TY - JOUR
AU - Peyron, Christelle
AB - Abstract Narcolepsy type 1 is a disabling disorder with four primary symptoms: excessive-daytime-sleepiness, cataplexy, hypnagogic hallucinations, and sleep paralysis. The later three symptoms together with a short rapid eye movement (REM) sleep latency have suggested impairment in REM sleep homeostatic regulation with an enhanced propensity for (i.e. tendency to enter) REM sleep. To test this hypothesis, we challenged REM sleep homeostatic regulation in a recognized model of narcolepsy, the orexin knock-out (Orex-KO) mice and their wild-type (WT) littermates. We first performed 48 hr of REM sleep deprivation using the classic small-platforms-over-water method. We found that narcoleptic mice are similarly REM sleep deprived to WT mice. Although they had shorter sleep latency, Orex-KO mice recovered similarly to WT during the following 10 hr of recovery. Interestingly, Orex-KO mice also had cataplexy episodes immediately after REM sleep deprivation, anticipating REM sleep rebound, at a time of day when cataplexy does not occur in baseline condition. We then evaluated REM sleep propensity using our new automated method of deprivation that performs a specific and efficient REM sleep deprivation. We showed that REM sleep propensity is similar during light phase in Orex-KO and WT mice. However, during the dark phase, REM sleep propensity was not suppressed in Orex-KO mice when hypocretin/orexin neuropeptides are normally released. Altogether our data suggest that in addition to the well-known wake-promoting role of hypocretin/orexin, these neuropeptides would also suppress REM sleep. Therefore, hypocretin/orexin deficiency would facilitate the occurrence of REM sleep at any time of day in an opportunistic manner as seen in human narcolepsy. narcolepsy, orexin, cataplexy, neuropeptides, hypocretin, REM sleep homeostasis, sleep deprivation, hypersomnia, anxiety Statement of Significance This study shows that rapid eye movement (REM) sleep dysregulation in narcolepsy is not due to an impairment of the homeostatic regulation of REM sleep. We showed in mice-rendered narcoleptic by genetically removing the gene encoding for hypocretin/orexin neuropeptides that REM sleep need develops similarly in narcoleptic mice and wild-type littermates, and that they similarly recover from specific REM sleep deprivation. However, REM sleep would occur throughout the day in an opportunistic manner with lack of inhibition due to the absence of the hypocretin/orexin neuropeptides, similar to human narcolepsy. Our data provide a better understanding of narcolepsy with new insights into its physiopathology. Introduction Narcolepsy type 1 (NT1) is an orphan neurological disorder that affects 0.002 per cent of the population worldwide [1]. Patients experience excessive daytime sleepiness, cataplexy (a sudden loss of muscle tone during active wake triggered by positive emotions), hypnagogic hallucinations, and sleep paralysis [1]. The later three symptoms share similarities with rapid eye movement (REM) sleep, also called paradoxical sleep, and together with the very short latency to enter REM sleep have supported the idea of an impairment of REM sleep regulation in narcolepsy. It is well established that REM sleep is finely tuned in mammals [2–4]. REM sleep is under a strong circadian control occurring in the second half of the night in humans and in its majority during the light phase in nocturnal rodents. REM sleep is also homeostatically regulated so that any debt of REM sleep is precisely compensated for in a proportional manner by REM sleep hypersomnia (i.e. rebound) during the following recovery period [5, 6] to maintain equilibrium. In patients with narcolepsy, the circadian time-keeping system is preserved [7, 8]. However, in a 3 day nap protocol where participants are on a 90 min sleep/wake schedule, patients with narcolepsy showed REM sleep throughout the day, in contrast to controls [2]. This observation may reflect a lack of circadian control over REM sleep or may be the consequence of a stronger homeostatic drive for REM sleep. To assess this question, Vu and colleagues [9] performed a selective REM sleep deprivation protocol on six patients with narcolepsy and six control participants. They found that the number of interventions per night required to selectively REM sleep–deprived participants was significantly higher in patients with narcolepsy suggesting a stronger REM sleep propensity in narcolepsy. However, no REM sleep rebound was observed during the recovery night post two consecutive nights of deprivation. This observation may be due to impairment of homeostatic regulation or to the fact that the patients performed a multiple sleep latency test (MSLT, five naps of 20 min scheduled at 2 hr intervals) during the day, between the second night of REM sleep deprivation and the recovery night. Due to the complexity of the REM sleep deprivation procedure in humans, animal models may be of help to provide additional insights. NT1 is due to the specific loss of a small group of neurons located in the tuberal hypothalamus that produces orexin/hypocretin neuropeptides [10, 11]. A well-recognized model of human NT1 known as the orexin Knock-Out mice (Orex-KO) [12] was created by the genetic deletion of the preprohypocretin gene and displays highly fragmented sleep and behavioral arrests considered equivalent to human cataplexy. As the neurons are still present, this Orex-KO model provides an excellent tool to reveal the roles played by the orexin/hypocretin neuropeptides specifically, rather than the entire neuronal contribution, in REM sleep regulation. Thus, to bring more light on the homeostatic regulation of REM sleep in narcolepsy as well as determining the role played by the orexin neuropeptides in this regulation, we specifically challenged REM sleep in Orex-KO mice and their wild-type (WT) littermates. We first used a standard method of 48 hr of selective REM sleep deprivation using the platforms-over-water method [4] and thoroughly investigated REM sleep during the following recovery period. Then, to objectively measure REM sleep propensity throughout deprivation, we used a newly developed innovative automatic deprivation method [13]. Finally, we performed behavioral tests and corticosterone measurements in Orex-KO mice and controls to consider eventual differences in anxiety and mild stress coping between genotypes in our analyses. Materials and Methods All experiments were conducted in accordance to the European Community guidelines for the use of animals for research and approved by the Ethics Research Committee of University-Lyon1 (DR2015-03). Experiments were conducted on a total of 46 male C57Bl/6J Orex-KO mice and 46 WT littermates [12]. WT and Orex-KO mice weighed, respectively, 25–32 and 30–40 g and were between 12 and 17 weeks of age at recordings. Animals were housed under constant 12:12 hr light/dark cycle (lights on at 08:00 am). Room temperature was maintained at 23 ± 1°C and food and water were available ad libitum throughout the experiment. Polysomnography Surgery Sixteen WT and 16 Orex-KO mice were implanted for electroencephalographic (EEG) and electromyographic (EMG) recordings. Mice were anesthetized by intraperitoneal (ip) administration of a ketamine:xylazine mixture (100:10 mg/kg) and fixed in a stereotaxic frame (David Kopf Instruments). Following incision of the scalp, three stainless steel screws were inserted above the frontal (1.5 mm anterior to Bregma, 1.7 mm lateral to midline), parietal (2.5 mm posterior to Bregma, 1.7 mm lateral to midline), and cerebellar (6 mm posterior to Bregma) cortices. Two gold-coated electrodes were also slipped between the neck muscles to record EMG activity. All EMG and EEG electrodes were then fixed to the skull using acrylic cement (Superbond, C&B Sun Medical) and connected to a miniature plug (Plastics One, Bilaney, Germany) that was additionally fixed using dental cement (Paladur, Heraeus Kulzer). After surgery, mice were allowed to recover for at least 1 week during which they were weighed daily. Recordings Mice were placed in a Plexiglas recording barrel and connected to a cable plugged to a rotating connector (Plastics One, Bilaney, Germany) to allow free movements during the entire experiment. Each frontal and parietal EEG signal was referenced to the cerebellar EEG electrode, and muscle tone was assessed by a differential EMG signal. EEG and EMG signals were then amplified (EEG: 2000×; EMG: 5000×) with 16-Channel Model 3500 AM System (United States). Signals were band filtered (EEG: 0.3–100 Hz; EMG: 1–100 Hz), digitalized at 512 Hz with NI Usb 6343 card (National Instrument, Austin, USA) and collected using Sleepscore software (Viewpoint, Lyon, France) [14]. Quantification of the sleep–wake cycle Vigilance states were scored using a 5 s window frame according to standard criteria using Sleepscore as described previously [4, 14]. Wake, nonrapid eye movement (NREM), and REM sleep were assigned using the standard mouse EEG/EMG scoring method, based on rules established in WT mice [15]. Each REM sleep episode recorded during the automatic deprivation protocol was rescored using a 1 s window frame to accurately evaluate residual amount of REM sleep, most of them lasting for less than 5 s. Circadian distribution of REM sleep was measured by calculating the circadian index (CI) as proposed previously [16], CI = (Mean quantity at night – Mean quantity at day)/Mean quantity in 24 hr. Quantification of cataplexy Cataplexy was scored according to the consensual definition [17] using both EEG/EMG and simultaneous video recordings. It always occurred outside the nest, in a period of high motor activity, following already reported behaviors such as grooming or digging [18]. They were analyzed during baseline and the recovery period that followed the 48 hr of platforms-over-water REM sleep deprivation. REM sleep deprivation with platforms-over-water As previously reported [4], mice were housed individually in a Plexiglas barrel (20 cm in diameter) containing two small platforms (2.5 cm in diameter, 2 and 3 cm high) for habituation in the recording chamber. Mice were plugged to the recording system and were left for 3 days of habituation followed by 2 days of baseline recordings. During REM sleep deprivation, woodchips were replaced by 1 cm deep of water (Figure 1A). REM sleep deprivation was started when REM sleep amount is high at 10:00 am (zeitgeber time 2 [ZT2]) and lasted for 48 hr. Then mice were returned to their habituation barrel, where they could sleep ad libitum for recovery. Figure 1. View largeDownload slide Illustration of REM sleep deprivation methodologies. Photographs illustrating selective REM sleep deprivation set-ups for (A) small-platforms-over-water protocol, or (B) automatic deprivation method. (C) Illustration of the polysomnographic signals (EEG, EMG) at transition into REM sleep during automatic REM sleep deprivation. Only stimulations occurring during the REM sleep episodes are counted (green arrow). Those occurring during wakefulness are not considered (black arrows). Figure 1. View largeDownload slide Illustration of REM sleep deprivation methodologies. Photographs illustrating selective REM sleep deprivation set-ups for (A) small-platforms-over-water protocol, or (B) automatic deprivation method. (C) Illustration of the polysomnographic signals (EEG, EMG) at transition into REM sleep during automatic REM sleep deprivation. Only stimulations occurring during the REM sleep episodes are counted (green arrow). Those occurring during wakefulness are not considered (black arrows). Automatic REM sleep deprivation method Mice were housed individually in a 30 cm Plexiglass diameter barrel (Figure 1B). Each barrel contained a platform resting on an air piston that is set in motion by an electromagnet [13]. After 3 days of habituation, mice were recorded during 24 hr of baseline and 48 hr of REM sleep deprivation. As automatic analysis of vigilance states is required during REM sleep deprivation, an algorithm file was computed for each mouse by manual scoring based on the 48 hr of baseline recordings. Then, during deprivation, when REM sleep was detected, a Transistor–Transistor Logic (TTL) signal was sent by the computer to an electromagnet to briefly move the platform up (25 ms, 2 cm of amplitude), once per second, until the mouse woke up. Between stimulations, the platform gently returned to its initial position thanks to gravity and the presence of an air spring to break the platform fall. Each of the stimulation that occurred during a REM sleep episode was automatically considered in the offline analysis (Figure 1C). Behavioral tests Mice were sibling groups housed in a ventilated chamber under a 12:12 hr light/dark cycle (80 lux; light on at 08:00 am). During the 7 days habituation period, mice were handled daily for 10 min by the experimenter. Thirty minutes before tests, mice were individually housed to familiarize to isolation conditions. The open-field and light-dark-box tests were conducted in this sequence, separated by 3 days of rest. All tests were performed between 2:00 pm and 5:00 pm. Mazes were cleaned with water after each trial. Open-field test The test was performed in a quiet dark room to assess animal coping to a nonanxiogenic novel environment using an open-field apparatus (50 × 50 × 45 cm; length × width × height) in which a 20 × 20 cm center space was numerically defined (Viewpoint, France). Two Orex-KO and two WT mice were recorded simultaneously for each trial. Each mouse was placed in the center of one of the four apparatus and locomotion was recorded for 45 min using a video-tracking system (Viewpoint, France). Time spent, distance traveled, and latency to enter the center of the arena were measured and used as indexes of anxiety. Light-dark box test The test evaluates mouse behavior when confronted to a novel and aversive environment. The apparatus used is a box (20 × 20 × 30 cm) equally divided into two compartments separated by a black wall containing a small open door (6 × 6 cm) in its center (Viewpoint, France). One compartment is brightly illuminated (250 lux), whereas the other compartment is completely dark. This test is based on competition between two spontaneous behaviors in mice: an innate aversion to brightly illuminated areas and a spontaneous inclination to explore a new environment. Mice were placed in the dark compartment with their back facing the open door and were video-tracked for 5 min. The total number of transitions between the two compartments, time spent in the brightly illuminated side and latency to the first entrance into the bright compartment, were recorded. Measure of plasma corticosterone level After 3 days of habituation in the deprivation chambers and 48 hr of experimentation (REM sleep deprivation with the platforms-over-water method or home cage condition), mice were immediately and deeply anaesthetized (sodium pentobarbital, 150 mg/kg ip; Ceva Santé Animale, Libourne, France). Blood samples (>500 µL) were collected from heart ventricles. Samples were spun immediately (5000 g for 5 min at 4°C) and 40 µL aliquots of plasma were stored at −20°C until use. Quantitative measurement of plasma corticosterone levels was assessed simultaneously on all samples using an enzyme-linked immunosorbent assay (ELISA) kit following manufacturer’s instructions (DetectX_Corticosterone Immunoassay kit; Arbor Assays, Ann Arbor, MI, USA). Experimental design and statistical analysis To align with the 3Rs, we minimized the number of mice per group (less than 15 per condition). After testing for normality using the Shapiro–Wilk test, we used nonparametric tests in our analyses when comparing two independent samples (Orex-KO versus WT mice). We thus used the Mann and Whitney test, with Holm’s correction when multiple comparisons were done. Tests were performed using Excell routines (www.anastats.fr). All results are expressed as mean ± sem. For all statistical procedures, significance was set at 0.05. In the first experiment, nine Orex-KO and nine WT littermates were REM sleep deprived using the small-platforms-over-water technique. We used the Mann and Whitney test with Holm’s correction to compare genotypes for quantities per hour of each vigilance state, number and mean duration of episodes per hour, latency to enter NREM and REM sleep after deprivation, number and duration of cataplexy episodes, and power spectra. To compare power spectra frequency and amplitude at peaks, we used the wilcoxon test for paired measures. In the second experiment, corticosterone measurements were performed on 18 Orex-KO and 17 WT mice. Mice from each genotype were separated into two groups (n = 8–9): a group REM sleep deprived with the small-platforms-over-water method and a control group. Four mice, one from each group per genotype, were tested in parallel. Note that 10 of these 35 mice also performed the open-field and light-dark box tests at least 2 weeks before the current protocol. Corticosterone levels for those mice were in the same range as those of the naive mice (p = 0.21). The four independent groups were analyzed using the Kruskal–Wallis test and groups were compared two by two using Dunn’s procedure. For the third set of experiments, 13 naïve Orex-KO and 13 WT mice without EEG/EMG implantation were tested in pairs (one WT and one Orex-KO) in the open-field and light-dark box tests. An additional seven WT and eight Orex-KO mice were tested in light-dark box immediately after a 48 hr of REM sleep deprivation using the platforms-over-water method in order to evaluate their anxiety level after REM sleep deprivation. Comparisons were made between genotypes for locomotion, duration of stay and number of transitions between compartments or zones, and latency to enter the bright light compartment, using the Mann and Whitney test. In the fourth experiment, seven Orex-KO and seven WT littermates underwent REM sleep deprivation using the automated method. Mice were recorded in pairs of one Orex-KO and one WT. Comparison of sleep quantities per hour, number and mean duration of episodes per hour, and number of stimulation per hour between independent samples (genotypes) were performed using Mann & Whitney test with Holm’s correction. Results Baseline recordings Quantitative analysis of sleep states and wakefulness assessed from simultaneous EEG/EMG polysomnography and video recordings is reported in Table 1. Briefly, Orex-KO and WT mice showed similar daily amounts of REM sleep, NREM sleep, and wakefulness as described previously [12] (Table 1). Table 1. Sleep Data during Baseline Period (with Video Recording) REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - Baseline 24 hrs  Total time (min) 78.7 ± 2.5 81.3 ± 3.5 590.7 ± 22.1 625.4 ± 16.1 770.5 ± 23.5 724.2 ± 16.8 8.2 ± 2.3  Number of bouts 85.4 ± 3.2 97.0 ± 7.6 355.4 ± 21.8 408.9 ± 35.5 357.9 ± 21.1 418.6 ± 35.9 7.1 ± 1.5  Mean Duration (sec) 55.5 ± 1.1 52.3 ± 2.8* 101.4 ± 3.5 97.6 ± 6.3 134.1 ± 9.1 109.9 ± 6.7 76.1 ± 18.8 Light Period  Total time (min) 57.9 ± 1.3 52.2 ± 2.0* 397.9 ± 9.9 401.8 ± 8.0 264.1 ± 10.1 265.2 ± 7.8 0.4 ± 0.3  Number of bouts 61.2 ± 1.4 62.7 ± 4.9 245.8 ± 11.6 236.9 ± 18.9 242.9 ± 11.8 235.1 ± 19.3 0.3 ± 0.2  Mean Duration (sec) 56.9 ± 0.7 52.7 ± 3.8* 98.5 ± 3.2 108.7 ± 7.5 67.3 ± 4.2 71.2 ± 4.4 68.8 ± 8.8 Dark Period  Total time (min) 20.8 ± 2.0 29.1 ± 2.4* 192.8 ± 18.0 223.5 ± 10.9 506.4 ± 19.5 459 ± 11.8* 7.8 ± 2.1  Number of bouts 24.2 ± 2.6 34.3 ± 3.5** 109.7 ± 12.9 172 ± 17.7** 115 ± 11.9 183.4 ± 17.9** 6.8 ± 1.3  Mean Duration (sec) 52.7 ± 2.4 52.2 ± 2.3 108.7 ± 5.2 82.8 ± 5.5** 299.8 ± 35.5 161.0 ± 11.0** 76.3 ± 18.9 REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - Baseline 24 hrs  Total time (min) 78.7 ± 2.5 81.3 ± 3.5 590.7 ± 22.1 625.4 ± 16.1 770.5 ± 23.5 724.2 ± 16.8 8.2 ± 2.3  Number of bouts 85.4 ± 3.2 97.0 ± 7.6 355.4 ± 21.8 408.9 ± 35.5 357.9 ± 21.1 418.6 ± 35.9 7.1 ± 1.5  Mean Duration (sec) 55.5 ± 1.1 52.3 ± 2.8* 101.4 ± 3.5 97.6 ± 6.3 134.1 ± 9.1 109.9 ± 6.7 76.1 ± 18.8 Light Period  Total time (min) 57.9 ± 1.3 52.2 ± 2.0* 397.9 ± 9.9 401.8 ± 8.0 264.1 ± 10.1 265.2 ± 7.8 0.4 ± 0.3  Number of bouts 61.2 ± 1.4 62.7 ± 4.9 245.8 ± 11.6 236.9 ± 18.9 242.9 ± 11.8 235.1 ± 19.3 0.3 ± 0.2  Mean Duration (sec) 56.9 ± 0.7 52.7 ± 3.8* 98.5 ± 3.2 108.7 ± 7.5 67.3 ± 4.2 71.2 ± 4.4 68.8 ± 8.8 Dark Period  Total time (min) 20.8 ± 2.0 29.1 ± 2.4* 192.8 ± 18.0 223.5 ± 10.9 506.4 ± 19.5 459 ± 11.8* 7.8 ± 2.1  Number of bouts 24.2 ± 2.6 34.3 ± 3.5** 109.7 ± 12.9 172 ± 17.7** 115 ± 11.9 183.4 ± 17.9** 6.8 ± 1.3  Mean Duration (sec) 52.7 ± 2.4 52.2 ± 2.3 108.7 ± 5.2 82.8 ± 5.5** 299.8 ± 35.5 161.0 ± 11.0** 76.3 ± 18.9 *P < 0.05 and ** P < 0.01 Orex-KO (-/-) vs. WT (+/+). View Large Table 1. Sleep Data during Baseline Period (with Video Recording) REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - Baseline 24 hrs  Total time (min) 78.7 ± 2.5 81.3 ± 3.5 590.7 ± 22.1 625.4 ± 16.1 770.5 ± 23.5 724.2 ± 16.8 8.2 ± 2.3  Number of bouts 85.4 ± 3.2 97.0 ± 7.6 355.4 ± 21.8 408.9 ± 35.5 357.9 ± 21.1 418.6 ± 35.9 7.1 ± 1.5  Mean Duration (sec) 55.5 ± 1.1 52.3 ± 2.8* 101.4 ± 3.5 97.6 ± 6.3 134.1 ± 9.1 109.9 ± 6.7 76.1 ± 18.8 Light Period  Total time (min) 57.9 ± 1.3 52.2 ± 2.0* 397.9 ± 9.9 401.8 ± 8.0 264.1 ± 10.1 265.2 ± 7.8 0.4 ± 0.3  Number of bouts 61.2 ± 1.4 62.7 ± 4.9 245.8 ± 11.6 236.9 ± 18.9 242.9 ± 11.8 235.1 ± 19.3 0.3 ± 0.2  Mean Duration (sec) 56.9 ± 0.7 52.7 ± 3.8* 98.5 ± 3.2 108.7 ± 7.5 67.3 ± 4.2 71.2 ± 4.4 68.8 ± 8.8 Dark Period  Total time (min) 20.8 ± 2.0 29.1 ± 2.4* 192.8 ± 18.0 223.5 ± 10.9 506.4 ± 19.5 459 ± 11.8* 7.8 ± 2.1  Number of bouts 24.2 ± 2.6 34.3 ± 3.5** 109.7 ± 12.9 172 ± 17.7** 115 ± 11.9 183.4 ± 17.9** 6.8 ± 1.3  Mean Duration (sec) 52.7 ± 2.4 52.2 ± 2.3 108.7 ± 5.2 82.8 ± 5.5** 299.8 ± 35.5 161.0 ± 11.0** 76.3 ± 18.9 REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - Baseline 24 hrs  Total time (min) 78.7 ± 2.5 81.3 ± 3.5 590.7 ± 22.1 625.4 ± 16.1 770.5 ± 23.5 724.2 ± 16.8 8.2 ± 2.3  Number of bouts 85.4 ± 3.2 97.0 ± 7.6 355.4 ± 21.8 408.9 ± 35.5 357.9 ± 21.1 418.6 ± 35.9 7.1 ± 1.5  Mean Duration (sec) 55.5 ± 1.1 52.3 ± 2.8* 101.4 ± 3.5 97.6 ± 6.3 134.1 ± 9.1 109.9 ± 6.7 76.1 ± 18.8 Light Period  Total time (min) 57.9 ± 1.3 52.2 ± 2.0* 397.9 ± 9.9 401.8 ± 8.0 264.1 ± 10.1 265.2 ± 7.8 0.4 ± 0.3  Number of bouts 61.2 ± 1.4 62.7 ± 4.9 245.8 ± 11.6 236.9 ± 18.9 242.9 ± 11.8 235.1 ± 19.3 0.3 ± 0.2  Mean Duration (sec) 56.9 ± 0.7 52.7 ± 3.8* 98.5 ± 3.2 108.7 ± 7.5 67.3 ± 4.2 71.2 ± 4.4 68.8 ± 8.8 Dark Period  Total time (min) 20.8 ± 2.0 29.1 ± 2.4* 192.8 ± 18.0 223.5 ± 10.9 506.4 ± 19.5 459 ± 11.8* 7.8 ± 2.1  Number of bouts 24.2 ± 2.6 34.3 ± 3.5** 109.7 ± 12.9 172 ± 17.7** 115 ± 11.9 183.4 ± 17.9** 6.8 ± 1.3  Mean Duration (sec) 52.7 ± 2.4 52.2 ± 2.3 108.7 ± 5.2 82.8 ± 5.5** 299.8 ± 35.5 161.0 ± 11.0** 76.3 ± 18.9 *P < 0.05 and ** P < 0.01 Orex-KO (-/-) vs. WT (+/+). View Large In the light phase, the so-called resting phase, sleep parameters were similar between genotypes except for REM sleep where total time and mean bouts duration were significantly shorter in Orex-KO mice (p = 0.03, p = 0.04, respectively) (Table 1). In the dark (active) phase, however, the number of REM sleep bouts was increased in Orex-KO mice resulting in a higher REM sleep quantity and a reduced circadian distribution of REM sleep (circadian index of 0.29 ± 0.04 vs 0.48 ± 0.29 in WT mice; p = 0.0096). NREM sleep and wake quantities were unchanged in Orex-KO mice despite a strong fragmentation (increased number of shorter NREM sleep and wake bouts) (Table 1). We observed cataplexy episodes in Orex-KO mice only (Table 1). They mostly occurred during the dark phase with a few observed around light transitions. As cataplexy occurs during wakefulness, it contributed to the fragmentation of this latter state. Challenging REM sleep regulation using the small-platforms-over water method In agreement with previous data [4], deprivation of REM sleep was efficient (77 ± 5.5% and 68.9 ± 3.8% REM sleep loss over 48 hr in Orex-KO and WT, respectively) (Table 2). The amount of REM sleep lost during the 48 hr of deprivation was similar between genotypes (118.2 ± 8.9 min in Orex-KO vs. 121.3 ± 10.3 min in WT mice, p = 0.35) (Figure 2; Table 2), indicating that REM sleep was suppressed with the same efficacy in all mice. Using this method, NREM sleep and wakefulness were fragmented with more bouts of shorter duration in both genotypes (Table 2). Table 2. Sleep Data during and after REM sleep deprivation with small-platforms-over water method REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - SMALL-PLATFORM-OVER-WATER DEPRIVATION  48 hrs   Total time (min) 35.9 ± 8.6 49.0 ± 4.8 932.7 ± 80.6 1048.1 ± 79.0 1891.2 ± 89.7 1781.8 ± 83.3   Number of bouts 110.9 ± 21.6 208.7 ± 19.7** 1167.6 ± 99.2 1594.4 ± 90.6** 1185.2 ± 97.7 1608.9 ± 87.8**   Mean Duration (sec) 17.7 ± 1.8 14.3 ± 1.0 48.4 ± 3.2 39.6 ± 2.6* 102.7 ± 11.8 68.9 ± 6.5*  1st 24hrs   Total time (min) 16.2 ± 4.1 22.6 ± 3.1 429.1 ± 56.2 570.1 ± 34.8* 994.6 ± 58.4 846.5 ± 37.1*   Number of bouts 43.3 ± 11.0 96.9 ± 14.0** 526.6 ± 85.1 793.0 ± 49.2** 535.4 ± 83.7 798.4 ± 48.8**   Mean Duration (sec) 19.0 ± 3.5 14.3 ± 1.4 50.6 ± 4.0 44.0 ± 3.4 147.1 ± 33.1 65.7 ± 5.3**  2nd 24hrs   Total time (min) 19.7 ± 5.0 26.4 ± 2.6 503.6 ± 35.6 478.0 ± 51.4 896.6 ± 41.1 935.2 ± 52.9   Number of bouts 67.6 ± 12.5 111.8 ± 11.3** 641 ± 32.8 801.4 ± 65.8* 649.8 ± 32.3 810.4 ± 63.7*   Mean Duration (sec) 16.2 ± 1.9 14.7 ± 1.4 47.7 ± 3.6 35.6 ± 2.4** 84.9 ± 6.2 76.5 ± 12.6  Light Period   Total time (min) 24.0 ± 4.9 29.8 ± 3.6 556.2 ± 46.5 592.6 ± 39.7 859.6 ± 50.4 817.3 ± 42.0   Number of bouts 74.0 ± 13.3 117.7 ± 10.3** 679.1 ± 53 816.7 ± 49.7 685.1 ± 52.0 820.6 ± 48.9   Mean Duration (sec) 19.0 ± 2.1 15.0 ± 1.2 49.7 ± 3.5 43.9 ± 2.8 80.6 ± 9.7 62.4 ± 6.7  Dark Period   Total time (min) 12.9 ± 4.4 20.7 ± 3.4 418.6 ± 42.1 497.4 ± 48.8 1108.3 ± 49.2 1041.0 ± 51.2   Number of bouts 41.0 ± 10.2 99.0 ± 17.1** 538.4 ± 59.1 851.0 ± 72.9** 550.9 ± 58 862.4 ± 71.5**   Mean Duration (sec) 14.6 ± 2.6 12.3 ± 1.1 47.2 ± 2.8 35.4 ± 2.5** 133.9 ± 17.6 78.1 ± 9.4** RECOVERY  10 hrs   Total time (min) 60.2 ± 3.5 59.9 ± 2.6 230.6 ± 15.0 208.4 ± 11.1 309.2 ± 17.0 322.0 ± 10.6 9.7 ± 2.9   Number of bouts 44.2 ± 3.6 43.7 ± 2.9 126.4 ± 16.9 121.1 ± 13.7 131.6 ± 16.1 131.4 ± 13.7 5.3 ± 1.6   Mean Duration (sec) 83.4 ± 4.2 84.1 ± 4.4 118.4 ± 9.5 113.1 ± 15.0 162.7 ± 24.8 160.0 ± 16.2 101.5 ± 14.0   Latency (min) 123.8 ± 16.3 85.7 ± 7.1 104.7 ± 14.1 66.8 ± 6.8 0 0 55.3 ± 26.6  Rebound 150 min   Total time (min) 22.4 ± 1.5 20.8 ± 1.2 74.7 ± 6.1 57.5 ± 5.2* 53.0 ± 6.9 70.1 ± 5.0* 1.6 ± 0.9   Number of bouts 15.7 ± 0.9 15.0 ± 1.0 39.9 ± 5.9 37.4 ± 3.7 40.4 ± 5.5 39.4 ± 4.0 1.1 ± 0.6   Mean Duration (sec) 87.1 ± 7.1 85.1 ± 5.4 125.1 ± 12.7 103.4 ± 19.4 97.0 ± 20.5 112.8 ± 10.2 74.4 ± 9.8  Rebound 360 min   Total time (min) 44.8 ± 2.1 42.2 ± 2.4 164.4 ± 10.1 133.4 ± 11.5* 150.8 ± 10.5 180.1 ± 12.1 4.2 ± 1.6   Number of bouts 32.2 ± 2.2 32.0 ± 2.5 88.6 ± 11.3 80.9 ± 9.4 91.1 ± 10.4 86.3 ± 9.4 2.8 ± 1.1   Mean Duration (sec) 85.0 ± 4.5 81.3 ± 4.7 120.6 ± 10.3 108.5 ± 16.0 112.9 ± 16.8 140.6 ± 19.4 102.3 ± 23.3 REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - SMALL-PLATFORM-OVER-WATER DEPRIVATION  48 hrs   Total time (min) 35.9 ± 8.6 49.0 ± 4.8 932.7 ± 80.6 1048.1 ± 79.0 1891.2 ± 89.7 1781.8 ± 83.3   Number of bouts 110.9 ± 21.6 208.7 ± 19.7** 1167.6 ± 99.2 1594.4 ± 90.6** 1185.2 ± 97.7 1608.9 ± 87.8**   Mean Duration (sec) 17.7 ± 1.8 14.3 ± 1.0 48.4 ± 3.2 39.6 ± 2.6* 102.7 ± 11.8 68.9 ± 6.5*  1st 24hrs   Total time (min) 16.2 ± 4.1 22.6 ± 3.1 429.1 ± 56.2 570.1 ± 34.8* 994.6 ± 58.4 846.5 ± 37.1*   Number of bouts 43.3 ± 11.0 96.9 ± 14.0** 526.6 ± 85.1 793.0 ± 49.2** 535.4 ± 83.7 798.4 ± 48.8**   Mean Duration (sec) 19.0 ± 3.5 14.3 ± 1.4 50.6 ± 4.0 44.0 ± 3.4 147.1 ± 33.1 65.7 ± 5.3**  2nd 24hrs   Total time (min) 19.7 ± 5.0 26.4 ± 2.6 503.6 ± 35.6 478.0 ± 51.4 896.6 ± 41.1 935.2 ± 52.9   Number of bouts 67.6 ± 12.5 111.8 ± 11.3** 641 ± 32.8 801.4 ± 65.8* 649.8 ± 32.3 810.4 ± 63.7*   Mean Duration (sec) 16.2 ± 1.9 14.7 ± 1.4 47.7 ± 3.6 35.6 ± 2.4** 84.9 ± 6.2 76.5 ± 12.6  Light Period   Total time (min) 24.0 ± 4.9 29.8 ± 3.6 556.2 ± 46.5 592.6 ± 39.7 859.6 ± 50.4 817.3 ± 42.0   Number of bouts 74.0 ± 13.3 117.7 ± 10.3** 679.1 ± 53 816.7 ± 49.7 685.1 ± 52.0 820.6 ± 48.9   Mean Duration (sec) 19.0 ± 2.1 15.0 ± 1.2 49.7 ± 3.5 43.9 ± 2.8 80.6 ± 9.7 62.4 ± 6.7  Dark Period   Total time (min) 12.9 ± 4.4 20.7 ± 3.4 418.6 ± 42.1 497.4 ± 48.8 1108.3 ± 49.2 1041.0 ± 51.2   Number of bouts 41.0 ± 10.2 99.0 ± 17.1** 538.4 ± 59.1 851.0 ± 72.9** 550.9 ± 58 862.4 ± 71.5**   Mean Duration (sec) 14.6 ± 2.6 12.3 ± 1.1 47.2 ± 2.8 35.4 ± 2.5** 133.9 ± 17.6 78.1 ± 9.4** RECOVERY  10 hrs   Total time (min) 60.2 ± 3.5 59.9 ± 2.6 230.6 ± 15.0 208.4 ± 11.1 309.2 ± 17.0 322.0 ± 10.6 9.7 ± 2.9   Number of bouts 44.2 ± 3.6 43.7 ± 2.9 126.4 ± 16.9 121.1 ± 13.7 131.6 ± 16.1 131.4 ± 13.7 5.3 ± 1.6   Mean Duration (sec) 83.4 ± 4.2 84.1 ± 4.4 118.4 ± 9.5 113.1 ± 15.0 162.7 ± 24.8 160.0 ± 16.2 101.5 ± 14.0   Latency (min) 123.8 ± 16.3 85.7 ± 7.1 104.7 ± 14.1 66.8 ± 6.8 0 0 55.3 ± 26.6  Rebound 150 min   Total time (min) 22.4 ± 1.5 20.8 ± 1.2 74.7 ± 6.1 57.5 ± 5.2* 53.0 ± 6.9 70.1 ± 5.0* 1.6 ± 0.9   Number of bouts 15.7 ± 0.9 15.0 ± 1.0 39.9 ± 5.9 37.4 ± 3.7 40.4 ± 5.5 39.4 ± 4.0 1.1 ± 0.6   Mean Duration (sec) 87.1 ± 7.1 85.1 ± 5.4 125.1 ± 12.7 103.4 ± 19.4 97.0 ± 20.5 112.8 ± 10.2 74.4 ± 9.8  Rebound 360 min   Total time (min) 44.8 ± 2.1 42.2 ± 2.4 164.4 ± 10.1 133.4 ± 11.5* 150.8 ± 10.5 180.1 ± 12.1 4.2 ± 1.6   Number of bouts 32.2 ± 2.2 32.0 ± 2.5 88.6 ± 11.3 80.9 ± 9.4 91.1 ± 10.4 86.3 ± 9.4 2.8 ± 1.1   Mean Duration (sec) 85.0 ± 4.5 81.3 ± 4.7 120.6 ± 10.3 108.5 ± 16.0 112.9 ± 16.8 140.6 ± 19.4 102.3 ± 23.3 *P<0.05 and ** P<0.01 Orex-KO (-/-) vs. WT (+/+). View Large Table 2. Sleep Data during and after REM sleep deprivation with small-platforms-over water method REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - SMALL-PLATFORM-OVER-WATER DEPRIVATION  48 hrs   Total time (min) 35.9 ± 8.6 49.0 ± 4.8 932.7 ± 80.6 1048.1 ± 79.0 1891.2 ± 89.7 1781.8 ± 83.3   Number of bouts 110.9 ± 21.6 208.7 ± 19.7** 1167.6 ± 99.2 1594.4 ± 90.6** 1185.2 ± 97.7 1608.9 ± 87.8**   Mean Duration (sec) 17.7 ± 1.8 14.3 ± 1.0 48.4 ± 3.2 39.6 ± 2.6* 102.7 ± 11.8 68.9 ± 6.5*  1st 24hrs   Total time (min) 16.2 ± 4.1 22.6 ± 3.1 429.1 ± 56.2 570.1 ± 34.8* 994.6 ± 58.4 846.5 ± 37.1*   Number of bouts 43.3 ± 11.0 96.9 ± 14.0** 526.6 ± 85.1 793.0 ± 49.2** 535.4 ± 83.7 798.4 ± 48.8**   Mean Duration (sec) 19.0 ± 3.5 14.3 ± 1.4 50.6 ± 4.0 44.0 ± 3.4 147.1 ± 33.1 65.7 ± 5.3**  2nd 24hrs   Total time (min) 19.7 ± 5.0 26.4 ± 2.6 503.6 ± 35.6 478.0 ± 51.4 896.6 ± 41.1 935.2 ± 52.9   Number of bouts 67.6 ± 12.5 111.8 ± 11.3** 641 ± 32.8 801.4 ± 65.8* 649.8 ± 32.3 810.4 ± 63.7*   Mean Duration (sec) 16.2 ± 1.9 14.7 ± 1.4 47.7 ± 3.6 35.6 ± 2.4** 84.9 ± 6.2 76.5 ± 12.6  Light Period   Total time (min) 24.0 ± 4.9 29.8 ± 3.6 556.2 ± 46.5 592.6 ± 39.7 859.6 ± 50.4 817.3 ± 42.0   Number of bouts 74.0 ± 13.3 117.7 ± 10.3** 679.1 ± 53 816.7 ± 49.7 685.1 ± 52.0 820.6 ± 48.9   Mean Duration (sec) 19.0 ± 2.1 15.0 ± 1.2 49.7 ± 3.5 43.9 ± 2.8 80.6 ± 9.7 62.4 ± 6.7  Dark Period   Total time (min) 12.9 ± 4.4 20.7 ± 3.4 418.6 ± 42.1 497.4 ± 48.8 1108.3 ± 49.2 1041.0 ± 51.2   Number of bouts 41.0 ± 10.2 99.0 ± 17.1** 538.4 ± 59.1 851.0 ± 72.9** 550.9 ± 58 862.4 ± 71.5**   Mean Duration (sec) 14.6 ± 2.6 12.3 ± 1.1 47.2 ± 2.8 35.4 ± 2.5** 133.9 ± 17.6 78.1 ± 9.4** RECOVERY  10 hrs   Total time (min) 60.2 ± 3.5 59.9 ± 2.6 230.6 ± 15.0 208.4 ± 11.1 309.2 ± 17.0 322.0 ± 10.6 9.7 ± 2.9   Number of bouts 44.2 ± 3.6 43.7 ± 2.9 126.4 ± 16.9 121.1 ± 13.7 131.6 ± 16.1 131.4 ± 13.7 5.3 ± 1.6   Mean Duration (sec) 83.4 ± 4.2 84.1 ± 4.4 118.4 ± 9.5 113.1 ± 15.0 162.7 ± 24.8 160.0 ± 16.2 101.5 ± 14.0   Latency (min) 123.8 ± 16.3 85.7 ± 7.1 104.7 ± 14.1 66.8 ± 6.8 0 0 55.3 ± 26.6  Rebound 150 min   Total time (min) 22.4 ± 1.5 20.8 ± 1.2 74.7 ± 6.1 57.5 ± 5.2* 53.0 ± 6.9 70.1 ± 5.0* 1.6 ± 0.9   Number of bouts 15.7 ± 0.9 15.0 ± 1.0 39.9 ± 5.9 37.4 ± 3.7 40.4 ± 5.5 39.4 ± 4.0 1.1 ± 0.6   Mean Duration (sec) 87.1 ± 7.1 85.1 ± 5.4 125.1 ± 12.7 103.4 ± 19.4 97.0 ± 20.5 112.8 ± 10.2 74.4 ± 9.8  Rebound 360 min   Total time (min) 44.8 ± 2.1 42.2 ± 2.4 164.4 ± 10.1 133.4 ± 11.5* 150.8 ± 10.5 180.1 ± 12.1 4.2 ± 1.6   Number of bouts 32.2 ± 2.2 32.0 ± 2.5 88.6 ± 11.3 80.9 ± 9.4 91.1 ± 10.4 86.3 ± 9.4 2.8 ± 1.1   Mean Duration (sec) 85.0 ± 4.5 81.3 ± 4.7 120.6 ± 10.3 108.5 ± 16.0 112.9 ± 16.8 140.6 ± 19.4 102.3 ± 23.3 REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - SMALL-PLATFORM-OVER-WATER DEPRIVATION  48 hrs   Total time (min) 35.9 ± 8.6 49.0 ± 4.8 932.7 ± 80.6 1048.1 ± 79.0 1891.2 ± 89.7 1781.8 ± 83.3   Number of bouts 110.9 ± 21.6 208.7 ± 19.7** 1167.6 ± 99.2 1594.4 ± 90.6** 1185.2 ± 97.7 1608.9 ± 87.8**   Mean Duration (sec) 17.7 ± 1.8 14.3 ± 1.0 48.4 ± 3.2 39.6 ± 2.6* 102.7 ± 11.8 68.9 ± 6.5*  1st 24hrs   Total time (min) 16.2 ± 4.1 22.6 ± 3.1 429.1 ± 56.2 570.1 ± 34.8* 994.6 ± 58.4 846.5 ± 37.1*   Number of bouts 43.3 ± 11.0 96.9 ± 14.0** 526.6 ± 85.1 793.0 ± 49.2** 535.4 ± 83.7 798.4 ± 48.8**   Mean Duration (sec) 19.0 ± 3.5 14.3 ± 1.4 50.6 ± 4.0 44.0 ± 3.4 147.1 ± 33.1 65.7 ± 5.3**  2nd 24hrs   Total time (min) 19.7 ± 5.0 26.4 ± 2.6 503.6 ± 35.6 478.0 ± 51.4 896.6 ± 41.1 935.2 ± 52.9   Number of bouts 67.6 ± 12.5 111.8 ± 11.3** 641 ± 32.8 801.4 ± 65.8* 649.8 ± 32.3 810.4 ± 63.7*   Mean Duration (sec) 16.2 ± 1.9 14.7 ± 1.4 47.7 ± 3.6 35.6 ± 2.4** 84.9 ± 6.2 76.5 ± 12.6  Light Period   Total time (min) 24.0 ± 4.9 29.8 ± 3.6 556.2 ± 46.5 592.6 ± 39.7 859.6 ± 50.4 817.3 ± 42.0   Number of bouts 74.0 ± 13.3 117.7 ± 10.3** 679.1 ± 53 816.7 ± 49.7 685.1 ± 52.0 820.6 ± 48.9   Mean Duration (sec) 19.0 ± 2.1 15.0 ± 1.2 49.7 ± 3.5 43.9 ± 2.8 80.6 ± 9.7 62.4 ± 6.7  Dark Period   Total time (min) 12.9 ± 4.4 20.7 ± 3.4 418.6 ± 42.1 497.4 ± 48.8 1108.3 ± 49.2 1041.0 ± 51.2   Number of bouts 41.0 ± 10.2 99.0 ± 17.1** 538.4 ± 59.1 851.0 ± 72.9** 550.9 ± 58 862.4 ± 71.5**   Mean Duration (sec) 14.6 ± 2.6 12.3 ± 1.1 47.2 ± 2.8 35.4 ± 2.5** 133.9 ± 17.6 78.1 ± 9.4** RECOVERY  10 hrs   Total time (min) 60.2 ± 3.5 59.9 ± 2.6 230.6 ± 15.0 208.4 ± 11.1 309.2 ± 17.0 322.0 ± 10.6 9.7 ± 2.9   Number of bouts 44.2 ± 3.6 43.7 ± 2.9 126.4 ± 16.9 121.1 ± 13.7 131.6 ± 16.1 131.4 ± 13.7 5.3 ± 1.6   Mean Duration (sec) 83.4 ± 4.2 84.1 ± 4.4 118.4 ± 9.5 113.1 ± 15.0 162.7 ± 24.8 160.0 ± 16.2 101.5 ± 14.0   Latency (min) 123.8 ± 16.3 85.7 ± 7.1 104.7 ± 14.1 66.8 ± 6.8 0 0 55.3 ± 26.6  Rebound 150 min   Total time (min) 22.4 ± 1.5 20.8 ± 1.2 74.7 ± 6.1 57.5 ± 5.2* 53.0 ± 6.9 70.1 ± 5.0* 1.6 ± 0.9   Number of bouts 15.7 ± 0.9 15.0 ± 1.0 39.9 ± 5.9 37.4 ± 3.7 40.4 ± 5.5 39.4 ± 4.0 1.1 ± 0.6   Mean Duration (sec) 87.1 ± 7.1 85.1 ± 5.4 125.1 ± 12.7 103.4 ± 19.4 97.0 ± 20.5 112.8 ± 10.2 74.4 ± 9.8  Rebound 360 min   Total time (min) 44.8 ± 2.1 42.2 ± 2.4 164.4 ± 10.1 133.4 ± 11.5* 150.8 ± 10.5 180.1 ± 12.1 4.2 ± 1.6   Number of bouts 32.2 ± 2.2 32.0 ± 2.5 88.6 ± 11.3 80.9 ± 9.4 91.1 ± 10.4 86.3 ± 9.4 2.8 ± 1.1   Mean Duration (sec) 85.0 ± 4.5 81.3 ± 4.7 120.6 ± 10.3 108.5 ± 16.0 112.9 ± 16.8 140.6 ± 19.4 102.3 ± 23.3 *P<0.05 and ** P<0.01 Orex-KO (-/-) vs. WT (+/+). View Large Figure 2. View largeDownload slide REM sleep debt accumulates similarly in WT and Orex-KO mice. Graph illustrating the cumulative amount of REM sleep compared with baseline amounts all along the REM sleep deprivation using the small-platforms-over-water method and the 8 following hours of recovery period, in Orex-KO and WT mice. The inclination of the curves indicates a loss of REM sleep during deprivation and a gain of REM sleep during recovery. The gray shaded areas represent the dark phase. The red line indicates the time when animals were transferred to the recovery chamber to sleep ad libitum. Figure 2. View largeDownload slide REM sleep debt accumulates similarly in WT and Orex-KO mice. Graph illustrating the cumulative amount of REM sleep compared with baseline amounts all along the REM sleep deprivation using the small-platforms-over-water method and the 8 following hours of recovery period, in Orex-KO and WT mice. The inclination of the curves indicates a loss of REM sleep during deprivation and a gain of REM sleep during recovery. The gray shaded areas represent the dark phase. The red line indicates the time when animals were transferred to the recovery chamber to sleep ad libitum. Recovery from REM sleep deprivation was recorded for the following 10 hr (ZT2-ZT12) until lights-off. As previously reported [4], WT mice spent 104.7 ± 14.1 min grooming, moving, and digging before entering sleep after the end of REM sleep deprivation using this deprivation method. Interestingly, REM sleep latency, calculated from the end of deprivation to the first episode of REM sleep, was much shorter in Orex-KO mice than in WT mice (Table 2; Figure 3B). However, REM sleep latency calculated from the start of the first NREM sleep episode to the first REM sleep bout was the same in Orex-KO and WT mice (18.9 ± 4.4 min vs. 19.1 ± 5.2 min, respectively; p = 0.08). Figure 3. View largeDownload slide REM sleep rebound after small-platforms-over-water REM sleep deprivation. Illustrations of sleep parameters during the recovery period in Orex-KO (blue) and WT (green) mice after 48 hr of REM sleep deprivation using the small-platforms-over-water method. (A) Graph representing the percentage of REM sleep per hour during the recovery period starting at 10AM (ZT2). (B) Bar chart representing the mean REM sleep latency in Orex-KO and WT. It is calculated as the time spent between the end of REM sleep deprivation and the occurrence of the first REM sleep episode. (C) and (D) Graphs illustrating the profile of REM sleep rebound reported as the percentage of REM sleep per hour in WT (C) and Orex-KO (D) mice when it is set to start from the first episode of REM sleep. (E) REM sleep power spectra during the first 6 hr of recovery (red line) and its corresponding baseline (black line). The power spectra being identical in WT and Orex-KO mice, and there were merged to show differences between conditions. (F) NREM sleep power spectra from the pool of WT and Orex-KO mice during the first 2 hr of recovery (red line) and its corresponding baseline (black line). (G) and (H) Graph illustrating the percentage per hour of NREM (G) and wakefulness (H) during the 10 hr of recovery period. *p < 0.05; ***p < 0.001. Figure 3. View largeDownload slide REM sleep rebound after small-platforms-over-water REM sleep deprivation. Illustrations of sleep parameters during the recovery period in Orex-KO (blue) and WT (green) mice after 48 hr of REM sleep deprivation using the small-platforms-over-water method. (A) Graph representing the percentage of REM sleep per hour during the recovery period starting at 10AM (ZT2). (B) Bar chart representing the mean REM sleep latency in Orex-KO and WT. It is calculated as the time spent between the end of REM sleep deprivation and the occurrence of the first REM sleep episode. (C) and (D) Graphs illustrating the profile of REM sleep rebound reported as the percentage of REM sleep per hour in WT (C) and Orex-KO (D) mice when it is set to start from the first episode of REM sleep. (E) REM sleep power spectra during the first 6 hr of recovery (red line) and its corresponding baseline (black line). The power spectra being identical in WT and Orex-KO mice, and there were merged to show differences between conditions. (F) NREM sleep power spectra from the pool of WT and Orex-KO mice during the first 2 hr of recovery (red line) and its corresponding baseline (black line). (G) and (H) Graph illustrating the percentage per hour of NREM (G) and wakefulness (H) during the 10 hr of recovery period. *p < 0.05; ***p < 0.001. Furthermore, the overall amount of REM sleep recovered was the same in both genotypes (p = 0.48) following a similar profile between genotypes (Figures 2 and 3A). When REM sleep is evaluated setting analysis from the first REM sleep episode of the recovery period to overcome the difference in REM sleep latency, WT and Orex-KO mice showed, respectively, a REM sleep hypersomnia of +46.6 ± 6.3% (p = 1.5E-3) and +63.15 ± 10.6% (p = 1.7E-4) compared to their corresponding baseline (Figure 3C and D). REM sleep rebounds followed a different profile with a lower peak of REM sleep hypersomnia in Orex-KO mice (17.0 ± 1.4% vs. 21.7 ± 1.8% in WT; p = 0.028) that lasted longer (2 vs. 1 hr in WT mice), possibly due to the difference in sleep latency and thus its occurrence at different circadian time. The REM sleep power spectrum was identical between Orex-KO and WT mice during the first 6 hr of the recovery period as well as during the corresponding baseline. However, the frequency at the peak was faster during recovery than baseline (Figure 3E). We also looked at the NREM sleep power spectrum during the first 2 and 6 hr of recovery and found no significant difference in peak frequency or amplitude between genotypes or conditions (baseline vs. recovery) (Figure 3F). Altogether, these data indicate that REM sleep homeostatic regulation is intact in narcoleptic Orex-KO mice. To our surprise, we also found that the REM sleep deprivation protocol induced an important peak of cataplexy in the first hour of the recovery period (4.7 ± 1.7% of total time at ZT2), at a time of day when cataplexy does not occur in baseline condition (Figure 4). These induced episodes were seen in all Orex-KO mice with a maximal amount per hour reaching 2.8 ± 1.0 min (vs. 1.5 ± 0.7 min during the dark phase in baseline, p = 0.23) (Figure 4C). The mean duration of induced cataplexy episodes (Figure 4C) was not significantly different to the mean duration of cataplexy episodes observed in baseline (p = 0.09) (Tables 1 and 2). They occurred with a latency of 55.3 ± 26.6 min, foreseeing the first episode of sleep and contributing to the fragmentation of wakefulness (Table 2). Note that cataplexy episodes were seen during the first hour of recovery when mice are active with grooming and digging. After this first hour, mice were less active as seen on the video recordings, entered sleep states as observed by polysomnography starting REM sleep rebound and stopping cataplexy (Figure 4). Figure 4. View largeDownload slide Cataplexy is induced in the first hour of recovery after REM sleep deprivation with small-platforms-over-water method. (A–C) Graphs representing the amount (A), the number (B), and the mean bouts duration (C) of cataplexy episodes per hour during the recovery period following 48 hr of REM sleep deprivation using the small-platforms-over-water method. (D) Illustration of polysomnographic signals (EEG and EMG) during cataplexy in Orex-KO mice. *p < 0.05. Figure 4. View largeDownload slide Cataplexy is induced in the first hour of recovery after REM sleep deprivation with small-platforms-over-water method. (A–C) Graphs representing the amount (A), the number (B), and the mean bouts duration (C) of cataplexy episodes per hour during the recovery period following 48 hr of REM sleep deprivation using the small-platforms-over-water method. (D) Illustration of polysomnographic signals (EEG and EMG) during cataplexy in Orex-KO mice. *p < 0.05. Evaluation of anxiety levels in Orex-KO narcoleptic and WT mice The shortened sleep latency we observed after small-platforms-over-water REM sleep deprivation in Orex-KO mice may reflect a stronger sleep drive or a better coping with the mild chronic stress caused by the environmental challenges of the procedure. To determine the potential contribution of stress as a confounding factor, we measured plasma corticosterone levels after 48 hr of REM sleep deprivation or home-cage control conditions for both genotypes. Orex-KO and WT mice had similar plasma corticosterone levels in control condition (31.0 ± 7.2 and 39.7 ± 12.3 ng/mL, respectively; p = 0.46). Both genotypes showed a significant increase in corticosterone levels after small-platforms-over-water REM sleep deprivation compared with control condition (Figure 5A). However, there was a tendency towards a reduced increase in plasmatic corticosterone level in Orex-KO mice (WT: 121.7 ± 14.9 and Orex-KO: 92.2 ± 6.8 ng/mL; p = 0.055), although it is not significant. To verify whether this observed tendency was physiologically relevant, we performed behavioral tests to explore this further. Figure 5. View largeDownload slide Level of anxiety and mild-stress coping are similar between narcoleptic and WT mice. (A) The level of plasmatic corticosterone is increased in WT and Orex-KO mice after REM sleep deprivation using the small-platforms-over-water method. (B) Orex-KO and WT mice behavior in the open-field apparatus presented over the 45 min of the test (B1, B3, B5) or by bins of 5 min (B2, B4, B6). WT mice have more locomotor activity in the open-field than Orex-KO mice all along the test (B1 and B2). Mice of both genotypes decreased their exploration activity between the first and the last 5 min of the test (KO: −51.6 ± 5.4% of LMA, WT: −52.4 ± 2.9%; p = 0.4). Mice of both genotypes spent the same amount of time in the center part of the open-field (B3 and B4). They similarly entered and exit the center part of the apparatus (B5 and B6). (C and D) Orex-KO and WT mice behavior during the 5 min light-dark box test in basal condition (C) and after REM sleep deprivation using the small-platforms-over-water method (D). Bar charts represent the total traveled distance (C-1 and D-1), the latency to first entrance into the light compartment (C-2 and D-2), the time spent in the light compartment (C-3 and D-3), and the number of transition in and out the light compartment (C-4 and D-4). **p < 0.01; ***p < 0.001. Figure 5. View largeDownload slide Level of anxiety and mild-stress coping are similar between narcoleptic and WT mice. (A) The level of plasmatic corticosterone is increased in WT and Orex-KO mice after REM sleep deprivation using the small-platforms-over-water method. (B) Orex-KO and WT mice behavior in the open-field apparatus presented over the 45 min of the test (B1, B3, B5) or by bins of 5 min (B2, B4, B6). WT mice have more locomotor activity in the open-field than Orex-KO mice all along the test (B1 and B2). Mice of both genotypes decreased their exploration activity between the first and the last 5 min of the test (KO: −51.6 ± 5.4% of LMA, WT: −52.4 ± 2.9%; p = 0.4). Mice of both genotypes spent the same amount of time in the center part of the open-field (B3 and B4). They similarly entered and exit the center part of the apparatus (B5 and B6). (C and D) Orex-KO and WT mice behavior during the 5 min light-dark box test in basal condition (C) and after REM sleep deprivation using the small-platforms-over-water method (D). Bar charts represent the total traveled distance (C-1 and D-1), the latency to first entrance into the light compartment (C-2 and D-2), the time spent in the light compartment (C-3 and D-3), and the number of transition in and out the light compartment (C-4 and D-4). **p < 0.01; ***p < 0.001. Two behavioral tests were performed: the open-field and light-dark box, both used to evaluate anxiety and stress levels [19]. We first conducted the open-field test after home-cage control condition. As previously reported [12], Orex-KO mice had a lowered locomotor activity (LMA) than WT (p = 1.8E-4) (Figure 5B1 and B2). We then assessed the time spent (duration), the locomotion (distance traveled normalized to the total traveled distance), and the number of transitions in and out of the central area. None of these parameters were significantly different between genotypes (Figure 5B3–B6). We then performed light/dark box tests after home-cage control condition (Figure 5C) or after 48 hr of REM sleep deprivation (Figure 5D) using the small-platforms-over-water setup. All mice showed a preference for the dark chamber and no significant difference was found between genotypes for all parameters measured (i.e. time spent in the bright compartment, distance traveled, latency to enter the bright compartment, and number of transitions) (Figure 5C and D). When comparing between conditions, the latency to enter the bright compartment was highly increased after REM sleep deprivation compared with home-cage condition (WT: 124.1 ± 24.9 vs. 35.5 ± 9.4 s, respectively, p = 0.003; KO: 117.2 ± 15.9 vs 39.2 ± 15.1 s, respectively, p = 6E-4), signaling the suitability of the test to evaluate anxiety level in mice (Figure 5C2 and D2). Altogether these data indicate that Orex-KO and WT mice have similar endogenous anxiety traits and behave similarly when challenged with a stressful environment. Thus, the difference in latency to enter sleep after REM sleep deprivation may rather be attributable to a difference in REM sleep propensity between genotypes. REM sleep pressure in Orex-KO narcoleptic mice To objectively measure REM sleep propensity, defined as the tendency to enter REM sleep, we utilized a new semiautomated REM sleep deprivation method developed in house [13]. Another series of mice underwent REM sleep deprivation using this novel automated method, with the results shown in Table 3. Baseline data from this experiment (Table 3) were not significantly different to that obtained with the first series of experiments (Table 1). This new method of REM sleep deprivation was efficient at depriving mice of REM sleep, with REM sleep reductions of 68.7 ± 3.8% and 59.6 ± 4.2% seen compared with baseline in WT and Orex-KO mice, respectively (p = 8,7E-4 for both genotypes). Table 3. Sleep Data during Automated REM Sleep deprivation REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - BASELINE (without Video Recording)  24 hrs   Total time (min) 70.2 ± 1.9 82.3 ± 4.0** 575.5 ± 35.4 612.2 ± 27.6 792.9 ± 36.2 739.4 ± 26.6 4.5 ± 1.8   Number of bouts 86.1 ± 7.7 108.1 ± 9.6* 356.0 ± 34.6 448.4 ± 52.1 362.1 ± 34.8 452.3 ± 52.6 2.7 ± 1.1   Mean Duration (sec) 51.0 ± 4.2 47.4 ± 3.9 104.1 ± 15.6 87.7 ± 9.2 140.4 ± 16.3 106.3 ± 12.6 102.6 ± 17.1  Light Period   Total time (min) 51.4 ± 1.4 53.1 ± 2.8 377.4 ± 22.3 402.2 ± 11.3 290.5 ± 23.3 263.9 ± 10.3 0.1 ± 0.1   Number of bouts 63.0 ± 5.5 69.7 ± 5.2 232.4 ± 22.6 245.7 ± 28 232.6 ± 22.6 243.1 ± 28.3 0.1 ± 0.1   Mean Duration (sec) 51.0 ± 4.2 46.7 ± 3.2 103.4 ± 13.7 107.1 ± 13.6 80.5 ± 12.2 70.0 ± 7.4 40.0  Dark Period   Total time (min) 18.8 ± 1.4 29.2 ± 1.4** 198.0 ± 21.5 210 ± 19.3 502.4 ± 22.3 475.5 ± 19.7 4.4 ± 1.8   Number of bouts 23.1 ± 3.4 38.4 ± 5.0* 123.6 ± 17.2 202.7 ± 25.5* 129.6 ± 17.3 209.1 ± 25.7* 2.6 ± 1.0   Mean Duration (sec) 52.8 ± 5.2 49.2 ± 5.4 106.9 ± 19.8 65.0 ± 6.0** 274.0 ± 54.0 149.7 ± 19.9* 105.7 ± 17.1 AUTOMATED DEPRIVATION  48 hrs   Total time (min) 43.5 ± 4.6 66.7 ± 8.5** 1131.6 ± 64.5 1192.4 ± 49.3 1702.0 ± 65.8 1618.2 ± 49.9 -   Number of bouts 643.7 ± 76.0 988 ± 68.5** 1120.6 ± 85.8 1531.9 ± 83.5** 1127.3 ± 85.1 1551.6 ± 85.7** -   Mean Duration (sec) 4.1 + 1.0 4.0 + 0.4 61.9 + 4.5 47.5 + 3.3** 94.2 + 8.4 63.9 + 4.4*  Day 1   Total time (min) 11.7 ± 1.8 27.1 ± 4.7** 546.7 ± 30.6 580.5 ± 28.1 880.2 ± 31.5 831.0 ± 27.9 -   Number of bouts 188.7 ± 18.0 408.6 ± 30.6*** 440.6 ± 25.6 680.4 ± 39.7*** 444.4 ± 24.6 684.9 ± 39.7*** -   Mean Duration (sec) 3.8 + 0.5 4.0 + 0.6 75.8 + 5.8 52.3 + 4.3** 121.5 + 9.1 74.4 + 5.1**  Day 2   Total time (min) 31.8 ± 3.2 39.6 ± 4.0 584.9 ± 35.1 611.9 ± 25.0 821.9 ± 35.4 787.2 ± 25.4 -   Number of bouts 455.0 ± 64.5 579.4 ± 43.9 680.0 ± 68.0 851.4 ± 52.5* 682.9 ± 68.8 866.7 ± 52.9* -   Mean Duration (sec) 5.3 ± 1.7 4.2 ± 0.4 53.9 ± 5.3 43.9 ± 2.7 77.8 ± 9.7 56.0 ± 4.4  Light Period   Total time (min) 31.1 ± 5.2 35.8 ± 4.7 752.3 ± 32.2 766.1 ± 24.1 655.2 ± 33.7 636.7 ± 23.2 -   Number of bouts 444.9 ± 66.9 544.4 ± 42.1 744.0 ± 55.1 838.0 ± 38.7 742.0 ± 56.6 835.9 ± 40.5 -   Mean Duration (sec) 5.0 ± 1.5 3.9 ± 0.4 62.1 ± 4.1 55.7 ± 3.5 55.3 ± 5.5 46.3 ± 2.6  Dark Period   Total time (min) 12.4 ± 1.3 31.0 ± 4.7*** 379.3 ± 38.8 426.3 ± 41.9 1046.9 ± 39.8 981.4 ± 44.4 -   Number of bouts 198.9 ± 22.9 443.6 ± 43.0*** 376.6 ± 47.8 693.9 ± 62.9** 385.3 ± 45.1 715.7 ± 61.0** -   Mean Duration (sec) 3.8 ± 0.2 4.2 ± 0.4 62.7 ± 6.0 37.5 ± 3.2** 181.5 ± 28.3 87.3 ± 10.1** REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - BASELINE (without Video Recording)  24 hrs   Total time (min) 70.2 ± 1.9 82.3 ± 4.0** 575.5 ± 35.4 612.2 ± 27.6 792.9 ± 36.2 739.4 ± 26.6 4.5 ± 1.8   Number of bouts 86.1 ± 7.7 108.1 ± 9.6* 356.0 ± 34.6 448.4 ± 52.1 362.1 ± 34.8 452.3 ± 52.6 2.7 ± 1.1   Mean Duration (sec) 51.0 ± 4.2 47.4 ± 3.9 104.1 ± 15.6 87.7 ± 9.2 140.4 ± 16.3 106.3 ± 12.6 102.6 ± 17.1  Light Period   Total time (min) 51.4 ± 1.4 53.1 ± 2.8 377.4 ± 22.3 402.2 ± 11.3 290.5 ± 23.3 263.9 ± 10.3 0.1 ± 0.1   Number of bouts 63.0 ± 5.5 69.7 ± 5.2 232.4 ± 22.6 245.7 ± 28 232.6 ± 22.6 243.1 ± 28.3 0.1 ± 0.1   Mean Duration (sec) 51.0 ± 4.2 46.7 ± 3.2 103.4 ± 13.7 107.1 ± 13.6 80.5 ± 12.2 70.0 ± 7.4 40.0  Dark Period   Total time (min) 18.8 ± 1.4 29.2 ± 1.4** 198.0 ± 21.5 210 ± 19.3 502.4 ± 22.3 475.5 ± 19.7 4.4 ± 1.8   Number of bouts 23.1 ± 3.4 38.4 ± 5.0* 123.6 ± 17.2 202.7 ± 25.5* 129.6 ± 17.3 209.1 ± 25.7* 2.6 ± 1.0   Mean Duration (sec) 52.8 ± 5.2 49.2 ± 5.4 106.9 ± 19.8 65.0 ± 6.0** 274.0 ± 54.0 149.7 ± 19.9* 105.7 ± 17.1 AUTOMATED DEPRIVATION  48 hrs   Total time (min) 43.5 ± 4.6 66.7 ± 8.5** 1131.6 ± 64.5 1192.4 ± 49.3 1702.0 ± 65.8 1618.2 ± 49.9 -   Number of bouts 643.7 ± 76.0 988 ± 68.5** 1120.6 ± 85.8 1531.9 ± 83.5** 1127.3 ± 85.1 1551.6 ± 85.7** -   Mean Duration (sec) 4.1 + 1.0 4.0 + 0.4 61.9 + 4.5 47.5 + 3.3** 94.2 + 8.4 63.9 + 4.4*  Day 1   Total time (min) 11.7 ± 1.8 27.1 ± 4.7** 546.7 ± 30.6 580.5 ± 28.1 880.2 ± 31.5 831.0 ± 27.9 -   Number of bouts 188.7 ± 18.0 408.6 ± 30.6*** 440.6 ± 25.6 680.4 ± 39.7*** 444.4 ± 24.6 684.9 ± 39.7*** -   Mean Duration (sec) 3.8 + 0.5 4.0 + 0.6 75.8 + 5.8 52.3 + 4.3** 121.5 + 9.1 74.4 + 5.1**  Day 2   Total time (min) 31.8 ± 3.2 39.6 ± 4.0 584.9 ± 35.1 611.9 ± 25.0 821.9 ± 35.4 787.2 ± 25.4 -   Number of bouts 455.0 ± 64.5 579.4 ± 43.9 680.0 ± 68.0 851.4 ± 52.5* 682.9 ± 68.8 866.7 ± 52.9* -   Mean Duration (sec) 5.3 ± 1.7 4.2 ± 0.4 53.9 ± 5.3 43.9 ± 2.7 77.8 ± 9.7 56.0 ± 4.4  Light Period   Total time (min) 31.1 ± 5.2 35.8 ± 4.7 752.3 ± 32.2 766.1 ± 24.1 655.2 ± 33.7 636.7 ± 23.2 -   Number of bouts 444.9 ± 66.9 544.4 ± 42.1 744.0 ± 55.1 838.0 ± 38.7 742.0 ± 56.6 835.9 ± 40.5 -   Mean Duration (sec) 5.0 ± 1.5 3.9 ± 0.4 62.1 ± 4.1 55.7 ± 3.5 55.3 ± 5.5 46.3 ± 2.6  Dark Period   Total time (min) 12.4 ± 1.3 31.0 ± 4.7*** 379.3 ± 38.8 426.3 ± 41.9 1046.9 ± 39.8 981.4 ± 44.4 -   Number of bouts 198.9 ± 22.9 443.6 ± 43.0*** 376.6 ± 47.8 693.9 ± 62.9** 385.3 ± 45.1 715.7 ± 61.0** -   Mean Duration (sec) 3.8 ± 0.2 4.2 ± 0.4 62.7 ± 6.0 37.5 ± 3.2** 181.5 ± 28.3 87.3 ± 10.1** *P<0.05, ** P<0.01 and *** P<0.001 Orex-KO (-/-) vs. WT (+/+). View Large Table 3. Sleep Data during Automated REM Sleep deprivation REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - BASELINE (without Video Recording)  24 hrs   Total time (min) 70.2 ± 1.9 82.3 ± 4.0** 575.5 ± 35.4 612.2 ± 27.6 792.9 ± 36.2 739.4 ± 26.6 4.5 ± 1.8   Number of bouts 86.1 ± 7.7 108.1 ± 9.6* 356.0 ± 34.6 448.4 ± 52.1 362.1 ± 34.8 452.3 ± 52.6 2.7 ± 1.1   Mean Duration (sec) 51.0 ± 4.2 47.4 ± 3.9 104.1 ± 15.6 87.7 ± 9.2 140.4 ± 16.3 106.3 ± 12.6 102.6 ± 17.1  Light Period   Total time (min) 51.4 ± 1.4 53.1 ± 2.8 377.4 ± 22.3 402.2 ± 11.3 290.5 ± 23.3 263.9 ± 10.3 0.1 ± 0.1   Number of bouts 63.0 ± 5.5 69.7 ± 5.2 232.4 ± 22.6 245.7 ± 28 232.6 ± 22.6 243.1 ± 28.3 0.1 ± 0.1   Mean Duration (sec) 51.0 ± 4.2 46.7 ± 3.2 103.4 ± 13.7 107.1 ± 13.6 80.5 ± 12.2 70.0 ± 7.4 40.0  Dark Period   Total time (min) 18.8 ± 1.4 29.2 ± 1.4** 198.0 ± 21.5 210 ± 19.3 502.4 ± 22.3 475.5 ± 19.7 4.4 ± 1.8   Number of bouts 23.1 ± 3.4 38.4 ± 5.0* 123.6 ± 17.2 202.7 ± 25.5* 129.6 ± 17.3 209.1 ± 25.7* 2.6 ± 1.0   Mean Duration (sec) 52.8 ± 5.2 49.2 ± 5.4 106.9 ± 19.8 65.0 ± 6.0** 274.0 ± 54.0 149.7 ± 19.9* 105.7 ± 17.1 AUTOMATED DEPRIVATION  48 hrs   Total time (min) 43.5 ± 4.6 66.7 ± 8.5** 1131.6 ± 64.5 1192.4 ± 49.3 1702.0 ± 65.8 1618.2 ± 49.9 -   Number of bouts 643.7 ± 76.0 988 ± 68.5** 1120.6 ± 85.8 1531.9 ± 83.5** 1127.3 ± 85.1 1551.6 ± 85.7** -   Mean Duration (sec) 4.1 + 1.0 4.0 + 0.4 61.9 + 4.5 47.5 + 3.3** 94.2 + 8.4 63.9 + 4.4*  Day 1   Total time (min) 11.7 ± 1.8 27.1 ± 4.7** 546.7 ± 30.6 580.5 ± 28.1 880.2 ± 31.5 831.0 ± 27.9 -   Number of bouts 188.7 ± 18.0 408.6 ± 30.6*** 440.6 ± 25.6 680.4 ± 39.7*** 444.4 ± 24.6 684.9 ± 39.7*** -   Mean Duration (sec) 3.8 + 0.5 4.0 + 0.6 75.8 + 5.8 52.3 + 4.3** 121.5 + 9.1 74.4 + 5.1**  Day 2   Total time (min) 31.8 ± 3.2 39.6 ± 4.0 584.9 ± 35.1 611.9 ± 25.0 821.9 ± 35.4 787.2 ± 25.4 -   Number of bouts 455.0 ± 64.5 579.4 ± 43.9 680.0 ± 68.0 851.4 ± 52.5* 682.9 ± 68.8 866.7 ± 52.9* -   Mean Duration (sec) 5.3 ± 1.7 4.2 ± 0.4 53.9 ± 5.3 43.9 ± 2.7 77.8 ± 9.7 56.0 ± 4.4  Light Period   Total time (min) 31.1 ± 5.2 35.8 ± 4.7 752.3 ± 32.2 766.1 ± 24.1 655.2 ± 33.7 636.7 ± 23.2 -   Number of bouts 444.9 ± 66.9 544.4 ± 42.1 744.0 ± 55.1 838.0 ± 38.7 742.0 ± 56.6 835.9 ± 40.5 -   Mean Duration (sec) 5.0 ± 1.5 3.9 ± 0.4 62.1 ± 4.1 55.7 ± 3.5 55.3 ± 5.5 46.3 ± 2.6  Dark Period   Total time (min) 12.4 ± 1.3 31.0 ± 4.7*** 379.3 ± 38.8 426.3 ± 41.9 1046.9 ± 39.8 981.4 ± 44.4 -   Number of bouts 198.9 ± 22.9 443.6 ± 43.0*** 376.6 ± 47.8 693.9 ± 62.9** 385.3 ± 45.1 715.7 ± 61.0** -   Mean Duration (sec) 3.8 ± 0.2 4.2 ± 0.4 62.7 ± 6.0 37.5 ± 3.2** 181.5 ± 28.3 87.3 ± 10.1** REM NREM AWAKE CATAPLEXY + / + - / - + / + - / - + / + - / - - / - BASELINE (without Video Recording)  24 hrs   Total time (min) 70.2 ± 1.9 82.3 ± 4.0** 575.5 ± 35.4 612.2 ± 27.6 792.9 ± 36.2 739.4 ± 26.6 4.5 ± 1.8   Number of bouts 86.1 ± 7.7 108.1 ± 9.6* 356.0 ± 34.6 448.4 ± 52.1 362.1 ± 34.8 452.3 ± 52.6 2.7 ± 1.1   Mean Duration (sec) 51.0 ± 4.2 47.4 ± 3.9 104.1 ± 15.6 87.7 ± 9.2 140.4 ± 16.3 106.3 ± 12.6 102.6 ± 17.1  Light Period   Total time (min) 51.4 ± 1.4 53.1 ± 2.8 377.4 ± 22.3 402.2 ± 11.3 290.5 ± 23.3 263.9 ± 10.3 0.1 ± 0.1   Number of bouts 63.0 ± 5.5 69.7 ± 5.2 232.4 ± 22.6 245.7 ± 28 232.6 ± 22.6 243.1 ± 28.3 0.1 ± 0.1   Mean Duration (sec) 51.0 ± 4.2 46.7 ± 3.2 103.4 ± 13.7 107.1 ± 13.6 80.5 ± 12.2 70.0 ± 7.4 40.0  Dark Period   Total time (min) 18.8 ± 1.4 29.2 ± 1.4** 198.0 ± 21.5 210 ± 19.3 502.4 ± 22.3 475.5 ± 19.7 4.4 ± 1.8   Number of bouts 23.1 ± 3.4 38.4 ± 5.0* 123.6 ± 17.2 202.7 ± 25.5* 129.6 ± 17.3 209.1 ± 25.7* 2.6 ± 1.0   Mean Duration (sec) 52.8 ± 5.2 49.2 ± 5.4 106.9 ± 19.8 65.0 ± 6.0** 274.0 ± 54.0 149.7 ± 19.9* 105.7 ± 17.1 AUTOMATED DEPRIVATION  48 hrs   Total time (min) 43.5 ± 4.6 66.7 ± 8.5** 1131.6 ± 64.5 1192.4 ± 49.3 1702.0 ± 65.8 1618.2 ± 49.9 -   Number of bouts 643.7 ± 76.0 988 ± 68.5** 1120.6 ± 85.8 1531.9 ± 83.5** 1127.3 ± 85.1 1551.6 ± 85.7** -   Mean Duration (sec) 4.1 + 1.0 4.0 + 0.4 61.9 + 4.5 47.5 + 3.3** 94.2 + 8.4 63.9 + 4.4*  Day 1   Total time (min) 11.7 ± 1.8 27.1 ± 4.7** 546.7 ± 30.6 580.5 ± 28.1 880.2 ± 31.5 831.0 ± 27.9 -   Number of bouts 188.7 ± 18.0 408.6 ± 30.6*** 440.6 ± 25.6 680.4 ± 39.7*** 444.4 ± 24.6 684.9 ± 39.7*** -   Mean Duration (sec) 3.8 + 0.5 4.0 + 0.6 75.8 + 5.8 52.3 + 4.3** 121.5 + 9.1 74.4 + 5.1**  Day 2   Total time (min) 31.8 ± 3.2 39.6 ± 4.0 584.9 ± 35.1 611.9 ± 25.0 821.9 ± 35.4 787.2 ± 25.4 -   Number of bouts 455.0 ± 64.5 579.4 ± 43.9 680.0 ± 68.0 851.4 ± 52.5* 682.9 ± 68.8 866.7 ± 52.9* -   Mean Duration (sec) 5.3 ± 1.7 4.2 ± 0.4 53.9 ± 5.3 43.9 ± 2.7 77.8 ± 9.7 56.0 ± 4.4  Light Period   Total time (min) 31.1 ± 5.2 35.8 ± 4.7 752.3 ± 32.2 766.1 ± 24.1 655.2 ± 33.7 636.7 ± 23.2 -   Number of bouts 444.9 ± 66.9 544.4 ± 42.1 744.0 ± 55.1 838.0 ± 38.7 742.0 ± 56.6 835.9 ± 40.5 -   Mean Duration (sec) 5.0 ± 1.5 3.9 ± 0.4 62.1 ± 4.1 55.7 ± 3.5 55.3 ± 5.5 46.3 ± 2.6  Dark Period   Total time (min) 12.4 ± 1.3 31.0 ± 4.7*** 379.3 ± 38.8 426.3 ± 41.9 1046.9 ± 39.8 981.4 ± 44.4 -   Number of bouts 198.9 ± 22.9 443.6 ± 43.0*** 376.6 ± 47.8 693.9 ± 62.9** 385.3 ± 45.1 715.7 ± 61.0** -   Mean Duration (sec) 3.8 ± 0.2 4.2 ± 0.4 62.7 ± 6.0 37.5 ± 3.2** 181.5 ± 28.3 87.3 ± 10.1** *P<0.05, ** P<0.01 and *** P<0.001 Orex-KO (-/-) vs. WT (+/+). View Large The efficiency of REM sleep deprivation was decreased when comparing day 1 with day 2 in both genotypes (WT: p = 9E-4; Orex-KO: p = 0.013; Figure 6A) indicating that REM sleep pressure increased during deprivation. An increased residual amount of REM sleep was found in Orex-KO than WT mice during deprivation (66.7 ± 8.5 vs. 43.5 ± 4.6 min, respectively, p = 9E-3), but this last observation is likely due to the higher number of attempts to enter REM sleep in Orex-KO than in WT mice (988.1 ± 68.5 and 643.7 ± 76.0, respectively; p = 0.004; Figure 6B). Indeed, with this method of deprivation, the mouse has to enter REM sleep for 1 s before REM sleep is detected and stimulations sent (Figure 1C). REM sleep episodes were abolished with the same number of stimulations in both genotypes (0.83 ± 0.02 vs. 0.89 ± 0.03 stimulations/REM sleep episode in WT and Orex-KO, respectively, p = 0.11; Figure 6D) and attempts to enter REM sleep had the same mean duration (4.1 ± 1.0 vs. 4.0 ± 0.4 s in WT and Orex-KO mice, respectively; Figure 6C). Altogether, these data indicate that Orex-KO mice show similar levels of REM sleep deprivation compared with WT mice, but interventions are more frequently needed throughout deprivation. Figure 6. View largeDownload slide REM sleep deprivation using the automated deprivation method is efficient in Orex-KO and WT mice. (A) Amount of REM sleep during 24 hr of baseline, the first, and the second day of REM sleep deprivation. (B) Number of REM sleep episodes during the entire 48 hr of REM sleep deprivation in both genotypes. (C) Mean duration of residual REM sleep bouts during deprivation. (D) Mean number of stimulation needed to wake up the mice. Note that the mean number is inferior to one stimulation per REM sleep episode because some entrances into REM sleep do not stabilize and mice come out of REM sleep before any stimulation is sent. *p < 0.05 and **p < 0.01 in Orex-KO versus WT mice; #p < 0.05 in day 1 versus baseline; +p < 0.05 and +++p < 0.0001 in day 2 versus day 1. Figure 6. View largeDownload slide REM sleep deprivation using the automated deprivation method is efficient in Orex-KO and WT mice. (A) Amount of REM sleep during 24 hr of baseline, the first, and the second day of REM sleep deprivation. (B) Number of REM sleep episodes during the entire 48 hr of REM sleep deprivation in both genotypes. (C) Mean duration of residual REM sleep bouts during deprivation. (D) Mean number of stimulation needed to wake up the mice. Note that the mean number is inferior to one stimulation per REM sleep episode because some entrances into REM sleep do not stabilize and mice come out of REM sleep before any stimulation is sent. *p < 0.05 and **p < 0.01 in Orex-KO versus WT mice; #p < 0.05 in day 1 versus baseline; +p < 0.05 and +++p < 0.0001 in day 2 versus day 1. Moreover, REM sleep deprivation with this new method was selective for REM sleep. Total amounts and amounts per hour of NREM sleep and wake were unchanged in both genotypes compared with their corresponding baseline (except in the first hour of deprivation where wakefulness was highly increased), whereas REM sleep quantities were largely decreased (Figure 7; Table 3). Wake and NREM sleep were significantly fragmented as indicated by the increased number of NREM and wake bouts compared with their corresponding baseline (Table 3). Figure 7. View largeDownload slide Deprivation using our automatic deprivation method is selectively of REM sleep. REM sleep (A-1 and A-2), NREM (B-1 and B-2), and Wake (C-1 and C-2) amounts in 2 hr bins across the 48 hr of deprivation using the automated method of deprivation. *p < 0.05 versus baseline. Figure 7. View largeDownload slide Deprivation using our automatic deprivation method is selectively of REM sleep. REM sleep (A-1 and A-2), NREM (B-1 and B-2), and Wake (C-1 and C-2) amounts in 2 hr bins across the 48 hr of deprivation using the automated method of deprivation. *p < 0.05 versus baseline. Orex-KO and WT mice did not attempt to enter REM sleep at lights off. This lack of REM sleep attempts was associated with a peak of wakefulness (Figure 7), possibly illustrating the well-known awakening effect of lights turning off [15]. These data further indicate that the orexin/hypocretin neuropeptides are not necessary to mediate this light effect. Finally, REM sleep propensity can be evaluated by counting the number of attempts to enter REM sleep or of stimulations needed to suppress REM sleep. In WT mice, the number of stimulations per hour followed the circadian occurrence of REM sleep observed during baseline, with more stimulations during the light phase than the dark active phase (383.1 ± 59.3 vs 155.3 ± 21.8 stimulations, respectively; p = 0.002) (Figure 8). Interestingly, both groups of mice received the same number of stimulations, with the same distribution over time during the light phases of days 1 and 2, suggesting that REM sleep propensity is similar in Orex-KO mice and their WT littermates (Figure 8B). However, in contrary to WT mice, Orex-KO mice were equally stimulated during the light and dark phases (492.7 ± 45.8 vs. 389.9 ± 40.9 stimulations, respectively, p = 0.112) (Figure 8B). This difference in number of stimulations during the dark phases between genotypes was particularly visible in day 1 (196.1 ± 26.6 in Orex-KO vs. 54.0 ± 8.9 in WT mice; p = 8.7E-4) but was also seen in day 2 (194.7 ± 20.1 in Orex-KO vs. 101.3 ± 15.3 in WT; p = 0.01 with a power of analysis of less than 50 per cent due to higher inter-individual variability). This could reflect the less pronounced circadian distribution of REM sleep in Orex-KO mice and/or a lack of REM sleep inhibition in absence of hypocretin/orexin signal. Figure 8. View largeDownload slide Illustration of REM sleep propensity during the 48 hr of REM sleep deprivation using the automated method. (A) Graph reporting the number of stimulations per hour needed all along the deprivation procedure. (B) Bar chart of the mean number of stimulations made during REM sleep attempts during the light phases (Day 1 + Day 2) and the dark phases (Day 1 + Day 2). *p < 0.05, **p < 0.01, n.s. = not significant. Figure 8. View largeDownload slide Illustration of REM sleep propensity during the 48 hr of REM sleep deprivation using the automated method. (A) Graph reporting the number of stimulations per hour needed all along the deprivation procedure. (B) Bar chart of the mean number of stimulations made during REM sleep attempts during the light phases (Day 1 + Day 2) and the dark phases (Day 1 + Day 2). *p < 0.05, **p < 0.01, n.s. = not significant. Discussion This study is the first to challenge the homeostatic regulation of REM sleep in an animal model of narcolepsy by depriving mice specifically of REM sleep. We demonstrate that the homeostatic regulation of REM sleep is preserved in narcoleptic Orex-KO mice lacking hypocretin/orexin transmission, and in a same magnitude as their WT littermates. Indeed, the same quantity of REM sleep is lost over the 2 days of REM sleep deprivation in both genotypes. Orex-KO mice showed a similar rebound of REM sleep after deprivation compared with WT mice. However, sleep latency was shortened in Orex-KO mice. This difference between genotypes is not due to alteration in experienced stress levels as no difference was seen in response to the anxiogenic environment of REM sleep deprivation using the small-platforms-over water method. Looking at REM sleep propensity with our new automated deprivation method, REM sleep pressure built up similarly during the light resting-phase in both genotypes. However, we show that REM sleep propensity is not suppressed during the dark phase in Orex-KO mice. Technical considerations In agreement with all previous reports, we found a fragmentation of all vigilance states in Orex-KO mice compared with their WT littermates [12, 16, 20–22]. In addition, we found an increased amount of REM sleep during the dark active phase in Orex-KO mice that translates to a decreased circadian index as described previously [12, 20, 22]. Remarkably, we were able to induce cataplexy during the first hour of recovery after the 48 hr of REM sleep deprivation using the small-platforms-over-water method, before REM sleep rebound took place. Cataplexy is quite rare in baseline condition. Triggering cataplexy in narcoleptic mice is a challenge of great value in order to study the neurobiological mechanisms of this symptom. Several emotional stimuli such as social interaction, palatable food, or running wheel have been shown to induce cataplexy during the dark active phase [20, 23, 24]. REM sleep deprivation is however the first stimulus that induces cataplexy during the light phase at a time of day (ZT2) when no cataplexy occurs in other conditions. Patients report a tendency to have more cataplexy when tired or sleep deprived [1], but Vu et al. [9] did not observe cataplexy after two nights of REM sleep deprivation in patients. As cataplexy is difficult to induce during interviews at the clinics and that patients were tested for sleepiness with MSLT right after deprivation, it is difficult to objectively conclude whether REM sleep deprivation facilitates cataplexy or not in humans. We also noticed that right after REM sleep deprivation, mice showed increased motor activity, grooming, and digging, and such behaviors have been described to induce cataplexy in mice [23]. To this point, we cannot assert whether cataplexy is triggered by an increase of such motor activities or the increase of REM sleep pressure or both. Another point to mention is that Orex-KO mice were possibly stimulated during cataplexy as well as REM sleep during the automated REM sleep deprivation protocol. As we had no video during these recordings, we could not reliably differentiate between the two. Nevertheless, it is to note that these mice only had 2.7 ± 1.1 episodes of cataplexy during the preceding 24 hr of baseline recordings and 108.1 ± 9.6 episodes of REM sleep (Table 3). We thus believe that the number of stimulations attributable to cataplexy is probably marginal compared with those clearly attributable to REM sleep episodes (i.e. always following NREM sleep) during REM sleep deprivation in Orex-KO mice. Coping with REM sleep deprivation One of the first studies on the hypocretin/orexin neuropeptides showed that intracerebroventricular (icv) injections of orexin-A provoked an increased release of corticosterone in a dose-dependent manner [25]. In agreement with previous reports [26, 27], we found no significant difference in corticosterone level and anxiety-like behaviors between genotypes in baseline condition. Furthermore, sleep deprivation is seen as being stressful, with difference in stress levels depending on the method used. We thus evaluated stress-induction and stress-coping in Orex-KO mice and their WT littermates combining biochemical (corticosterone measurement) and behavioral (Light-Dark-box test) approaches. In agreement with Emmerzaal and colleagues [27] who reported that plasma corticosterone levels were similarly increased in Orex-KO mice and WT mice after an acute stress of 2 hr at 4°C, we show here that the mild chronic stress induced by REM sleep deprivation with small-platforms-over-water method [4] is dealt with in a similar manner by both genotypes as the tendency for a reduced increase in plasmatic corticosterone in Orex-KO mice compared with WT mice after REM sleep deprivation did not translate to the behavioral test. REM sleep homeostasis In this current study, the homeostatic regulation of REM sleep was found to be intact in our narcoleptic mice. Indeed, REM sleep loss due to our selective deprivation method was compensated for by a REM sleep hypersomnia in a similar proportion in both genotypes. Mochizuki and colleagues [21] had explored REM sleep rebound in Orex-KO mice but only following 8 hr of total sleep deprivation. Although of interest, such a protocol introduced a confounding factor of competition between NREM and REM sleep recovery, with the REM sleep rebound being delayed by several hours possibly due to NREM sleep recovery occurring first. Nevertheless, in agreement with our current data, the authors mentioned that WT and Orex-KO mice recovered their REM sleep deficits at the same rate and to the same extent when analyzed over the 16 hr of recovery period and normalized to baseline. By challenging REM sleep homeostatic regulation using the automatic deprivation method, we were able to visualize REM sleep propensity as revealed by the number of attempts to enter REM sleep and see that it increased throughout the duration of the deprivation procedure. Interestingly, when focusing on the light phase of days 1 and 2, REM sleep propensity increased at a similar and linear rate between genotypes indicating that REM sleep requirements are similar in Orex-KO and WT mice. However, during the dark active phase, differences in REM sleep propensity were seen between Orex-KO and WT mice. In Orex-KO mice, REM sleep propensity increased with the same rate as during the light phase while REM sleep propensity was strongly reduced in WT mice, indicating that in contrary to WT mice, Orex-KO mice were unable to suppress REM sleep during the active phase. Supporting our observations, Mochizuki and colleagues [21] had mentioned that Orex-KO mice had slightly more REM sleep than WT mice during the few hours of recovery that took place during the dark period. The suppression of REM sleep during the active phase is further supported by REM sleep deprivation studies in rats showing that REM sleep rebound is displaced by several hours when rats were released from deprivation at mid-active phase but not when released at mid-resting phase although the amount of REM sleep recovered over 24 hr was equal between groups [3]. Hypocretin/orexin neuropeptides are mainly released during the dark phase when rodents are most active and awake [28, 29]. The wake-promoting role of hypocretin/orexin neuropeptides is now well established, thanks to the multiple pharmacological, electrophysiological, chemogenetic, and optogenetic studies (see for review, Ref. [30]). The higher amount of REM sleep during the dark phase seen in Orex-KO mice in the current study may thus reflect an opportunistic occurrence of REM sleep during the dark phase in the absence of hypocretin/orexin neurotransmission rather than a higher need for REM sleep. Finally, we found similar amounts of wakefulness between Orex-KO mice and WT littermates during the dark phase of REM sleep deprivation. Thus, the higher REM sleep propensity during the dark phase in Orex-KO mice cannot be attributed to a lack of wake drive. Furthermore, Hagan et al. [25] showed that icv injection of Orexin-A in rats induces wakefulness during 2 hr and an inhibition of REM sleep for 8 hr even when long bouts of NREM sleep were taking place. Altogether our data evaluated with the literature indicate that the hypocretin/orexin neuropeptides play a key inhibitory role on REM sleep, independent of its wake-promoting function, repressing the occurrence REM sleep during the active phase when not appropriate. In the absence of hypocretin/orexin neuropeptides, REM sleep would occur throughout the day in an opportunistic manner, as seen in patients with narcolepsy. Based on our knowledge of hypocretin/orexin neuropeptides connectomics [31] and on the neuronal network controlling REM sleep [32], we speculate that hypocretin/orexin would inhibit REM sleep by its excitatory projection onto the GABAergic neurons of the ventrolateral periaqueductal gray (vlPAG), those neurons being inhibitory of the sublaterodorsal nucleus generating REM sleep. This proposed mode of action is further supported by lesion studies of the GABAergic neurons of the vlPAG using microinjection of hypocretin 2 conjugated to saporin in Orex-KO and WT mice showing that such lesions induce a strong and specific increase in REM sleep quantity during the dark phase [33]. Funding This study was funded by ARC2 région Rhône-Alpes, ANR optoREM (ANR-13-BSV4-0003-01), Société Française de Recherche et de Médecine du Sommeil (SFRMS), and Centre National de Recherche Scientifique (CNRS). S.M. is now affiliated to the University of Exeter and Bristol funded by the MRC GW4 BioMed DTP. Notes Conflict of interest statement. None declared. Acknowledgments The authors thank Jean-Michel Vicat and Clémence Brando at Alecs-SPF platform-Lyon for taking good care of mice and Virginie Rappeneau for her advices in setting up the behavioral experiments. References 1. Dauvilliers Y et al. Narcolepsy with cataplexy . Lancet . 2007 ; 369 ( 9560 ): 499 – 511 . Google Scholar CrossRef Search ADS PubMed 2. Dantz B et al. Circadian rhythms in narcolepsy: studies on a 90 minute day . Electroencephalogr Clin Neurophysiol . 1994 ; 90 ( 1 ): 24 – 35 . Google Scholar CrossRef Search ADS PubMed 3. Wurts SW et al. 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TI - The inappropriate occurrence of rapid eye movement sleep in narcolepsy is not due to a defect in homeostatic regulation of rapid eye movement sleep
JF - SLEEP
DO - 10.1093/sleep/zsy046
DA - 2018-03-07
UR - https://www.deepdyve.com/lp/oxford-university-press/the-inappropriate-occurrence-of-rapid-eye-movement-sleep-in-narcolepsy-wYx6l0Z8Uk
SP - 1
VL - Advance Article
IS - 6
DP - DeepDyve
ER -