleep deprivation [PSD]), Stress (no footshock stress [NFS], single footshock stress [SFS] and multiple footshock stress [MFS]) and Day (repeated measure: Baseline, Recovery day 1 [R1], Recovery day 2 [R2] and Recovery day 3 [R3]). Sleep was also recorded during the sleep deprivation period; however these data will not be presented because no relevant effects of footshock were observed. For EKG data, ANOVA was performed in two steps: during the deprivation period, the main factors were Group (CTL, PSD), Stress (NFS, MFS) and Day (repeated measure: Baseline, Day 1 [D1], Day 2 [D2], Day 3 [D3] and Day 4 [D4]); during the rebound period, the factors were Group, Stress (no footshock stress [NFS], single footshock stress [SFS] and multiple footshock stress [MFS]) and Day (repeated measure: Baseline, Recovery day 1 [R1], Recovery day 2 [R2] and Recovery day 3 [R3]). This two-step analysis was necessary because CTL + SFS and PSD + SFS groups were not recorded during the sleep deprivation period, since these groups were exactly the same as CTL + NFS and PSD + NFS. Hormone values of non-stressed groups (basal levels) were initially analyzed by a Student’s t-test. Hormonal and neurochemical data were carried out by a two-way ANOVA, with main factors Group (CTL, PSD) and Stress (NFS, SFS, MFS). Post-hoc analysis was performed by the Newman—Keuls test. Finally, Pearson’s correlation tests were performed between the percentage of PS obtained in the first 12-h of recovery period and each one of the hormone levels for CTL and PS-deprived animals, separately; between plasma hormone concentrations and monoamine contents in the hypothalamus, and between monoamine contents and sleep parameters. The level of significance was set at p 0.05.

R2

2.7. Statistical analyses

288.21 11.6 300.21 8.9 289.06 7.0

R3

MO) and the solvents (HPLC grade) were purchased from Carlo Erba (Italy). Although monoamines were measured in various brain regions, only data regarding those areas directly involved with the behavioral, physiological and endocrine changes observed in the present study will be presented.

325.63 5.3 324.17 3.0 311.82 3.2

R.B. Machado et al. 289.13 3.0 297.14 8.9 288.59 2.6

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Chronic stress during paradoxical sleep deprivation increases rebound of paradoxical sleep

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that PSD rats exhibited more SWS in R1 compared to baseline ( p 0.0001), but were not different from CTL rats.

Figure 1 Sleep latency (time elapsed between the onset of sleep recording and sleep onset, taken as the first three epochs of continuous sleep) measured in R1, immediately after the end of PSD or at the corresponding time for CTL rats or immediately after the end of footshock session (CTL + SFS, CTL + MFS, PSD + SFS, PSD + MFS). The latency is presented in min as mean S.E.M. of 8—10 animals/group. (*) Different from baseline values; threeway ANOVA for repeated measures, followed by the Newman— Keuls test. CLT, home-cage controls; PSD, paradoxical sleepdeprived; NFS, non-footshock stress; SFS, single footshock stress, given immediately after PSD period, preceding the recovery period; MFS, multiple footshock stress, given at 7:00 h and 19:00 h, with the light shift, everyday during the deprivation period including an additional session before the recovery period.

3.2. Sleep parameters 3.2.1. Latency to sleep (Fig. 1) Analysis of the light phase showed a three-way interaction F (6,138) = 2.6376; p 0.02, in which SFS delayed the sleep onset of CTL rats compared to baseline ( p 0.01), but did not alter this parameter in PS-deprived rats. During the dark phase there was a three way interaction F (6,138) = 2.905; p 0.01. Analysis of this interaction showed that the sleep latency of CTL + NFS rats was reduced in all recovery nights, when compared to baseline ( p 0.01).

3.2.4. Paradoxical sleep 3.2.4.1. Latency to paradoxical sleep. Similarly to the effects described for sleep latency, an interaction between Stress and Day was detected F (3,138) = 5.682; p 0.001 in the light phase, indicating that single footshock stress induced a delayed onset of PS ( p 0.005), with a subsequent return to baseline latency afterwards (R2). In the dark phase there was a main effect of Day F (3,138) = 3.858; p 0.01, in which PS latency in the first dark period was shortened compared to baseline ( p 0.01). 3.2.4.2. Percentage of paradoxical sleep (Fig. 2A). Light phase: During the rebound period, there was a three-way interaction F (6,138) = 7.207; p 0.001. All PS-deprived rats exhibited more paradoxical sleep than CTL rats ( p 0.001) and than their respective basal levels ( p 0.0005) in R1; however, the association of sleep deprivation with repeated footshock stress produced a further increase in this parameter when compared to the other PS-deprived groups ( p 0.0005); this increase persisted until R2 ( p 0.001, compared to baseline). Dark phase: An interaction Stress by Group by Day was shown F (6,138) = 4.789; p 0.001. All PS-deprived groups exhibited more PS during R1 than baseline ( p 0.001); however, this increase was even higher in PSD + MFS rats than in PSD + SFS, PSD + NFS and CTL + MFS rats ( p 0.001).

3.2.2. Total sleep time (Table 2) A three-way interaction was revealed during the light phase F (6,138) = 3.531; p 0.005. PSD resulted in greater sleep time during recovery day 1 (R1) than baseline; in addition, SFS applied immediately before the rebound period prevented the increase in sleep time, whereas MFS led to the highest sleep efficiency in R1 ( p 0.01); this increase was still significant during R2 ( p 0.05). In the dark phase there was a Day by Group interaction F (3,138) = 9.643; p 0.001, in which PS-deprived rats slept more than control animals in R1 ( p 0.001).

3.2.4.3. Events of paradoxical sleep (Fig. 2B). Light phase: Interactions between Day and Group F (3,138) = 29.119; p 0.001 and between Day and Stress F (3,127) = 3.127; p 0.01 were obtained. Post hoc analysis indicated that PS-deprived rats exhibited more events of PS in R1 and R2, than baseline and R3 ( p 0.05). Compared to control groups, PS-deprived rats showed more PS events in R1 ( p 0.05). Both stress regimens reduced the number of PS events in R1, compared to non footshock stress ( p 0.05). For NFS rats, PS events were higher in R1 than in all other days ( p 0.05). Rats treated with MFS exhibited more PS events in R1 and R2 than baseline and R3 ( p 0.05). Dark phase: A three way interaction during sleep recovery period was observed F (6,138) = 2.637; p 0.05. The Newman—Keuls analysis revealed that PSD + MFS rats exhibited more events of PS in R1 than in all other recovery days ( p 0.001) and than all other groups ( p 0.01), and this increase persisted until R2 ( p 0.001). In addition, the group PSD + NFS also displayed more events of PS in R1 than in all other days ( p 0.05).

3.2.3. Slow wave sleep (Table 2) Because the statistical analysis did not indicate any substantial impact of the stress procedures on HSWS and LSWS, both fractions were pooled together to represent total slow wave sleep. During the light phase, an interaction between Stress and Day was observed F (6,138) = 8.879; p 0.0001, and rats submitted to MFS exhibited less slow wave sleep than those which were not submitted to footshock ( p 0.001). During the dark phase, a Group by Day interaction was detected F (6,138) = 3.005; p 0.05 and the post hoc analysis revealed

3.2.4.4. Length of PS events (Fig. 2C). Light phase: A threeway interaction was obtained F (6,138) = 9.525; p 0.001. Analysis of this interaction showed that all PS-deprived groups exhibited longer PS events during R1 than baseline, R2 and R3 ( p 0.005). During the first day of recovery, the length of PS events was higher in PSD + MFS rats than in PSD + NFS and PSD + SFS and CTL + MFS rats ( p 0.005). Dark phase: Interactions between Day and Group F (6,138) = 3.116; p 0.05 and between Day and Stress F (6,138) = 2.684; p 0.01 were observed. For CTL groups, the length of PS events was reduced in R3, compared to

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Table 2 Group

CTL

Total sleep time (%ST) and slow wave sleep time (%SWS) obtained in approximately 11 h recording periods, in the light and dark phases Stress

NFS SFS MFS

PSD

NFS SFS MFS

Sleep parameter

Baseline

R1

R2

R3

Light

Dark

Light

Dark

Light

Dark

Light

Dark

%ST %SWS %ST %SWS %ST %SWS

60.01 3.0 49.04 2.6 56.47 3.1 45.90 2.6 54.02 3.0 44.46 2.3

30.64 1.8 27.18 1.3 33.20 1.4 29.68 1.0 31.96 2.4 28.80 2.0

64.56 2.3 52.88 1.9 a 53.42 2.3 44.94 4.0 49.29 5.3 39.59 2.2 c

36.36 3.1 33.43 2.8 35.77 2.0 31.86 1.9 30.06 2.5 26.45 2.1

59.28 2.2 48.43 1.6 49.16 1.9 41.56 1.6 58.95 2.5 48.10 1.8

30.00 2.3 28.20 2.4 30.43 2.2 26.76 1.7 30.38 2.3 27.59 2.0

55.67 1.6 44.82 1.3 60.89 2.3 49.58 2.2 58.96 1.8 50.08 1.9

32.24 2.2 29.70 1.9 34.11 1.2 29.99 1.0 28.45 2.1 25.96 1.8

%ST %SWS %ST %SWS %ST %SWS

56.06 3.3 46.61 2.6 50.87 2.5 42.42 1.7 48.45 1.9 39.31 1.9

30.76 2.3 27.61 2.0 27.66 1.6 24.90 1.2 28.61 1.6 25.06 1.4

75.62 3.0 a 52.49 2.0 a 63.88 3.5 42.48 2.4 76.77 2.3a, b 37.61 2.3 c

43.92 5.1a, b 36.31 3. 8 a 38.34 3.2a, b 31.56 2.6 a 47.84 1.8a, b 32.81 1.5 a

55.15 3.1 45.00 2.4 56.98 1.6 44.82 1.9 68.07 1.7 50.05 1.8

31.32 4.0 27.40 3.5 28.92 2.5 25.53 2.3 36.38 3.2 30.68 2.2

57.00 2.4 48.44 1.9 51.20 3.3 42.06 2.1 57.51 3.4 46.84 2.0

31.12 2.8 27.48 2.3 32.11 1.8 27.88 1.3 29.19 3.0 26.21 2.4

The values are presented as mean S.E.M. of 8—10 animals/group. ANOVA followed by Newman—Keuls; p 0.05. CLT, home-cage controls; PSD, paradoxical sleep-deprived; NFS, non-footshock stress; SFS, single footshock stress; MFS, multiple footshock stress; R, recovery period. a Different from baseline values. b Different from respective CTL group. c Different from NFS counterpart.

R.B. Machado et al.


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Figure 2 Effects of acute or chronic footshock associated to paradoxical sleep deprivation on: (A) time of paradoxical sleep (% of sleep time, expressed as mean S.E.M. of 8—10 rats/group); (B) events of paradoxical sleep (mean absolute number S.E.M. of 8— 10 rats/group); and (C) length of PS events in minutes (mean S.E.M. of 8—10 rats/group). Data were obtained in recording periods of approximately 11 h during the light and dark phases. (*) Different from baseline; (y) different from respective CTL group; (z) different from respective NFS group; (#) different from respective MFS group; three-way ANOVA for repeated measures, followed by the Newman—Keuls test. CLT, home-cage controls; PSD, paradoxical sleep-deprived; NFS, non-footshock stress; SFS, single footshock stress, given immediately after PSD period, preceding the recovery period; MFS, multiple footshock stress, given at 7:00 h and 19:00 h, with the light shift, everyday during the deprivation period including an additional session before the recovery period.

R1, whilst PS-deprived rats exhibited longer events in R1, compared to the other days ( p 0.01). Finally, in R1, rats submitted to MFS exhibited longer PS events than their NFS and SFS counterparts ( p 0.05).

3.3. Hormone plasma concentrations (Table 3) 3.3.1. ACTH Levels of non-stressed animals were higher in PSD than in CTL group (t = 7.618; p 0.001). Main effects of Group F (1,46) = 18.489, p 0.00007, Stress F (2,46) = 21.967, p 0.0000001, and an interaction between these factors F (2,46) = 9.654, p 0.0004 were shown. Analysis of the interaction revealed that both SFS and MFS resulted in augmented secretion of ACTH in CTL rats compared to non-stressed animals ( p 0.001), whereas in PS-deprived animals only MFS led to increased ACTH levels ( p 0.01). Both in CTL and PSD groups, ACTH levels were higher in animals submitted to MFS than in those submitted to SFS ( p 0.05). Moreover, ACTH levels were lower in PSD + SFS and PSD + MFS than in their control counterparts ( p 0.001). 3.3.2. Corticosterone Levels of non-stressed PS-deprived rats were higher than those of CTL counterparts (t = 2.075; p 0.05). Main effects of Group F (1,46) = 13.598, p 0.001 and Stress F (2,46) =

168.929, p 0.000001, and an interaction between these factors F (2,46) = 19.666, p 0.000001 were revealed. Post hoc analysis showed that SFS and MFS produced a similar increase in CORT levels in both CTL and PSD groups, compared to their respective non-stressed counterparts ( p 0.001). These levels, however, were lower in PSdeprived than in CTL rats ( p 0.005). 3.3.3. Prolactin Comparison between non-stressed groups revealed that PSD stimulated prolactin secretion (t = 2.454; d.f. = 10; p 0.05). ANOVA detected a main effect of Stress F (2,31) = 34.803, p 0.0001, and an interaction between the factors F (2,31) = 9.281, p 0.001. Post hoc analysis showed that CTL rats submitted to either single or multiple footshocks exhibited higher prolactin levels than CTL + NFS rats ( p 0.001). For PS-deprived rats, MFS resulted in the highest prolactin levels, compared to both PSD + NFS and PSD + SFS groups ( p 0.001), which did not differ from each other, and compared to CTL + MFS group ( p 0.05). 3.3.4. Correlations between hormone levels and paradoxical sleep In PS-deprived rats, there was a positive correlation between CORT levels and the length of PS episodes during the first 12 h of sleep recovery (r = 0.53, N = 26). This correlation was even

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R.B. Machado et al.

Table 3 Plasma levels of ACTH, corticosterone, prolactin and catecholamines in control (CLT) or PS-deprived (PSD) rats submitted to single (SFS), multiple sessions (MFS) of footshock stress or non-footshock stress (NFS) Group

Stress

ACTH (pg/mL)

Corticosterone (mg/dL)

Prolactin (ng/mL)

CLT

NFS SFS MFS

21.80 3.4 839.57 77.5 a 1066.20 187.0a, b

8.88 2.0 76.26 3.6 a 80.29 3.2 a

4.38 0.8 61.48 15.5 a 65.23 10.8 a

PSD

NFS SFS MFS

134.18 14.3 c 244.64 60.7 c 433.80 27.7c,a, b

23.04 4.8 c 53.21 5.0c, a 59.00 3.9c, a

24.12 15.6 c 26.47 13.6 c 89.30 23.0c,a, b

Values are presented as mean S.E.M. of 6—10 animals/group. CLT, home-cage controls; PSD, paradoxical sleep-deprived; NFS, nonfootshock stress; SFS, single footshock stress; MFS, multiple footshock stress. a Different from respective NFS animals. b Different from respective SFS rats. c Different from respective CTL group.

stronger for prolactin levels (r = 0.71, N = 19). Moreover, there was a significant correlation between prolactin levels and total time of paradoxical sleep during the first 12 h of sleep rebound (r = 0.53, N = 19).

3.4. Monoamine concentrations in discrete brain areas (Fig. 3 and Table 4) 3.4.1. Medulla A main effect of group was detected for DOPAC/DA ratio (F (1,46) = 12.6287, p 0.001), HVA/DA ratio (F (1,46) = 8.06549, p 0.01) and 5-HIAA/5-HT ratio (F (1,45) = 6.7713, p 0.05). For all indexes, PSD rats exhibited higher turnover than CTL rats ( p 0.01). 3.4.2. Pons No effects were observed in NA and 5-HT levels and in the DOPAC/DA ratio. DA: A main effect of stress was detected F (2,46) = 5.2860, p 0.01, and rats submitted to SFS showed higher levels of this neurotransmitter than NFS rats, regardless of the group (+86.48%; p 0.01). DOPAC: ANOVA showed a main effect of stress F (2,46) = 4.0688, p 0.05; in this case, rats exposed to MFS showed an 85.11% increase above NFS levels ( p < 0.05). HVA: Main effects of group F (1,46) = 4.18543, p 0.05 and stress F (2,46) = 3.67107, p 0.05 were revealed. PSD rats showed higher levels of HVA than CTL rats (+62.1%; p 0.05) and animals submitted to both forms of stress exhibited higher levels than non-stressed rats ( p 0.05). 5-HIAA: There was an effect of group F (1,46) = 6.1135, p 0.05 on this metabolite, and again PSD rats exhibited higher levels than CTL rats (+37.69%; p 0.05). HVA/DA ratio: A main effect of Group was detected F (1,46) = 4.16770, p 0.05, and PSD rats showed increased dopamine turnover when compared to CTL rats (+68.27%; p 0.05). 5-HIAA/5-HT ratio: Again, a main effect of Group was revealed F (1,46) = 21.6665, p 0.0005 and augmented serotonin turnover was shown in PSD compared to CTL rats (+46.91%). 3.4.3. Hypothalamus No changes were observed in NA, DOPAC and 5-HT concentrations and in the DOPAC/DA ratio.

DA: Only a main effect of stress was detected F (2,46) = 3.9701, p 0.05. Rats submitted to MFS showed higher concentrations of the neurotransmitter than rats submitted to SFS (+41.64%; p 0.05), which in turn were higher than NFS rats (+34.52%; p < 0.05). HVA: A main effect of group was revealed F (1,46) = 9.82643, p 0.005; in this case, PSD rats exhibited higher levels of the metabolite than CTL rats (+139.33%). 5-HIAA: A main effect of group was detected F (1,46) = 4.9575, p 0.05, in which metabolite levels were higher in PSD than CTL rats (+42.11%). HVA/DA ratio: A main effect of Group was shown F (1,46) = 8.33912, p 0.01. Once again, PSD rats exhibited higher ratio than CTL rats (+169.65%). 5-HIAA/5-HT ratio: ANOVA detected a main effect of Group F (1,45) = 7.2032, p 0.01, in which PSD rats exhibited increased serotonergic turnover compared to CTL rats (+39.87%). 3.4.4. Hippocampus No differences were found in NA, DA and HVA levels, and in the HVA/DA ratio. DOPAC: Main effect of group was shown F (1,46) = 5.62480, p 0.05. Higher metabolite levels were found in PSD than in CTL rats (+64.35%). 5-HT: There was a significant interaction between group and stress F (2,46) = 4.4155, p 0.05). Post-hoc analysis detected a reduction of 5-HT levels in the hippocampus of stressed animals (CTL + SFS, PSD + SFS, CTL + MFS and PSD + MFS) compared to CTL + NFS animals ( p 0.05). DOPAC/DA ratio: ANOVA revealed a significant interaction between the factors F (2,46) = 3.7324, p 0.05). Newman— Keuls analysis of this interaction showed that PSD + SFS exhibited an increase in dopaminergic turnover compared to PSD + NFS and CTL + SFS groups (+55.98% and + 122.88%, respectively; p 0.05). 5-HIAA/5-HT: Main effect of group was shown F (1,46) = 3.9099, p 0.05, with PSD rats showing increased serotonergic turnover than CTL rats (+39.94%). 3.4.5. Correlations between concentrations of hypothalamic neurotransmitters and pituitary hormones Pearson’s correlation tests were carried out for each group, individually. Significant correlations in CTL + NFS group were


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Figure 3 Catecholamines and indolamines concentrations (ng/mg of tissue) and the ratio between monoamines and their metabolites in discrete brain regions involved with heart rate (medulla and pons) and hormone regulation (hypothalamus and hippocampus) of control (CTL) and paradoxical sleep-deprived (PSD) rats, submitted to no-footshock (NFS), single footshock (SFS) or multiple footshock stress (MFS). Values are presented as mean S.E.M. of 8—10 rats/group. Samples were obtained at the end of the sleep deprivation period or at the correspondent time-point for control rats. (*) Different from respective CTL group (lines above bars indicates the group main effect); (#) different from respective NFS group; ( ) different from CTL + NFS animals (lines above bars indicates main effect of group). NE, norepineprine; DA, dopamine; DOPAC, 3,4-hydroxyphenilacetic acid; HVA, homovalinic acid; 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleacetic acid.

revealed between 5-HT and ACTH (r = 0.72; N = 9; p 0.05) and between 5-HT and PRL (r = 0.89, N = 6; p 0.05). For PSD + NFS rats, significant correlations were shown between HVA/DA turnover and PRL (r = 0.95, N = 6; p 0.005) and between 5-HIAA/5-HT turnover and ACTH (r = 0.83, N = 9; p 0.01). Finally, significant correlations were detected in CTL + SFS between 5-HIAA/5-HT turnover and PRL (0.92, N = 6; p 0.01).

4. Discussion The results of the present study showed that sleep homeostasis in sleep-deprived rats can be changed by an additional stressful stimulus, but this alteration depends on the regimen of stress exposure; thus, multiple sessions of footshock during the deprivation period produced a further increase of PS rebound, but impaired slow wave sleep rebound. In response

1220 Table 4 group

R.B. Machado et al. Summary of the findings of neurotransmitter concentrations in several brain areas, determined by HPLC in 10 animals/ Pons

Medulla

Hypothalamus

Hippocampus

— — —

— Stress: MFS > SFS > NFS Group: PSD > CTL

— — —

DOPAC 5-HT

— Stress: MFS > NFS Group: PSD > CTL; Stress: SFS and MFS > NFS Stress: MFS > NFS —

— —

— —

5-HIAA HVA/DA DOPAC/DA

Group: PSD > CTL Group: PSD > CTL —

— Group: PSD > CTL Group: PSD > CTL

Group: PSD > CTL Group: PSD > CTL —

5-HIAA/5-HT

Group: PSD > CTL

Group: PSD > CTL

Group: PSD > CTL

Group: PSD > CTL Interaction: PSD + SFS and PSD + MFS < CTL + NFS; CTL + MFS and SFS < CTL + NFS Group: PSD > CTL — Interaction: PSD + SFS > PS + NFS and CTL + SFS Group: PSD > CTL

NE DA HVA

CTL, home-cage controls; PSD, paradoxical sleep-deprived; NFS, non-footshock stress; SFS, single footshock stress; MFS, multiple footshock stress; NE, norepinephrine; DA, dopamine; DOPAC, 3,4-hydroxyphenilacetic acid; HVA, homovalinic acid; 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleacetic acid.

to the additional forms of stress, PS-deprived rats displayed smaller ACTH and CORT responses than their CTL counterparts, despite the fact that PSD alone induced higher levels of these hormones compared to CTL non-stressed rats. Interestingly, the increase in heart rate induced by PSD was not altered by either single or multiple footshock stress, indicating that sleep deprivation might have induced a ceiling effect, which persisted during the first day of recovery. The persistent tachycardia herein reported is in accordance with previous publications using either prolonged PSD (De Mesquita and Hale, 1992) or short (48 h) periods of total sleep deprivation (Sgoifo et al., 2006). According to the authors of the latter study, sleep deprivation causes a blunted vagal tone following the sympathetic activation (Sgoifo et al., 2006). This is a possible explanation for our results, although we did not find increased catecholamines peripheral levels in PSD rats (data not shown). In the brain, however, PSD deprivation produced a significant increase in dopaminergic turnover in the medulla and pons (HVA/DA and DOPAC/DA), which seemed to be implicated in the augmented heart rate observed in all PS-deprived rats, regardless of the stress regimen. It has been shown that local administration of dopamine in the nucleus tratus solitarius produces tachycardia and hypertension in rats (Granata and Woodruff, 1982), possibly acting at the D2 receptors (Yang et al., 1990). Likewise, painful mechanical stimulation increases heart rate and mean arterial pressure and that microdyalisates taken from the rostral ventrolateral medulla show increased 5-HT and DA, and reduced NA concentrations (Karlsson et al., 2006). Moreover, local administration of serotonin in the rostral part of the ventrolateral medulla leads to tachycardia in rats (Lovick, 1989), although administration of 5-HT1A agonists in the intermediate part results in low blood pressure and bradycardia (Mandal et al., 1990). Regarding the sleep pattern, the main effect of chronic footshock during sleep deprivation was a robust increase in the homeostatic drive to paradoxical sleep, due especially to a major lengthening of PS events. During the first 3 h of rebound we observed events of approximately 12 min in average, which correspond to the duration of a whole sleep

cycle in naı¨ve rats (Zepelin, 1994). Such specific effect has only been reported after i.c.v. administration of corticotrophin-like intermediate lobe peptide (CLIP or ACTH18—39) (Chastrette et al., 1990; Wetzel et al., 1997), which is processed from the ACTH sequence of pro-opiomelanocortin (POMC) in two distinct locations of the brain, the nucleus tractus solitarius and the arcuate nucleus of the hypothalamus (Leger et al., 1990). The lower levels of ACTH seen in PSD + SFS and PSD + MFS could be due to a shift in proopiomelanocortin processing from ACTH to CLIP production. However, the hypothesis about the involvement of CLIP in the expression of PS rebound is speculative and will soon be tested. A more straightforward explanation for the lower ACTH levels in stressed PSD rats is the down-regulation of type 1 corticotropin-releasing factor (CRF) receptors (CRFR1) in the pituitary resulting in reduced pituitary responsiveness to CRF stimulation. In a recent study (unpublished data), we observed that chronic administration of CRF to PS-deprived rats yielded a similar pattern of ACTH secretion, whereas removal of the glucocorticoid negative feedback by means of metyrapone chronic administration did not alter the capacity of pituitary to secrete ACTH, suggesting that PSD may have a greater impact on CRF than on glucocorticoid receptors. These results are in agreement with Fadda and Fratta’s findings (1997) of a 38% reduction of CRF receptors in the pituitary of paradoxical sleep-deprived rats. Glucocorticoids possess permissive or suppressive effects on many functions, depending on the circulating levels. Optimal concentrations, i.e., sufficient to saturate high affinity mineralocorticoid receptors and approximately 50% of low affinity glucocorticoid receptors (Lupien et al., 2007), are required for optimal outcome. Thus, in response to stress, intermediate levels of circulating glucocorticoids, favor behavioral and physiological processes including learning and memory, the activity of the immune system and sleep, whereas both very low and very high concentrations can cause impairment (Sapolsky et al., 2000). This glucocorticoid-induced PS sleep phenomenon is clearly seen in rats submitted to different lengths of immobilization stress (Marinesco et al., 1999) and in Addison’s patients, whose impaired

Chronic stress during paradoxical sleep deprivation increases rebound of paradoxical sleep REM sleep is successfully corrected by administering hydrocortisone before bed time (Garcıá-Borreguero et al., 2000). In the present study, corticosterone plasma levels in PSdeprived rats were positively correlated with the length of PS episodes, indicating that corticosterone participates in the expression of PS rebound. The fact that in non sleepdeprived rats either low or high corticosterone concentrations did not influence the length of paradoxical sleep episodes, suggest that this hormone is involved in the sleep homeostatic process in animals with high propensity for paradoxical sleep (for instance, paradoxical sleep-deprived rats). Recent data from our laboratory showed that corticosterone supplementation during sleep deprivation resulted in increased length of PS episodes, corroborating the role of this hormone in the expression of PS in sleep-deprived rats (unpublished data). Nonetheless, prolactin was the hormone more strongly correlated with PS rebound, with a highly significant correlation with the length of PS episodes. Previous studies from our laboratory had shown that 96 h of PSD performed by the modified multiple platform method result in elevated levels of prolactin (Andersen et al., 2005). PSD alone induced higher prolactin levels than those seen in the CTL group, whereas chronic stress in PS-deprived rats led to a 3.7-fold increase compared to the effects of acute stress. In human beings, prolactin is mainly secreted during the second half of the night (Sassin et al., 1972), with a very clear circadian rhythm (Van Cauter, 1990). Experimental studies demonstrate that prolactin is, directly or indirectly, involved in the regulation of paradoxical sleep (Oba ´l et al., 1989, 1994; Roky et al., 1993). Thus, systemic administration of prolactin induces a clear increase of PS in rodents, mainly during the light phase (Roky et al., 1995), whilst anti-prolactin suppresses PS in rats (Oba ´l et al., 1997); prolactin deficient mice exhibit less PS (Oba ´l et al., 2005) and ether stress increases both prolactin and paradoxical sleep in rats (Bodosi et al., 2000). It is important to mention that in the present study, the strong association between PRL and PS was true only for PS-deprived rats, since in response to footshock, CTL rats displayed a major increase in PRL levels, but no changes in PS. A surprising finding in the present study was the lack of negative correlation between hypothalamic dopamine concentration and prolactin levels. In general, a main effect of stress on hypothalamic dopamine levels was observed, i.e., both forms of footshock increase hypothalamic dopamine levels, but this effect was not reflected on prolactin secretion. Classically, dopamine is considered to be the major inhibitor of prolactin release, acting via D2 receptors located in pituitary lactotrophs, whereas the activity of these dopaminergic neurons at the lactotroph is inhibited by D1 dopaminergic receptors (Freeman et al., 2000). In a previous study from our laboratory, 96 h of PSD did not result in changes in D1 or D2 receptors density in the hypothalamus, except for the suprachiasmatic nucleus. Unfortunately, dopaminergic receptors in the pituitary were not assessed, making difficult to draw any conclusion (Nunes et al., 1994). It is important to bare in mind, however, that 5-HT is the major stimulator of PRL secretion; this action seems to be modulated by vasoactive intestinal peptide — VIP (Balsa et al., 1998). Interestingly, PSD causes an accumulation of VIP levels in the cerebrospinal fluid (Jime ńez-Anguiano et al., 1993) and an upregulation of VIP receptors in several brain regions, some

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of which are closely related to PS (Jime ńez-Anguiano et al., 1996). Nevertheless, it is important to mention that within the constraints of the present protocol HPLC was performed in whole hypothalamus, thus providing a general idea of the monoamine concentrations in this brain region. It has been reported in a recent study, that in female rats, the prolactin surge that occurs during estrous is mediated by reduced dopamine concentrations and increased dopamine turnover in the mediobasal hypothalamus and the medial preoptic area (Szawka et al., 2007). Therefore, had we performed microdissections of the hyptothalamus or microdyalisis, we might have been able to estimate more accurately how paradoxical sleep deprivation and footshock stress altered the role of each biogenic amine in the hypothalamic regulation of prolactin secretion. From the results of the present study, it appears as though the most likely mediating process for PSD + MFS-induced such robust lengthening of PS episodes involves optimum corticosterone associated with maximum prolactin plasma levels, as is illustrated in Fig. 4. Corroborating this idea, a study by Sanford and co-workers report that mice displaying different levels of anxiety respond differently to footshock, i.e., the more anxious-type strain BALB/cJ mice exhibit intense PS inhibition following footshock and re-exposure to the stress chamber than the less anxious-type C57BL/C6 (Sanford et al., 2003b). Interestingly, it is also the less anxious-type strain which secretes more prolactin and exhibit more PS in response to a 1 h restraint stress (Meerlo et al., 2001b), despite a similar corticosterone response of both strains, suggesting that PRL plays a major role as a mediator of stress-induced PS rebound. Serotonin is involved with the stress response, sleep and behavioral adaptation to stress (Linthorst and Reul, 2008). Neurons in the dorsal raphe nucleus fire in a behavioral statedependent manner and hippocampal mycrodialysis studies have shown that maximum 5-HT release occurs during wake, followed by a reduction during slow wave sleep and a further reduction during paradoxical sleep (Park et al., 1999; Pen ãlva et al., 2003). Short-term sleep deprivation increases 5-HT levels and turnover in the hippocampus, which is rapidly reversed by sleep rebound (Asikainen et al., 1997; Pen ãlva et al., 2003), whereas long-term (96 h) paradoxical sleep deprivation increases 5-HIAA levels and serotonergic turnover in the hypothalamus and hippocampus (Youngblood et al., 1997). In the present study we observed the same changes as Youngblood et al.’s (1997), indicating higher activity of this system in PS-deprived than in control rats. These effects on serotonergic activity may be explained by the down-regulation of 5-HT1A receptors in the dorsal raphe nucleus (Evrard et al., 2006; Gardner et al., 1997) and subsensitivity to systemic administration of 8-OH-DPAT (Roman et al., 2005; Roman et al., 2006) produced by shortor long-term sleep deprivation. That dysfunctions of the hippocampal serotonergic system are involved with depression is corroborated by reports of reduced concentrations of 5-HIAA in a subpopulation of depressed patients and indications of altered expression of serotonergic receptors in post-mortem tissue studies of depressed patients (Maes and Meltzer, 1995). An active serotonergic system, therefore, is believed to underlie adaptive responses to chronic stress. One of the hallmarks of depression is the change in sleep architecture that includes

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R.B. Machado et al. that longer REM sleep episodes, and consequently a more consolidated REM sleep, may be an important protective feature against trauma-induced PTSD (Mellman et al., 2007). In summary the present study demonstrated that PSD produced a sustained tachycardia, resulting from increased dopaminergic and serotonergic turnover at the level of the medulla and pons. The association of PSD with a high intensity stressor (i.e. the footshock stress) induced significant changes in the pattern of sleep rebound, including an exuberant increase in paradoxical sleep, due to increased length of episodes. This sleep change was positively correlated with corticosterone and prolactin levels, and these effects were regulated, at least in part, by increased hypothalamic and hippocampal serotonergic turnover. The increment of hippocampal serotonergic turnover and the lengthening of paradoxical sleep events may play an important role in the adaptive response to the highly aversive situation represented by paradoxical sleep deprivation associated to repeated exposure to footshock sessions.

Role of the funding sources

Figure 4 Illustration of the relation between corticosterone (A) and prolactin secretion (B) and the mean length of paradoxical sleep (PS) episodes during the first day of sleep recovery in control (CTL) and paradoxical sleep-deprived (PSD) rats, submitted or not (NFS) to single (SFS) or multiple footshock stress (MFS). The illustration was assembled by ranking the hormones concentrations and plotting the correspondent length of PS episodes for each group.

increased percentage of REM sleep, reduced latency to REM sleep and reduced time spent in delta sleep, associated with REM sleep fragmentation (Duncan et al., 1979). The overexpression of paradoxical sleep as seen in PSD + MFS rats might be interpreted as being a sign of depressive-like state. However, the increase in paradoxical sleep was normalized by the second recording night, indicating that paradoxical sleep pressure faded away spontaneously, we did not observe change in latency to paradoxical sleep and, most important, paradoxical sleep was very consolidated, rather than fragmented, in this group. Among the many proposed functions for REM sleep (Siegel, 2005), possibly one of the most fascinating ones is that of emotion-related adaptive function, based on the postulation that REM sleep is important for the integration of traumatic memories (Cartwright and Lloyd, 1994). Recent studies have shown that posttraumatic stress disorder (PTSD) patients present sleep disturbances, including less sleep efficiency, longer sleep latency and excessive REM sleep disruptions (Breslau et al., 2004; Habukawa et al., 2007). The adaptive role of REM sleep in the context of PTSD is given by a recent study by Mellman et al. (2007), who evaluated the sleep of subjects who were injured and admitted to the hospital, where they were sleep-recorded. One to two months after discharge they were recalled for a psychiatric interview and evaluation of PTSD symptoms. The individuals who were exposed to traumatic events but did not show PTSD symptoms exhibited longer REM sleep episodes, whereas those who developed PTSD presented more REM episodes, suggesting

Funding for this study was provided by FAPESP Grant 98/ 14303-3; FAPESP had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Conflict of interest All authors of this manuscript declare that they have no potential conflict of interest.

Acknowledgements The authors would like to thank Dr. Dulce H. Casarini, Ms. Luciana Teixeira and Ms. Adriana Faria for their helpfully assistance on blood analysis and to Giovana Camila de Macedo for helping in the determination of brain monoamines content. This work was supported by Associaça õ Fundo de Incentivo à Psicofarmacologia (AFIP) and Fundaça õ de Amparo à Pesquisa do Estado de Sa õ Paulo (FAPESP/ CEPID#98/14303-3). Ricardo Borges Machado is the recipient of graduate fellowship from Fundaça õ de Amparo à Pesquisa do Estado de Sa õ Paulo (FAPESP#04/02213-2). Sergio Tufik and Deborah Suchecki are recipients of research fellowships from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo ´gico (CNPq).

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