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Effects of sleep loss on sleep architecture in Wistar rats: Gender-specific rebound sleep
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Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 975 – 983 www.elsevier.com/locate/pnpbp
Effects of sleep loss on sleep architecture in Wistar rats: Gender-specific rebound sleep M.L. Andersen ⁎, I.B. Antunes, A. Silva, T.A.F. Alvarenga, E.C. Baracat, S. Tufik Department of Psychobiology, Universidade Federal de São Paulo, Escola Paulista de Medicina (UNIFESP/EPM), R. Napoleão de Barros, 925, Vila Clementino 04024-002, São Paulo, SP, Brazil Received 10 October 2007; received in revised form 8 January 2008; accepted 8 January 2008 Available online 16 January 2008
Abstract This study was designed to examine the influence of gender on sleep rebound architecture after a 4-day paradoxical sleep deprivation period. After a 5-day baseline sleep recording, both male and female rats in different phases of the estrus cycle were submitted to paradoxical sleep deprivation for 96 h. After this period, the sleep rebound recording was evaluated for 5 days (one estrus cycle). The findings revealed that after paradoxical sleep deprivation, sleep efficiency and paradoxical sleep returned to baseline values on the second day of the light period, for all except the proestrus group. During the dark rebound period, only the female groups presented increased sleep efficiency on the first day. Paradoxical sleep returned to baseline values on the third day, except for males and the cycling females submitted to paradoxical sleep deprivation in the diestrus phase, whose baseline values returned to normal on the second day of rebound period. Thus, the females and males displayed distinct patterns as a result of sleep disruption. © 2008 Elsevier Inc. All rights reserved. Keywords: EEG; Estrous cycle; Female rats; Gender; Sleep deprivation; Sleep rebound
1. Introduction There is an increasing concern about risks related to sleepiness in our society, which is normally a consequence of chronic sleep loss. Although sleep deprivation has a dramatic impact on multiple physiological processes (Leibowitz et al., 2006), it is not yet clear which factors contribute the most to the sleep disturbances reported by women (Lindberg et al., 1997). Indeed, in sleep surveys, women report considerably more sleep problems than men (Lindberg et al., 1997).
Abbreviations: PSD, paradoxical sleep deprivation; R, rebound sleep; EEG, electroencephalograph; SWS, slow wave sleep; REM, rapid eye movement; NREM, non-rapid eye movement; ECoG, electrocorticogram; EMG, electromyogram; P, proestrus; E, estrus; D, diestrus; Dc, diestrus with regular cycles; Da, diestrus–anestrus; CTRL, control. ⁎ Corresponding author. Department of Psychobiology, Universidade Federal de São Paulo, Rua Napoleão de Barros, 925, Vila Clementino, SP-04024-002, São Paulo, Brazil. Tel.: +55 11 2149 0155; fax: +55 11 5572 5092. E-mail address: [email protected] (M.L. Andersen). 0278-5846/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.01.007
It is an established fact that sex hormones influence sleep and circadian rhythms. Sleep in turn affects the episodic secretion of gonadotropin hormones (Hall et al., 2005). Although some women may not experience premenstrual symptoms and secondary insomnia, the latter and its associated symptoms often occur at the onset of menses (Freeman et al., 2004). There is some evidence indicating that sleep alterations are associated with the menstrual cycle, as could be expected from hormonal variations during that cycle. These changes can be small or significant and they include changes in the stage 2 slow wave sleep (SWS), and the percentage of REM sleep, or its latency (Lee et al., 1990; Ishizuka et al., 1994; Driver et al., 1996). Moreover, subtle differences in sleep at different phases of the menstrual cycle have also been reported in the luteal phase when REM sleep is reduced (Baker et al., 1999). In rats, sleep alterations also occur during the estrous cycle (Kleinlogel, 1975; Schwierin et al., 1998) as noted by a marked reduction in the nocturnal amount of non-REM (NREM) and REM sleep, as well as in slow wave activity in NREM sleep in proestrus when compared to other phases (Colvin et al., 1968; Zhang et al.,
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1995; Schwierin et al., 1998). In contrast to these results, Fang and Fishbein (1996) reported no alterations in SWS during the estrous cycle. Although some studies have focused on the sleep patterns of female rats under baseline conditions, few have focused on sleep parameters after sleep deprivation. Since gender differences in brain organization influence the regulation of basic physiological processes such as electrical activity and sleep (Corsi-Cabrera et al., 2003), it seems reasonable to predict that male and female systems will present differing effects of sleep deprivation on sleep parameters in the recovery period. Investigation of sleep deprivation in females has focused on specific aspects of sleep, mainly those related to pathologies. In view of the current scenario, there is great relevance in examining the sleep patterns of male and female rats subjected to paradoxical sleep deprivation (PSD), while in different phases of the estrous cycle during the recovery period. 2. Materials and methods 2.1. Subjects The study was performed on ten-week old female and male Wistar rats from our vivarium. The animals were maintained in a temperature-controlled room (23 ± 1 °C) with a 12:12-hour light– dark cycle (lights on at 0700) from birth and allowed free access to food and water in standard polypropylene cages. The rats used in this study were maintained and treated in accordance with the Ethical and Practical Principles of the Use of Laboratory Animal guidelines and the experimental protocol was approved by the Ethical Committee of UNIFESP (CEP N. 0998/04). Maximum efforts were taken to reduce the number of animals used in the experiments while employing enough to ensure unambiguous and reliable statistical analysis and data interpretation. 2.2. Surgical preparation and electrocorticogram/electromyographic recording Electrodes were surgically implanted in the rats in order to monitor electrocorticographic (ECoG) and electromyographic (EMG) activities, thus permitting the assessment of the sleep– wake cycle. Prior to implantation of the electrodes, the animals were briefly anesthetized with diazepam (10 mg/kg i.p.) and ketamine (90 mg/kg i.p.). To record cortical ECoG with a minimum of theta activity, one pair of screw electrodes was placed through the skull ipsilaterally: 1 mm posterior to bregma, 3 mm lateral to the central suture; and 1 mm anterior to lambda, 4 mm lateral to the central suture. EMG electrodes were implanted in the neck muscles, soldered to a six-pin socket and covered with dental acrylic cement. Body temperature was maintained at 37 °C with a regulated electric heating pad (Harvard Apparatus, USA). After the surgical procedure, each rat received an i.m. injection of 0.5 mL/rat of antibiotic and sodium diclofenaco (0.1 mL/rat, vo). The rats were individually placed in rounded transparent plastic cages and allowed a 10-day surgery recovery period, followed by a 4-day adaptation period with the cable connected. The recordings were performed on a Nihon Koden Co. model QP 223A apparatus (Tokyo, Japan), using three channels
for each animal: two for ECoG and one for head-muscle activity (EMG). The ECoG signals were amplified and filtered with a low pass filter at 0.1 s (1.6 Hz) and EMG activity was filtered with a low pass filter at 0.03 s (5.3 Hz). All recordings were scored by only one researcher, thus assuring reliability of the data. Analysis of the recordings was performed manually in a blind fashion using the Polysmith Neurotronics™ Inc. program (Neurotronics®, FL, USA), mainly based on the predominant amplitude and frequency of the tracing (Timo-Iaria et al., 1970; Andersen et al., 2001). The analysis was done in 30 s epochs with the dominant tracing (waking, slow wave sleep or paradoxical sleep) for more than 50% of each epoch. When artifacts or noise did not allow characterization of an epoch, assessments were made based on immediate surrounding epochs. The recordings were performed in individual home-cages. The following sleep parameters were considered: sleep efficiency (percentage of total sleep time during the recording time), total SWS (percentage of all periods of deep sleep throughout the recording), and total paradoxical sleep (percentage of all periods of paradoxical sleep throughout the recording). 2.3. Vaginal cytology Vaginal smears were obtained between 0800 and 0900 h. The vaginal smear samples were performed with a 200 μL pipette. A few drops of distilled water were injected into the vagina (but not deeply), and then pipetted out (see Marcondes et al., 2002 for complete description). The water with its cellular contents was then transferred to a slide. The determination of the estrous cycle was based on the occurrence of three types of cells in the vaginal smears: leukocytes, cornified cells, and nucleated epithelial cells. The stages of the estrous cycle were identified according to the following criteria: proestrus (P) was characterized by many epithelial cells and few leukocytes; estrus (E) was characterized by many cornified cells and no leukocytes, and diestrus (D) was characterized by few epithelial cells and many leukocytes. 2.4. Paradoxical sleep deprivation (PSD) The PSD method used 14 narrow circular platforms (6.5 cm in diameter), protruding 1 cm above the surface in a 123 × 44 × 44-cm tank filled with water. The rats were placed on the platforms and could move around by leaping from one platform to another. Each estrous cycle phase rat was placed in one tank, in a way such that each water tank contained only one estrus phase female rat. When they reached the paradoxical sleep (PS) phase, they would fall into the water, due to muscle atonia, and would be awakened. The narrow platform procedure caused complete loss of PS during all 4 days and approximately 30% of SWS (Machado et al., 2004). Our intention was to analyze the alterations on sleep architecture caused by total suppression of PS over 4 days. Throughout the study, the experimental room was maintained under controlled temperature (23 ± 1 °C) and a light–dark cycle (lights on at 0700 h and off at 1900 h). Food and water were provided ad libitum by placing chow pellets and water bottles on a grid located on top of the tank. The water in the tank was changed daily throughout the PSD period. Finally,
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the cage control group of each estrous cycle phase was maintained in the same room as the experimental rats for the duration of their respective PSD periods. By housing the groups in the same room, we controlled for any difference in housing condition between the two groups. The period of 96 h of PSD was chosen since our previous data showed that the most dramatic alterations in hormone concentration (Andersen et al., 2005) occur at this time point. To evaluate the long lasting effects of PSD on the estrous cycle, the baseline (B) and sleep recovery (R1–R5) periods were evaluated for 5 days. 2.5. Experimental design After a 10-day surgery recovery period, followed by a 4-day adaptation period with the polysomnography cable connected, all rats were submitted to vaginal smears over a period of 10 days. The females were smeared daily and animals with regular cycles were selected. After this, a baseline sleep recording for 5 con-
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secutive days (one estrous cycle) was performed. Males underwent the same process for statistical comparison. Following this period, females in different phases of the estrous cycle (proestrus [P], estrus [E], and diestrus [D]), as well as the males were submitted to PSD for 96 h. The respective control rats were maintained in their home-cages (CTRL). Although the vaginal smear was performed daily throughout the PSD period, no sleep recording was performed in that time. After the PSD period, the sleep rebound recording was evaluated for 5 consecutive days representing one estrous cycle (n = 6–10 for each group). Since we had recently published that 60% of the females that were submitted to PSD in the diestrus phase presented constant diestrus (anestrus cycle) up to the ninth day of the recovery period, the females in this study that were submitted to PSD in the diestrus phase were distributed into two groups for recovery sleep analysis: females that showed a regular estrous cycle (Dc) and those that presented an anestrus cycle (Da) (Antunes et al., 2006). Considering that estrus and diestrus are representative of the
Fig. 1. Mean ± SEM percentage of sleep efficiency during the light period (panel A) and during the dark period (panel B) in females in proestrus (P), estrus (E), and diestrus (D) at baseline and 5 days rebound recording (R1–R5). Panel A: ⁎Denotes a difference from respective baseline; #denotes a difference from R2–R5 of the respective group; ¤denotes a difference from the R1 respective group. Panel B: ⁎Denotes a difference from respective baseline; †denotes a difference from Da; ‡denotes a difference from Dc; ¥denotes a difference from other groups at the same day; ¤denotes a difference from the R1 respective group; £denotes a difference from male group (ANOVA followed by Tukey's post-hoc test, see text for p-values).
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ovulatory and luteal phases in women, respectively, these are the phases that present hormonal statistical significance, and because the estrous cycle was unaltered after PSD at metestrus phase (Antunes et al., 2006), we elected not to include the estrous phase in the current study. The vaginal smear was performed daily during the baseline, PSD, and recovery periods. 2.6. Statistical analysis Data analysis was carried out using two-way ANOVA followed by the Tukey's post-hoc test. The level of significance was set at p b 0.05. Data are presented in the figures and text as means± SEM.
3. Results 3.1. Baseline 3.1.1. Light period The sleep recording performed during the 5 baseline days in female rats did not reveal statistical differences in sleep efficiency (Fig. 1A), SWS (Fig. 2A), and PS (Fig. 3A) in the estrous phases. In addition, no statistically significant differences were observed when these parameters were compared with those of the phases of the estrous cycle and those of the males.
Fig. 2. Mean ± SEM percentage of slow wave sleep (SWS) during the light period (panel A) and during the dark period (panel B) in females in proestrus (P), estrus (E), and diestrus (D) at baseline and 5 days rebound recording (R1–R5). Panel A: ⁎Denotes a difference from respective baseline; §denotes a difference from estrus group; † denotes a difference from Da; ‡denotes a difference from Dc; ¤denotes a difference from the R1 respective group. Panel B: †Denotes a difference from Da; ‡denotes a difference from Dc; £denotes a difference from male group; ¥denotes a difference from other groups at the same day; (ANOVA followed by Tukey's post-hoc test, see text for p-values).
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Fig. 3. Mean ± SEM percentage of paradoxical sleep during the light period (panel A) and during the dark period (panel B) in females in proestrus (P), estrus (E), and diestrus (D) at baseline and 5 days rebound recording (R1–R5). Panel A: ⁎Denotes a difference from respective baseline; #denotes a difference from R2–R5; §different from estrus group. Panel B: ⁎Denotes a difference from respective baseline; #denotes a difference from R2–R5; ¤denotes a difference from the R1 respective group (ANOVA followed by Tukey's post-hoc test, see text for p-values).
3.2.1. Light period
of rebound (R1) in all groups when compared with respective baseline recordings ( ps b 0.0001). The R1, in turn, differed from the respective remaining recovery days in estrus, Da females and male rats (estrus: ps b 0.01; Da: ps b 0.02; males: ps b 0.01). From R3 to R5, proestrus and Dc groups presented a significant reduction compared to the corresponding R1 recordings ( ps b 0.01). No statistically significant differences were observed between females and males on any of the recorded days (Fig. 1A).
3.2.1.1. Sleep efficiency. The ANOVA indicated a significant effect in the groups [F(28,166) = 11.26; p b 0.00001]. Tukey's test revealed that sleep efficiency was higher on the first day
3.2.1.2. Slow wave sleep. Statistically significant differences were revealed [F(28,166) = 6.93; p b 0.00001]. Compared to baseline, proestrus females presented a decrease in SWS during
3.1.2. Dark period No differences in sleep efficiency, SWS, or PS were detected among the estrous phases (Figs. 1B, 2B, and 3B). In comparison with male rats, a significant increase in SWS was observed in D females ( p b 0.01) (Fig. 2B). 3.2. Sleep recovery
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R1 ( p b 0.05). As depicted in Fig. 2A, the Dc and Da groups showed a statistical increase in SWS on the second and fourth days compared to the corresponding R1 ( ps b 0.05). At R4, proestrus rats showed a significant augmentation in SWS compared to R1 ( p b 0.01). On the third day of rebound, male SWS increased significantly in relation to the corresponding R1 ( p b 0.03). When gender effects were compared, males showed a significant reduction at R1 compared to estrus rats ( p b 0.02). There was a significant reduction in SWS during the R2 and R4 in male rats compared to Dc ( ps b 0.01). In addition, males differed from Da at R4 ( p b 0.01) (Fig. 2A). 3.2.1.3. Paradoxical sleep. A significant effect was observed [F(28,166) = 81.35; p b 0.001]. The post-hoc analysis revealed a significant increase on R1 in all groups compared to respective baseline recordings ( ps b 0.0001). The enhanced PS observed in R1 differed from respective R2 to R5 in all groups evaluated ( ps b 0.0001). At R2, proestrus females were the only group that presented an enhancement of PS compared to baseline recording ( p b 0.01). Tukey's test showed that this sleep stage was significantly lower in estrus compared to Da and male groups on the first day of the rebound period ( ps b 0.01) (Fig. 3A). 3.2.2. Dark period 3.2.2.1. Sleep efficiency. Analyses of variance [F(28,159) = 10.48; p b 0.00001] followed by Tukey's post-hoc test revealed a significant increase in R1 in all estrous phases compared to their respective baseline values ( ps b 0.05). The Dc rats showed a significant reduction in sleep efficiency from R3 to R5 days compared with R1 ( ps b 0.03). The augmentation of sleep time observed at R1 was statistically different from R4 and R5 in proestrus ( p b 0.02) and in males ( p b 0.04). The Da group showed an increase from R2 to R5 compared with males ( ps b 0.01). In addition, the Dc differed from males at R1, R2 and R5 ( ps b 0.03). The proestrus rats showed a significant increase at R3 compared to males as depicted in Fig. 1B ( p b 0.02). All females, regardless of estrous cycle phase, differed from males at R5 ( p b 0.02) (Fig. 1B). 3.2.2.2. Slow wave sleep. Significant effects were detected across groups [F(28,159) = 6.46; p b 0.00001]. The comparison between male and female rats revealed that at R2, the Da and Dc rats presented a significant increase in SWS ( p b 0.03 and p b 0.01, respectively). Except for estrus rats, all female groups presented increased SWS compared to males at R3 ( ps b 0.03). Additionally, at R5, all female groups differed from males ( ps b 0.01) (Fig. 2B). 3.2.2.3. Paradoxical sleep. The ANOVA results indicated a change in PS [F(28,159) = 15.35; p b 0.001]. There was a significant increase on R1 in all groups compared to their respective baseline ( ps b 0.0001). Moreover, R1 differed from R2 to R5 in all groups (females: ps b 0.0001 and males: p b 0.01), except for Da ( p N 0.05). Proestrus ( p b 0.01) and estrus ( p b 0.04) phases, and Da ( p b 0.01) maintained the increase of PS in R2 in relation to baseline. At R3, the proestrus rats still
maintained the increased PS compared to baseline ( p b 0.03). The Da showed a reduction at R3 ( p b 0.04) and R5 ( p b 0.01) in relation to R1 (Fig. 3B). 4. Discussion Our results showed that the estrous cycle had an undetectable influence on the sleep patterns during baseline recording. It also showed PSD induced marked alterations in females in the rebound period compared to males. Sleep efficiency and PS in all except the proestrus group returned to baseline values on the second day of the light period. Although PSD provoked a significant increase in PS on the first day of the rebound dark period in all groups, proestrus, estrus, and Da, in contrast to Dc and males, maintained this increase until the second day of the rebound dark period. As previously reported by Machado et al. (2004), males showed differences in all parameters except SWS, but only on the first day of the rebound period. In comparison with males, Dc presented a significant increase in SWS in the baseline dark period. Interestingly, Da was the only group in which PS remained enhanced at R2 in the dark recording. In contrast, the other groups presented a statistically significant reduction in PS on the second rebound day when compared to that of the first day. Thus far, little attention has been given to whether sleep is differentially regulated between genders and the magnitude of the consequences of sleep loss. Studies have documented that while healthy women appear to objectively have better sleep quality than men, women of all adult age groups report more sleep problems, including inadequate sleep time and insomnia (Bixler et al., 2002; Collop et al., 2004; Zhang and Wing, 2006). Moreover, nightmares were reported to be twice as frequent in women (Ohayon et al., 1997). The reasons for such discrepancies are attributed to hormonal fluctuations over the menstrual or estrous cycles, a factor that has been associated with sleep variations in both humans and rats (Hachul et al., 2006; Antunes et al., 2006; 2007). The loss of sleep that results from our modern lifestyle, increased work pressure, and psychosocial stress may have many unknown repercussions on health and general well-being (Meerlo et al., 2002; Leibowitz et al., 2006). This may be further aggravated in women due to additional child care and home tasks. The well documented constellation of consequences of sleep restriction established for males remains to be demonstrated in females. In fact, it is widely accepted that excessive daytime somnolence is markedly present in the male gender. However, few studies have reported the gender difference in sleep architecture after sleep deprivation. In particular, alterations in reproductive hormone release coincidental with sleep have been appraised as a manifestation of entrained links between central nervous regulation and endocrine function (Fehm et al., 1991). Notably, our results demonstrate that the hormonal oscillation present in the proestrus phase (Meites et al., 1980) is associated with alterations in sleep patterns recorded for 5 days after sleep deprivation (R1–R5). In fact, at light rebound recording, proestrus females were the only group that presented an enhancement of paradoxical sleep
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compared to baseline recording, whereas at the dark recording, these females still maintained the increased paradoxical sleep until the third day, indicating a long-term effect of PSD on these females. Additionally, only females with prolonged diestrus (anestrus) showed the increased time of paradoxical sleep at R2 in the dark recording, suggesting that hormonal alterations may influence sleep regulation after PSD. Although the current study did not evaluate the hormone concentrations, in our previous study we reported the hormonal variation in females during different phases of the estrous cycle and the effects of PSD on these levels. It is possible to infer that the marked alterations in hormonal concentrations in anestrus rats (Antunes et al., 2006) would influence their sleep architecture by prolonging the recovery effects of PSD. In accordance with our previous findings, in which we reported that both diestrus and proestrus rats when submitted to PSD paradigm presented a disruption of cyclicity within the first week of sleep recovery period, herein the data demonstrated that these females also had marked sleep changes. In addition to hormonal alterations, studies utilizing sleep deprivation have been conducted to comprehend the mechanisms involved in the regulation and maintenance of sleep patterns. Indeed, in rats, sleep deprivation induced by the platform technique involves numerous awakenings, which predominantly affect the paradoxical stage of sleep. We therefore reasoned that this procedure could mimic sleep fragmentation due to repeated awakenings, and thus would be a useful tool to investigate the effects of sleep loss on sleep patterns. It is well established that PSD in experimental animals results in behavioral (Tufik et al., 1978, 1987; Frussa-Filho et al., 2004; Andersen et al., 2002, 2003a, 2004a; Fukushiro et al., 2007; Martins et al., 2008) and hormonal changes (Spiegel et al., 1999; Andersen et al., 2003b, 2004b, 2005, 2006). In addition to hormonal alterations, there is evidence that PSD is related to biochemical factors involved in cardiovascular disease. For instance, PSD significantly increased LDL concentrations in males (Andersen et al., 2004b) and in ovariectomized females compared to intact females (Antunes et al., 2007). It is noteworthy that the ovariectomized group displayed higher levels of LDL and HDL when compared with the male rats after PSD. Some could argue the stress involved in this platform technique. This methodology results in a total suppression of paradoxical sleep and partial loss of SWS in male rats (Machado et al., 2004). However, we acknowledge that sleep deprivation is an inherently stressful procedure, so it may not be possible to completely extricate sleep deprivation effects from general stress effects. Yet, several aspects in our findings argue against the possibility that non-specific stress per se, could account for our observations (Andersen et al., 2000, 2004b) in which the PSD group differed from the other groups investigated. The many consequences of sleep deprivation have been well documented in males, but the data have yet to be replicated in females. Conceivably, this suggests that women are either more susceptible to clinical symptoms derived from inadequate sleep, or are more likely to report symptoms in general (Krishnan and Collop, 2006). For instance, there are many cases in which women play the dual role of the mother/housewife and pro-
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fessional, working during periods when she should be resting. This state of affairs renders those women in a constant state of stress/sleep restriction. Furthermore, several sleep abnormalities, including increased or decreased sleep duration, fragmentation of REM sleep, among others, have been associated with depression (Gillin, 1983). Furthermore, sleep deprivation, particularly in the later part of the night (when REM sleep takes place), has been associated with improved mood in depressed patients (Gillin, 1983), however the biological phenomena responsible for this effect is still unknown. Sleep deprivation may modulate the hormones released by altering hormonal–neurochemical mechanisms. This consequence of sleep loss was also exhibited in anestrus females, who presented high levels of corticosterone and progesterone (Antunes et al., 2006) both secreted by the adrenal gland that, in turn, is augmented after PSD (Coenen and van Luijtelaar, 1985). Of note, progesterone has a modulating influence on sleep (Andersen et al., 2006) and an ovulatory menstrual cycle is characterized by fluctuating levels of progesterone. However, sleep data on normal women during the menstrual cycle is still contradictory. Lee et al. (1990) reported shortened REM sleep latency, but no changes in the percent of REM sleep. Driver et al. (1996) did not find any changes in SWS or slow wave activity in non-REM sleep, but observed decreased REM sleep. Other studies did not find any significant changes in asymptomatic women (Manber and Armitage, 1999). Additionally, the scenarios become even more complex, when studies on sleep deprivation in women are very scarce. In 2003, Corsi-Cabrera reported that EEG changes recover pre-deprivation (38 h of total sleep deprivation) values after one night recovery sleep in men while the same amount of sleep was not enough to reverse all of the sleep deprivation effects in women. These authors stated the existence of compensatory mechanisms in women, probably postponing sleep recovery, suggesting that women need more sleep than men to recover. Of note, not only different deprivation protocols (total/ partial/REM sleep), but also distinct durations have been used. Currently, most sleep deprivation occurs in the REM phase that occurs in the last half of the night. Thus, many investigators have developed and employed different approaches to reduce or abolish paradoxical sleep. The PSD method simulates a near natural situation in the sense that often one experiences a certain amount of PS loss during one's day to day life. As for the recovery process in female rats, sleep deprivation for the first 6 h of the light period enhanced slow wave activity in SWS during the subsequent 6 h of rebound (Schwierin et al., 1998). In humans, there was an increase in SWS during the first recovery night after total sleep deprivation in men and women (Reynolds et al., 1986), but women showed a more dramatic increase in slow wave activity after 40 h of total sleep deprivation than age-matched men, thus showing a greater response to sleep deprivation in women (Armitage et al., 2001). In particular, our findings indicate that Wistar anestrus females, like females in proestrus, are more susceptible to PSD because both showed alterations in sleep patterns that increased the time needed to return to baseline values. Further studies are required to address the specific role of each strain on sleep architecture across estrous cycle.
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Our data showed that females and males had distinct pattern when sleep was disrupted. The selective PSD paradigm seems to be an efficient method of paradoxical sleep manipulation in experimental models and may prove to be an important contribution to the investigation of sleep regulation physiology. On this basis, more in-depth knowledge of the functional significance of sleep deprivation in women must be obtained to assist current endeavors to find ways of preserving and promoting better sleep throughout one's lifetime. Acknowledgements The authors would like to express their sincere thanks to Waldemarks Leite, Juliana Perry, Ligia Papale, and Marilde Costa. This work was supported by grants from the Associação Fundo de Incentivo à Psicofarmacologia (AFIP) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #04/ 03979-9 to I.B.A., #06/58274-5 to T.A.F.A. and CEPID #98/ 14303-3 to S.T.). References Andersen ML, Palma D, Rueda AD, Tufik S. The effects of acute cocaine administration in paradoxical sleep-deprived rats. Addict Biol 2000;5: 417–20. Andersen ML, Valle AC, Timo-Iaria IC, Tufik S. In: Balieiro CLR, editor. Implantação de elétrodos para o estudo eletrofisiológico do ciclo vigíliasono do rato. São Paulo: Univ Fed Sao Paulo; 2001. Andersen ML, Bignotto M, Machado RB, Tufik S. Does paradoxical sleep deprivation and cocaine induce penile erection and ejaculation in old rats? Addict Biol 2002;7:285–90. Andersen ML, Bignotto M, Tufik S. The effect of apomorphine on genital reflexes in male rats deprived of paradoxical sleep. Physiol Behav 2003a;80: 211–5. Andersen ML, Bignotto M, Tufik S. Influence of paradoxical sleep deprivation and cocaine on development of spontaneous penile reflexes in rats of different ages. Brain Res 2003b;4; 968:130–8. Andersen ML, Bignotto M, Tufik S. Hormone treatment facilitates penile erection in castrated rats after sleep deprivation and cocaine. J Neuroendocrinol 2004a;16:154–9. Andersen ML, Bignotto M, Machado RB, Tufik S. Different stress modalities result in distinct steroid hormone responses by male rats. Braz J Med Biol Res 2004b;37:791–7. Andersen ML, Martins PJF, D'Almeida V, Bignotto M, Tufik S. Endocrinological alterations during sleep deprivation and recovery in male rats. J Sleep Res 2005;14:83–90. Andersen ML, Antunes IB, Tufik S. Effects of paradoxical sleep deprivation on genital reflexes in five rat strains. Horm Behav 2006;49:173–80. Antunes IB, Andersen ML, Alvarenga TA, Tufik S. Effects of paradoxical sleep deprivation on blood parameters associated with cardiovascular risk in intact and ovariectomized rats compared with male rats. Behav Brain Res 2007;25: 187–92. Armitage R, Emslie GJ, Hoffmann RF, Rintelmann J, Rush AJ. Delta sleep EEG in depressed adolescent females and healthy controls. J Affect Disord 2001;63: 139–48. Antunes IB, Andersen ML, Baracat EC, Tufik S. The effects of paradoxical sleep deprivation on estrous cycles of the female rats. Horm Behav 2006;49: 433–40. Baker FC, Maloney S, Driver HS. A comparison of subjective estimates of sleep with objective polysomnographic data in healthy men and women. J Psychosom Res 1999;47:335–41. Bixler EO, Vgontzas AN, Lin HM, Vela-Bueno A, Kales A. Insomnia in central Pennsylvania. J Psychosom Res 2002;53:589–92.
Coenen AM, van Luijtelaar EL. Stress induced by three procedures of deprivation of paradoxical sleep. Physiol Behav 1985;35:501–4. Collop NA, Adkins D, Phillips BA. Gender differences in sleep and sleepdisordered breathing. Clin Chest Med 2004;25:257–68. Colvin GB, Whitmoyer DI, Lisk RD, Walter DO, Sawyer CH. Changes in sleep–wakefulness in female rats during circadian and estrous cycles. Brain Res 1968;7:173–81. Corsi-Cabrera M, Sanchez AI, del-Rio-Portilla Y, Villanueva Y, Perez-Garci E. Effect of 38 h of total sleep deprivation on the waking EEG in women: sex differences. Int J Phys 2003;50:213–24. Driver HE, Dijk DJ, Werth E, Biedermann K, Borbély AA. Sleep and the sleep electroencephalogram across the menstrual cycle in young healthy women. J Clin Endocrinol Metab 1996;81:728–35. Fang J, Fishbein W. Sex differences in paradoxical sleep: influences of estrus cycle and ovariectomy. Brain Res 1996;734:275–85. Fehm HL, Clausing J, Kern W, Pietrowsky R, Born J. Sleep-associated augmentation and synchronization of luteinizing hormone pulses in adult men. Neuroendocrinology 1991;54:192–5. Freeman EW, Sammel MD, Rinaudo PJ, Sheng L. Premenstrual syndrome as a predictor of menopausal symptoms. Obstet Gynecol 2004;103:960–6. Frussa-Filho R, Gonçalves MT, Andersen ML, de Araujo NP, Chinen CC, Tufik S. Paradoxical sleep deprivation potentiates amphetamine-induced behavioural sensitization by increasing its conditioned component. Brain Res 2004;1003:188–93. Fukushiro DF, Calzavara MB, Trombin TF, Lopez GB, Abílio VC, Andersen ML, et al. Effects of environmental enrichment and paradoxical sleep deprivation on open-field behavior of amphetamine-treated mice. Physiol Behav 2007;23:773–9. Gillin JC. The sleep therapies of depression. Prog. Neuro-Psychopharmacol Biol Psychiatry 1983;7:351–64. Hachul de Campos H, Brandão LC, D'Almeida V, Greco BH, Bittencourt LR, Tufik S, et al. Sleep disturbances, oxidative stress and cardiovascular risk parameters postmenopausal women complaining of insomnia. Climacteric 2006;9:312–9. Hall JE, Sullivan JP, Richardson GS. Brief wake episodes modulate sleepinhibited luteinizing hormone secretion in the early follicular phase. J Clin Endocrinol Metab 2005;90:2050–5. Ishizuka Y, Pollak CP, Shirakawa S, Usui A, Fukuzawa H, Kariya T, et al. Sleep spindle frequency changes during the menstrual cycle. J Sleep Res 1994;3: 26–9. Kleinlogel H. The rat's sleep in oestrous cycle. Experimental 1975;31: 712–3. Krishnan V, Collop NA. Gender differences in sleep disorders (sleep and respiratory neurobiology). Curr Opin Pulm Med 2006;12:383–9. Lee KA, Shaver JF, Giblin EC, Woods NF. Sleep patterns related to menstrual cycle phase and premenstrual affective symptoms. Sleep 1990;13:403–9. Leibowitz SM, Lopes MC, Andersen ML, Kushida CA. Sleep deprivation and sleepiness caused by sleep loss. Sleep Med Clin 2006;1:31–45. Lindberg E, Janson C, Gislason T, Bjomsson E, Hetta J, Boman G. Sleep disturbances in a young adult population: can gender differences be explained by differences in psychological status? Sleep 1997;20: 213–29. Machado RB, Hipólide DC, Benedito-Silva AA, Tufik S. Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery. Brain Res 2004;1004:45–51. Manber R, Armitage R. Sex, steroids, and sleep: a review. Sleep 1999;22: 540–55. Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol 2002;62:609–14. Martins RS, Andersen ML, Shih MC, Tufik S. The effects of cocaine, methamphetamine and modafinil challenge on sleep rebound after paradoxical sleep deprivation in rats. Braz J Med Biol Res 2008;41:68–77. Meerlo P, Koehl M, van der Borght K, Turek FW. Sleep restriction alters the hypothalamic–pituitary–adrenal response to stress. J Neuroendocrinol 2002;14:397–402. Meites J, Steger RW, Huang HHH. Relation of neuroendocrine system to the reproductive decline in aging rats and human subjects. Fed Proc 1980;39: 3168–72.
M.L. Andersen et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 975–983 Ohayon MM, Morselli PL, Guilleminault C. Prevalence of nightmares and their relationship to psychopathology and daytime functioning in insomnia subjects. Sleep 1997;20:340–8. Reynolds CF, Kupfer DJ, Hoch CC, Stack JA, Houck PR, Berman SR. Sleep deprivation in healthy elderly men and women: effects on mood and on sleep during recovery. Sleep 1986;9:492–501. Schwierin B, Borbely AA, Tobler I. Sleep homeostasis in the female rat during the estrous cycle. Brain Res 1998;811:96–104. Spiegel K, Leproult R, Van Cauter E. Impact of sleep debt on metabolic and endocrine function. Lancet 1999;354:1435–9. Timo-Iaria C, Negrão N, Schmidek WR, Hoshino K, Lobato de Menezes CE, Leme da Rocha T. Phases and states of sleep in the rat. Physiol Behav 1970;5: 1057–62.
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Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 975 – 983 www.elsevier.com/locate/pnpbp
Effects of sleep loss on sleep architecture in Wistar rats: Gender-specific rebound sleep M.L. Andersen ⁎, I.B. Antunes, A. Silva, T.A.F. Alvarenga, E.C. Baracat, S. Tufik Department of Psychobiology, Universidade Federal de São Paulo, Escola Paulista de Medicina (UNIFESP/EPM), R. Napoleão de Barros, 925, Vila Clementino 04024-002, São Paulo, SP, Brazil Received 10 October 2007; received in revised form 8 January 2008; accepted 8 January 2008 Available online 16 January 2008
Abstract This study was designed to examine the influence of gender on sleep rebound architecture after a 4-day paradoxical sleep deprivation period. After a 5-day baseline sleep recording, both male and female rats in different phases of the estrus cycle were submitted to paradoxical sleep deprivation for 96 h. After this period, the sleep rebound recording was evaluated for 5 days (one estrus cycle). The findings revealed that after paradoxical sleep deprivation, sleep efficiency and paradoxical sleep returned to baseline values on the second day of the light period, for all except the proestrus group. During the dark rebound period, only the female groups presented increased sleep efficiency on the first day. Paradoxical sleep returned to baseline values on the third day, except for males and the cycling females submitted to paradoxical sleep deprivation in the diestrus phase, whose baseline values returned to normal on the second day of rebound period. Thus, the females and males displayed distinct patterns as a result of sleep disruption. © 2008 Elsevier Inc. All rights reserved. Keywords: EEG; Estrous cycle; Female rats; Gender; Sleep deprivation; Sleep rebound
1. Introduction There is an increasing concern about risks related to sleepiness in our society, which is normally a consequence of chronic sleep loss. Although sleep deprivation has a dramatic impact on multiple physiological processes (Leibowitz et al., 2006), it is not yet clear which factors contribute the most to the sleep disturbances reported by women (Lindberg et al., 1997). Indeed, in sleep surveys, women report considerably more sleep problems than men (Lindberg et al., 1997).
Abbreviations: PSD, paradoxical sleep deprivation; R, rebound sleep; EEG, electroencephalograph; SWS, slow wave sleep; REM, rapid eye movement; NREM, non-rapid eye movement; ECoG, electrocorticogram; EMG, electromyogram; P, proestrus; E, estrus; D, diestrus; Dc, diestrus with regular cycles; Da, diestrus–anestrus; CTRL, control. ⁎ Corresponding author. Department of Psychobiology, Universidade Federal de São Paulo, Rua Napoleão de Barros, 925, Vila Clementino, SP-04024-002, São Paulo, Brazil. Tel.: +55 11 2149 0155; fax: +55 11 5572 5092. E-mail address: [email protected] (M.L. Andersen). 0278-5846/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.01.007
It is an established fact that sex hormones influence sleep and circadian rhythms. Sleep in turn affects the episodic secretion of gonadotropin hormones (Hall et al., 2005). Although some women may not experience premenstrual symptoms and secondary insomnia, the latter and its associated symptoms often occur at the onset of menses (Freeman et al., 2004). There is some evidence indicating that sleep alterations are associated with the menstrual cycle, as could be expected from hormonal variations during that cycle. These changes can be small or significant and they include changes in the stage 2 slow wave sleep (SWS), and the percentage of REM sleep, or its latency (Lee et al., 1990; Ishizuka et al., 1994; Driver et al., 1996). Moreover, subtle differences in sleep at different phases of the menstrual cycle have also been reported in the luteal phase when REM sleep is reduced (Baker et al., 1999). In rats, sleep alterations also occur during the estrous cycle (Kleinlogel, 1975; Schwierin et al., 1998) as noted by a marked reduction in the nocturnal amount of non-REM (NREM) and REM sleep, as well as in slow wave activity in NREM sleep in proestrus when compared to other phases (Colvin et al., 1968; Zhang et al.,
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1995; Schwierin et al., 1998). In contrast to these results, Fang and Fishbein (1996) reported no alterations in SWS during the estrous cycle. Although some studies have focused on the sleep patterns of female rats under baseline conditions, few have focused on sleep parameters after sleep deprivation. Since gender differences in brain organization influence the regulation of basic physiological processes such as electrical activity and sleep (Corsi-Cabrera et al., 2003), it seems reasonable to predict that male and female systems will present differing effects of sleep deprivation on sleep parameters in the recovery period. Investigation of sleep deprivation in females has focused on specific aspects of sleep, mainly those related to pathologies. In view of the current scenario, there is great relevance in examining the sleep patterns of male and female rats subjected to paradoxical sleep deprivation (PSD), while in different phases of the estrous cycle during the recovery period. 2. Materials and methods 2.1. Subjects The study was performed on ten-week old female and male Wistar rats from our vivarium. The animals were maintained in a temperature-controlled room (23 ± 1 °C) with a 12:12-hour light– dark cycle (lights on at 0700) from birth and allowed free access to food and water in standard polypropylene cages. The rats used in this study were maintained and treated in accordance with the Ethical and Practical Principles of the Use of Laboratory Animal guidelines and the experimental protocol was approved by the Ethical Committee of UNIFESP (CEP N. 0998/04). Maximum efforts were taken to reduce the number of animals used in the experiments while employing enough to ensure unambiguous and reliable statistical analysis and data interpretation. 2.2. Surgical preparation and electrocorticogram/electromyographic recording Electrodes were surgically implanted in the rats in order to monitor electrocorticographic (ECoG) and electromyographic (EMG) activities, thus permitting the assessment of the sleep– wake cycle. Prior to implantation of the electrodes, the animals were briefly anesthetized with diazepam (10 mg/kg i.p.) and ketamine (90 mg/kg i.p.). To record cortical ECoG with a minimum of theta activity, one pair of screw electrodes was placed through the skull ipsilaterally: 1 mm posterior to bregma, 3 mm lateral to the central suture; and 1 mm anterior to lambda, 4 mm lateral to the central suture. EMG electrodes were implanted in the neck muscles, soldered to a six-pin socket and covered with dental acrylic cement. Body temperature was maintained at 37 °C with a regulated electric heating pad (Harvard Apparatus, USA). After the surgical procedure, each rat received an i.m. injection of 0.5 mL/rat of antibiotic and sodium diclofenaco (0.1 mL/rat, vo). The rats were individually placed in rounded transparent plastic cages and allowed a 10-day surgery recovery period, followed by a 4-day adaptation period with the cable connected. The recordings were performed on a Nihon Koden Co. model QP 223A apparatus (Tokyo, Japan), using three channels
for each animal: two for ECoG and one for head-muscle activity (EMG). The ECoG signals were amplified and filtered with a low pass filter at 0.1 s (1.6 Hz) and EMG activity was filtered with a low pass filter at 0.03 s (5.3 Hz). All recordings were scored by only one researcher, thus assuring reliability of the data. Analysis of the recordings was performed manually in a blind fashion using the Polysmith Neurotronics™ Inc. program (Neurotronics®, FL, USA), mainly based on the predominant amplitude and frequency of the tracing (Timo-Iaria et al., 1970; Andersen et al., 2001). The analysis was done in 30 s epochs with the dominant tracing (waking, slow wave sleep or paradoxical sleep) for more than 50% of each epoch. When artifacts or noise did not allow characterization of an epoch, assessments were made based on immediate surrounding epochs. The recordings were performed in individual home-cages. The following sleep parameters were considered: sleep efficiency (percentage of total sleep time during the recording time), total SWS (percentage of all periods of deep sleep throughout the recording), and total paradoxical sleep (percentage of all periods of paradoxical sleep throughout the recording). 2.3. Vaginal cytology Vaginal smears were obtained between 0800 and 0900 h. The vaginal smear samples were performed with a 200 μL pipette. A few drops of distilled water were injected into the vagina (but not deeply), and then pipetted out (see Marcondes et al., 2002 for complete description). The water with its cellular contents was then transferred to a slide. The determination of the estrous cycle was based on the occurrence of three types of cells in the vaginal smears: leukocytes, cornified cells, and nucleated epithelial cells. The stages of the estrous cycle were identified according to the following criteria: proestrus (P) was characterized by many epithelial cells and few leukocytes; estrus (E) was characterized by many cornified cells and no leukocytes, and diestrus (D) was characterized by few epithelial cells and many leukocytes. 2.4. Paradoxical sleep deprivation (PSD) The PSD method used 14 narrow circular platforms (6.5 cm in diameter), protruding 1 cm above the surface in a 123 × 44 × 44-cm tank filled with water. The rats were placed on the platforms and could move around by leaping from one platform to another. Each estrous cycle phase rat was placed in one tank, in a way such that each water tank contained only one estrus phase female rat. When they reached the paradoxical sleep (PS) phase, they would fall into the water, due to muscle atonia, and would be awakened. The narrow platform procedure caused complete loss of PS during all 4 days and approximately 30% of SWS (Machado et al., 2004). Our intention was to analyze the alterations on sleep architecture caused by total suppression of PS over 4 days. Throughout the study, the experimental room was maintained under controlled temperature (23 ± 1 °C) and a light–dark cycle (lights on at 0700 h and off at 1900 h). Food and water were provided ad libitum by placing chow pellets and water bottles on a grid located on top of the tank. The water in the tank was changed daily throughout the PSD period. Finally,
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the cage control group of each estrous cycle phase was maintained in the same room as the experimental rats for the duration of their respective PSD periods. By housing the groups in the same room, we controlled for any difference in housing condition between the two groups. The period of 96 h of PSD was chosen since our previous data showed that the most dramatic alterations in hormone concentration (Andersen et al., 2005) occur at this time point. To evaluate the long lasting effects of PSD on the estrous cycle, the baseline (B) and sleep recovery (R1–R5) periods were evaluated for 5 days. 2.5. Experimental design After a 10-day surgery recovery period, followed by a 4-day adaptation period with the polysomnography cable connected, all rats were submitted to vaginal smears over a period of 10 days. The females were smeared daily and animals with regular cycles were selected. After this, a baseline sleep recording for 5 con-
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secutive days (one estrous cycle) was performed. Males underwent the same process for statistical comparison. Following this period, females in different phases of the estrous cycle (proestrus [P], estrus [E], and diestrus [D]), as well as the males were submitted to PSD for 96 h. The respective control rats were maintained in their home-cages (CTRL). Although the vaginal smear was performed daily throughout the PSD period, no sleep recording was performed in that time. After the PSD period, the sleep rebound recording was evaluated for 5 consecutive days representing one estrous cycle (n = 6–10 for each group). Since we had recently published that 60% of the females that were submitted to PSD in the diestrus phase presented constant diestrus (anestrus cycle) up to the ninth day of the recovery period, the females in this study that were submitted to PSD in the diestrus phase were distributed into two groups for recovery sleep analysis: females that showed a regular estrous cycle (Dc) and those that presented an anestrus cycle (Da) (Antunes et al., 2006). Considering that estrus and diestrus are representative of the
Fig. 1. Mean ± SEM percentage of sleep efficiency during the light period (panel A) and during the dark period (panel B) in females in proestrus (P), estrus (E), and diestrus (D) at baseline and 5 days rebound recording (R1–R5). Panel A: ⁎Denotes a difference from respective baseline; #denotes a difference from R2–R5 of the respective group; ¤denotes a difference from the R1 respective group. Panel B: ⁎Denotes a difference from respective baseline; †denotes a difference from Da; ‡denotes a difference from Dc; ¥denotes a difference from other groups at the same day; ¤denotes a difference from the R1 respective group; £denotes a difference from male group (ANOVA followed by Tukey's post-hoc test, see text for p-values).
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ovulatory and luteal phases in women, respectively, these are the phases that present hormonal statistical significance, and because the estrous cycle was unaltered after PSD at metestrus phase (Antunes et al., 2006), we elected not to include the estrous phase in the current study. The vaginal smear was performed daily during the baseline, PSD, and recovery periods. 2.6. Statistical analysis Data analysis was carried out using two-way ANOVA followed by the Tukey's post-hoc test. The level of significance was set at p b 0.05. Data are presented in the figures and text as means± SEM.
3. Results 3.1. Baseline 3.1.1. Light period The sleep recording performed during the 5 baseline days in female rats did not reveal statistical differences in sleep efficiency (Fig. 1A), SWS (Fig. 2A), and PS (Fig. 3A) in the estrous phases. In addition, no statistically significant differences were observed when these parameters were compared with those of the phases of the estrous cycle and those of the males.
Fig. 2. Mean ± SEM percentage of slow wave sleep (SWS) during the light period (panel A) and during the dark period (panel B) in females in proestrus (P), estrus (E), and diestrus (D) at baseline and 5 days rebound recording (R1–R5). Panel A: ⁎Denotes a difference from respective baseline; §denotes a difference from estrus group; † denotes a difference from Da; ‡denotes a difference from Dc; ¤denotes a difference from the R1 respective group. Panel B: †Denotes a difference from Da; ‡denotes a difference from Dc; £denotes a difference from male group; ¥denotes a difference from other groups at the same day; (ANOVA followed by Tukey's post-hoc test, see text for p-values).
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Fig. 3. Mean ± SEM percentage of paradoxical sleep during the light period (panel A) and during the dark period (panel B) in females in proestrus (P), estrus (E), and diestrus (D) at baseline and 5 days rebound recording (R1–R5). Panel A: ⁎Denotes a difference from respective baseline; #denotes a difference from R2–R5; §different from estrus group. Panel B: ⁎Denotes a difference from respective baseline; #denotes a difference from R2–R5; ¤denotes a difference from the R1 respective group (ANOVA followed by Tukey's post-hoc test, see text for p-values).
3.2.1. Light period
of rebound (R1) in all groups when compared with respective baseline recordings ( ps b 0.0001). The R1, in turn, differed from the respective remaining recovery days in estrus, Da females and male rats (estrus: ps b 0.01; Da: ps b 0.02; males: ps b 0.01). From R3 to R5, proestrus and Dc groups presented a significant reduction compared to the corresponding R1 recordings ( ps b 0.01). No statistically significant differences were observed between females and males on any of the recorded days (Fig. 1A).
3.2.1.1. Sleep efficiency. The ANOVA indicated a significant effect in the groups [F(28,166) = 11.26; p b 0.00001]. Tukey's test revealed that sleep efficiency was higher on the first day
3.2.1.2. Slow wave sleep. Statistically significant differences were revealed [F(28,166) = 6.93; p b 0.00001]. Compared to baseline, proestrus females presented a decrease in SWS during
3.1.2. Dark period No differences in sleep efficiency, SWS, or PS were detected among the estrous phases (Figs. 1B, 2B, and 3B). In comparison with male rats, a significant increase in SWS was observed in D females ( p b 0.01) (Fig. 2B). 3.2. Sleep recovery
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R1 ( p b 0.05). As depicted in Fig. 2A, the Dc and Da groups showed a statistical increase in SWS on the second and fourth days compared to the corresponding R1 ( ps b 0.05). At R4, proestrus rats showed a significant augmentation in SWS compared to R1 ( p b 0.01). On the third day of rebound, male SWS increased significantly in relation to the corresponding R1 ( p b 0.03). When gender effects were compared, males showed a significant reduction at R1 compared to estrus rats ( p b 0.02). There was a significant reduction in SWS during the R2 and R4 in male rats compared to Dc ( ps b 0.01). In addition, males differed from Da at R4 ( p b 0.01) (Fig. 2A). 3.2.1.3. Paradoxical sleep. A significant effect was observed [F(28,166) = 81.35; p b 0.001]. The post-hoc analysis revealed a significant increase on R1 in all groups compared to respective baseline recordings ( ps b 0.0001). The enhanced PS observed in R1 differed from respective R2 to R5 in all groups evaluated ( ps b 0.0001). At R2, proestrus females were the only group that presented an enhancement of PS compared to baseline recording ( p b 0.01). Tukey's test showed that this sleep stage was significantly lower in estrus compared to Da and male groups on the first day of the rebound period ( ps b 0.01) (Fig. 3A). 3.2.2. Dark period 3.2.2.1. Sleep efficiency. Analyses of variance [F(28,159) = 10.48; p b 0.00001] followed by Tukey's post-hoc test revealed a significant increase in R1 in all estrous phases compared to their respective baseline values ( ps b 0.05). The Dc rats showed a significant reduction in sleep efficiency from R3 to R5 days compared with R1 ( ps b 0.03). The augmentation of sleep time observed at R1 was statistically different from R4 and R5 in proestrus ( p b 0.02) and in males ( p b 0.04). The Da group showed an increase from R2 to R5 compared with males ( ps b 0.01). In addition, the Dc differed from males at R1, R2 and R5 ( ps b 0.03). The proestrus rats showed a significant increase at R3 compared to males as depicted in Fig. 1B ( p b 0.02). All females, regardless of estrous cycle phase, differed from males at R5 ( p b 0.02) (Fig. 1B). 3.2.2.2. Slow wave sleep. Significant effects were detected across groups [F(28,159) = 6.46; p b 0.00001]. The comparison between male and female rats revealed that at R2, the Da and Dc rats presented a significant increase in SWS ( p b 0.03 and p b 0.01, respectively). Except for estrus rats, all female groups presented increased SWS compared to males at R3 ( ps b 0.03). Additionally, at R5, all female groups differed from males ( ps b 0.01) (Fig. 2B). 3.2.2.3. Paradoxical sleep. The ANOVA results indicated a change in PS [F(28,159) = 15.35; p b 0.001]. There was a significant increase on R1 in all groups compared to their respective baseline ( ps b 0.0001). Moreover, R1 differed from R2 to R5 in all groups (females: ps b 0.0001 and males: p b 0.01), except for Da ( p N 0.05). Proestrus ( p b 0.01) and estrus ( p b 0.04) phases, and Da ( p b 0.01) maintained the increase of PS in R2 in relation to baseline. At R3, the proestrus rats still
maintained the increased PS compared to baseline ( p b 0.03). The Da showed a reduction at R3 ( p b 0.04) and R5 ( p b 0.01) in relation to R1 (Fig. 3B). 4. Discussion Our results showed that the estrous cycle had an undetectable influence on the sleep patterns during baseline recording. It also showed PSD induced marked alterations in females in the rebound period compared to males. Sleep efficiency and PS in all except the proestrus group returned to baseline values on the second day of the light period. Although PSD provoked a significant increase in PS on the first day of the rebound dark period in all groups, proestrus, estrus, and Da, in contrast to Dc and males, maintained this increase until the second day of the rebound dark period. As previously reported by Machado et al. (2004), males showed differences in all parameters except SWS, but only on the first day of the rebound period. In comparison with males, Dc presented a significant increase in SWS in the baseline dark period. Interestingly, Da was the only group in which PS remained enhanced at R2 in the dark recording. In contrast, the other groups presented a statistically significant reduction in PS on the second rebound day when compared to that of the first day. Thus far, little attention has been given to whether sleep is differentially regulated between genders and the magnitude of the consequences of sleep loss. Studies have documented that while healthy women appear to objectively have better sleep quality than men, women of all adult age groups report more sleep problems, including inadequate sleep time and insomnia (Bixler et al., 2002; Collop et al., 2004; Zhang and Wing, 2006). Moreover, nightmares were reported to be twice as frequent in women (Ohayon et al., 1997). The reasons for such discrepancies are attributed to hormonal fluctuations over the menstrual or estrous cycles, a factor that has been associated with sleep variations in both humans and rats (Hachul et al., 2006; Antunes et al., 2006; 2007). The loss of sleep that results from our modern lifestyle, increased work pressure, and psychosocial stress may have many unknown repercussions on health and general well-being (Meerlo et al., 2002; Leibowitz et al., 2006). This may be further aggravated in women due to additional child care and home tasks. The well documented constellation of consequences of sleep restriction established for males remains to be demonstrated in females. In fact, it is widely accepted that excessive daytime somnolence is markedly present in the male gender. However, few studies have reported the gender difference in sleep architecture after sleep deprivation. In particular, alterations in reproductive hormone release coincidental with sleep have been appraised as a manifestation of entrained links between central nervous regulation and endocrine function (Fehm et al., 1991). Notably, our results demonstrate that the hormonal oscillation present in the proestrus phase (Meites et al., 1980) is associated with alterations in sleep patterns recorded for 5 days after sleep deprivation (R1–R5). In fact, at light rebound recording, proestrus females were the only group that presented an enhancement of paradoxical sleep
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compared to baseline recording, whereas at the dark recording, these females still maintained the increased paradoxical sleep until the third day, indicating a long-term effect of PSD on these females. Additionally, only females with prolonged diestrus (anestrus) showed the increased time of paradoxical sleep at R2 in the dark recording, suggesting that hormonal alterations may influence sleep regulation after PSD. Although the current study did not evaluate the hormone concentrations, in our previous study we reported the hormonal variation in females during different phases of the estrous cycle and the effects of PSD on these levels. It is possible to infer that the marked alterations in hormonal concentrations in anestrus rats (Antunes et al., 2006) would influence their sleep architecture by prolonging the recovery effects of PSD. In accordance with our previous findings, in which we reported that both diestrus and proestrus rats when submitted to PSD paradigm presented a disruption of cyclicity within the first week of sleep recovery period, herein the data demonstrated that these females also had marked sleep changes. In addition to hormonal alterations, studies utilizing sleep deprivation have been conducted to comprehend the mechanisms involved in the regulation and maintenance of sleep patterns. Indeed, in rats, sleep deprivation induced by the platform technique involves numerous awakenings, which predominantly affect the paradoxical stage of sleep. We therefore reasoned that this procedure could mimic sleep fragmentation due to repeated awakenings, and thus would be a useful tool to investigate the effects of sleep loss on sleep patterns. It is well established that PSD in experimental animals results in behavioral (Tufik et al., 1978, 1987; Frussa-Filho et al., 2004; Andersen et al., 2002, 2003a, 2004a; Fukushiro et al., 2007; Martins et al., 2008) and hormonal changes (Spiegel et al., 1999; Andersen et al., 2003b, 2004b, 2005, 2006). In addition to hormonal alterations, there is evidence that PSD is related to biochemical factors involved in cardiovascular disease. For instance, PSD significantly increased LDL concentrations in males (Andersen et al., 2004b) and in ovariectomized females compared to intact females (Antunes et al., 2007). It is noteworthy that the ovariectomized group displayed higher levels of LDL and HDL when compared with the male rats after PSD. Some could argue the stress involved in this platform technique. This methodology results in a total suppression of paradoxical sleep and partial loss of SWS in male rats (Machado et al., 2004). However, we acknowledge that sleep deprivation is an inherently stressful procedure, so it may not be possible to completely extricate sleep deprivation effects from general stress effects. Yet, several aspects in our findings argue against the possibility that non-specific stress per se, could account for our observations (Andersen et al., 2000, 2004b) in which the PSD group differed from the other groups investigated. The many consequences of sleep deprivation have been well documented in males, but the data have yet to be replicated in females. Conceivably, this suggests that women are either more susceptible to clinical symptoms derived from inadequate sleep, or are more likely to report symptoms in general (Krishnan and Collop, 2006). For instance, there are many cases in which women play the dual role of the mother/housewife and pro-
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fessional, working during periods when she should be resting. This state of affairs renders those women in a constant state of stress/sleep restriction. Furthermore, several sleep abnormalities, including increased or decreased sleep duration, fragmentation of REM sleep, among others, have been associated with depression (Gillin, 1983). Furthermore, sleep deprivation, particularly in the later part of the night (when REM sleep takes place), has been associated with improved mood in depressed patients (Gillin, 1983), however the biological phenomena responsible for this effect is still unknown. Sleep deprivation may modulate the hormones released by altering hormonal–neurochemical mechanisms. This consequence of sleep loss was also exhibited in anestrus females, who presented high levels of corticosterone and progesterone (Antunes et al., 2006) both secreted by the adrenal gland that, in turn, is augmented after PSD (Coenen and van Luijtelaar, 1985). Of note, progesterone has a modulating influence on sleep (Andersen et al., 2006) and an ovulatory menstrual cycle is characterized by fluctuating levels of progesterone. However, sleep data on normal women during the menstrual cycle is still contradictory. Lee et al. (1990) reported shortened REM sleep latency, but no changes in the percent of REM sleep. Driver et al. (1996) did not find any changes in SWS or slow wave activity in non-REM sleep, but observed decreased REM sleep. Other studies did not find any significant changes in asymptomatic women (Manber and Armitage, 1999). Additionally, the scenarios become even more complex, when studies on sleep deprivation in women are very scarce. In 2003, Corsi-Cabrera reported that EEG changes recover pre-deprivation (38 h of total sleep deprivation) values after one night recovery sleep in men while the same amount of sleep was not enough to reverse all of the sleep deprivation effects in women. These authors stated the existence of compensatory mechanisms in women, probably postponing sleep recovery, suggesting that women need more sleep than men to recover. Of note, not only different deprivation protocols (total/ partial/REM sleep), but also distinct durations have been used. Currently, most sleep deprivation occurs in the REM phase that occurs in the last half of the night. Thus, many investigators have developed and employed different approaches to reduce or abolish paradoxical sleep. The PSD method simulates a near natural situation in the sense that often one experiences a certain amount of PS loss during one's day to day life. As for the recovery process in female rats, sleep deprivation for the first 6 h of the light period enhanced slow wave activity in SWS during the subsequent 6 h of rebound (Schwierin et al., 1998). In humans, there was an increase in SWS during the first recovery night after total sleep deprivation in men and women (Reynolds et al., 1986), but women showed a more dramatic increase in slow wave activity after 40 h of total sleep deprivation than age-matched men, thus showing a greater response to sleep deprivation in women (Armitage et al., 2001). In particular, our findings indicate that Wistar anestrus females, like females in proestrus, are more susceptible to PSD because both showed alterations in sleep patterns that increased the time needed to return to baseline values. Further studies are required to address the specific role of each strain on sleep architecture across estrous cycle.
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