Altered Sleep Regulation in Leptin Deficient Mice

Transcription

1 Articles in PresS. Am J Physiol Regul Integr Comp Physiol (November 17, 2005). doi: /ajpregu Altered Sleep Regulation in Leptin Deficient Mice Aaron D. Laposky 1,2 * Jonathan Shelton 1,2 Joseph Bass 1,2,3 Christine Dugovic 1,2 Nicholas Perrino 1 Fred W. Turek 1,2 Department of Neurobiology and Physiology, Northwestern University, Evanston, IL Center for Sleep and Circadian Biology, Northwestern University, Evanston, IL Department of Endocrinology, Evanston Northwestern Hospital, Evanston, IL Running Head: Sleep in leptin deficient, ob/ob mice Corresponding author* Aaron D. Laposky Northwestern University Center for Sleep and Circadian Biology 2205 Tech Drive Hogan Evanston, IL Ph (847) FAX (847) a-laposky@northwestern.edu Copyright 2005 by the American Physiological Society.

2 2 Abstract Recent epidemiological, clinical and experimental studies have demonstrated important links between sleep duration and architecture, circadian rhythms and metabolism, although the genetic pathways that may interconnect these processes are not well understood. Leptin is a circulating hormone and major adiposity signal involved in long-term energy homeostasis. In this study, we tested the hypothesis that leptin deficiency leads to impairments in sleep-wake regulation. Male ob/ob mice, a genetic model of leptin deficiency, had significantly disrupted sleep architecture with an elevated number of arousals from sleep (wt, 108.2±7.2 vs. ob/ob, 148.4±4.5, P<.001) and increased stage shifts (wt, 519.1±25.2 vs. ob/ob, 748.0±38.8, P<.001) compared to wild-type (wt) mice. Ob/ob mice also had more frequent, but shorter lasting sleep bouts compared to wt mice, indicating impaired sleep consolidation. Interestingly, ob/ob mice showed changes in sleep time, with increased amounts of 24-hr NREM sleep (wt, 601.5±10.8 vs. ob/ob, 669.2±13.4 minutes, P<.001). Ob/ob mice had overall lower body temperature (wt, 35.1±0.2 vs. ob/ob, 33.4±0.2 C, P<.001) and locomotor activity counts (wt, 25125±2137 vs. ob/ob, 5219±1759, P<.001). Ob/ob mice displayed an attenuated diurnal rhythm of sleep-wake stages, NREM delta power and locomotor activity. Following sleep deprivation, ob/ob mice had smaller amounts of NREM and REM recovery sleep, both in terms of the magnitude and the duration of the recovery response. In combination, these results indicate that leptin deficiency disrupts the regulation of sleep architecture and diurnal rhythmicity. Key words: obesity, sleep time, locomotor activity, sleep fragmentation, circadian rhythms, energy metabolism

3 3 Introduction: Two major health trends afflicting individuals that are impacting medicine and society are (1) a progressive decrease in nightly sleep time resulting in a state of chronic sleep debt, and (2) a continuous increase in the incidence and severity of obesity and the metabolic syndrome (1, 2, 15). Interestingly, experimental and epidemiological studies have demonstrated a close relationship between sleep time and metabolic regulation. For example, young healthy adults subjected to chronic partial sleep restriction exhibit decreased glucose utilization, diminished insulin release following a glucose challenge, activation of the hypothalamo-pituitary-adrenal (HPA) axis, increased sympathetic output, increased ghrelin levels and decreased leptin concentration and diurnal rhythmicity (43-45). Epidemiological and clinical data indicate that voluntary sleep curtailment (32, 46) and sleep disorders that impair sleep architecture, such as narcolepsy (25) and sleep disordered breathing (35), are associated with an increased incidence of disrupted metabolic regulation. In rats, chronic total sleep deprivation (TSD) leads to pronounced changes in energy regulation, including increased food intake, progressive weight loss, elevated sympathetic activation, hyperthermia and low thyroid hormone levels (4, 13). More recent studies have demonstrated effects of sleep deprivation on the levels of particular hormones and neuropeptides involved in metabolism. Short-term sleep deprivation in rats increases plasma ghrelin concentrations (5). Prolonged TSD results in decreased growth hormone, insulin-like growth factor-1 (IGF-1), prolactin and leptin concentrations (14). While it is clear that sleep loss results in adjustments in energy metabolism, limited data are available on how changes in energy metabolism affect sleep-wake patterns. Behavioral studies have shown that in mice, a high fat diet leads to increases in NREM sleep time (20) whereas in rats, food deprivation results in decreased sleep time (10, 27) or a more fragmented sleep pattern (6). In rats, the re-feeding period following food deprivation is accompanied by

4 4 elevated sleep time (39), possibly depending on the nutritional content of the food (27). Satiety inducing agents, such as cholecystokinin and insulin, increase sleep time in rodents (29). Acute leptin administration has been shown to decrease REM sleep and increase NREM sleep time in rats (42). Furthermore, substances such as histamine, orexin/hypocretin which are synthesized in and/or have receptor sites in the hypothalamus, have important roles in the control of both feeding and sleep-wake patterns (38, 41, 47, 52). The effect of sleep deprivation on leptin and the ability of leptin to modulate sleep-wake patterns is of particular interest since this anorectic hormone is produced in adipose cells and serves a critical function as a signal of adiposity and satiety to the hypothalamus, (3, 33), and may represent an important mechanistic link between sleep and metabolic regulation. The ob/ob mouse is a genetic model of leptin deficiency resulting from a spontaneous mutation in the gene (ob) encoding leptin (18, 56). These mice exhibit hyperphagia and early onset obesity, as well as hallmarks of the metabolic syndrome including hyperglycemia, insulin resistance and dyslipidemia. In order to explore the possible relationship between leptin deficiency and sleepwake regulatory processes, we characterized the sleep-wake phenotype in ob/ob mice. Here we report that genetic leptin deficiency in ob/ob mice results in altered sleep-wake organization under baseline conditions, affects the capacity to recover from sleep deprivation, and impairs the diurnal rhythms of sleep-wake states and locomotor activity.

5 5 Materials and Methods: A group of 12 week old male B6-Lep ob (ob/ob, N=8) mice and age matched male wild type (wt, N=9) C57Bl/6J controls were used for sleep-wake recordings. A separate group of 6 week old ob/ob (N=5) and age-matched wt (N=5) mice were used for locomotor activity recordings. All mice were purchased from Jackson Laboratories (Bar Harbor, ME). Upon arrival, animals were group housed in a light (12:12 LD, lights on 06:00) and temperature (23-24 C) controlled environment with free access to food (LabDiet, PMI Nutrition International, LLC; protein 18%, fat 6%, carbohydrate 55%, fiber 5%) and water. All procedures were in accordance with the policies and regulations outlined by the Animal Use and Care Committee at Northwestern University. Surgical Procedures At 12 weeks of age, ob/ob (48.7 ± 1.7 g) and wt (28.1 ± 1.5 g) mice were anesthetized with an intraperitoneal injection of ketamine (80mg/kg) and xylazine (9 mg/kg). An incision was made on the scalp to expose the skull. To record electroencephalographic (EEG) waveforms, stainless steel screws (Small Parts, Inc. Miami Lakes, Fl) were placed in two bilaterally pre-drilled holes in the skull. The first screw was located 1mm anterior to bregma and 2 mm lateral to the central suture, and the second was positioned 1mm anterior to lambda and 2.5 mm lateral to the central suture. To record electromyographic (EMG) activity, the exposed ends of two stainless steel Teflon coated wires (.002" in diameter, Medwire, Mt. Vernon, VA) were bilaterally inserted into the nuchal muscles in the dorsal neck region. All electrodes were connected to a plastic 1x4 pin grid array connector (Plastics One, Roanoke, VA) that was secured to the skull using VetBond and cyanoacrylamide resin. Following implantation of the electrodes, a 1cm incision was made in the right hind quarter region and a transducer (1.6 grams in weight, PDT-4000, E-mitter by Mini-Mitter) was implanted in the peritoneal cavity to record body temperature. A minimum of 14 days was allowed before beginning data collection.

6 6 Sleep recording and data analysis Following the recovery period, wt (N=9) and ob/ob (N=8) mice were housed individually in cylindrical (diameter 10 inches) sleep recording cages and placed into light (12:12 L:D, lights on 06:00), temperature (23-24 C) and sound controlled recording chambers with free access to food and water. EEG/EMG data were collected using a wire tether/commutator system (Plastics One, Roanoke, VA), which allowed animals unrestricted movement about the cage. After 7 days of acclimation to the recording environment, EEG/EMG waveforms were collected for 48 hours starting at lights on. At the time of recording, ob/ob mice weighed 59.7 ± 1.7 grams and wt mice were 30.1 ± 1.2 grams. EEG signals were amplified 10,000x with -6 db/oct high pass and low pass filters set at 1 and 30 Hz, respectively. EMG signals were amplified 5000x with high and low pass filtered at 30 and 100 HZ. Both signals were digitized at 100 Hz/channel by an analog to digital converter (model DT-01EZ; Data Translation Inc., Marlboro, MA) and stored on an IBM PC compatible computer, using specialized software for acquiring and processing sleep data in rodents (Multilevel, Actimetrics, Evanston, IL). After the baseline recording, animals were sleep deprived for 6 hrs during the last half of the light phase (12:00-18:00) by a gentle handling procedure. To keep an animal awake, an experimenter observed the EEG/EMG recordings for signs of sleep, and then used a progression of stimuli (cage tapping, cage shaking, gentle contact) to awaken the animal. At the beginning of dark onset (18:00), sleep deprivation was terminated and EEG/EMG waveforms were recorded for an 18-hour uninterrupted recovery sleep period. With the use of a custom designed software package (SleepReport, Actimetrics, Evanston, IL), EEG and EMG recordings were divided into 10-second epochs and scored via visual inspection as either wake (low voltage, high frequency EEG and high amplitude EMG), non rapid eye movement sleep (NREM) (high voltage, low frequency EEG and low amplitude EMG) or rapid eye movement sleep (REM) (low amplitude EEG constituted mainly by theta

7 7 wave activity and EMG atonia). SleepReport software was used for post-scoring analysis, which allowed the determination of sleep structure parameters, including sleep amount, distribution and consolidation as well as EEG spectral analysis. The distribution of sleep/wake parameters was quantified by determining the ratio of sleep/wake amounts between the 12-hr light and 12-hour dark periods (light:dark ratio). Sleep architecture was further examined by comparing wt and ob/ob mice on the number of arousals from sleep (NREM or REM sleep interrupted by a 10 second epoch of wakefulness) stage shifts (number of transitions between ten second epochs of wake, NREM, and REM), sleep/wake bouts (at least 2 consecutive epochs, 20 seconds, of wake NREM or REM) and the average duration (minutes) of sleep/wake bouts. For quantitative analysis of the EEG signal, each 10-second scoring epoch was divided into five 2-sec intervals and subjected to Fast Fourier Transformation, which included a range of 1-25 Hz with a frequency resolution of 0.5 Hz. For all epochs of NREM sleep, the EEG power in the delta (1-4 Hz), theta (4-8 Hz) and sigma (11-15 Hz) frequency ranges were calculated. Because absolute power density values can show substantial inter-individual variability, to analyze the time course of EEG power in each frequency band, the power values were normalized and expressed as a percentage of the individual 24- hour mean. Body temperature monitoring Body temperature was measured simultaneously with EEG/EMG recordings. Body temperature measurements were taken from a transducer surgically implanted in the peritoneal cavity (1.6 grams in weight, PDT-4000 E-Mitter by Mini-Mitter). These biotelemetry transducers are pre-calibrated to an accuracy of 0.1 C. The transducers are powered by an inductive solenoid coil and output signals are captured by a radiofrequency receiver that is located beneath each mouse cage. Body temperature was sampled every 10 seconds and analyzed at the end of the experiment with a custom designed software package (Multilevel, Actimetrics, Evanston, IL).

8 8 Problems with transmitter recordings occurred in 2 of the 9 wt mice, and these animals were removed from the analyses of body temperature data. Locomotor Activity Monitoring At 6 weeks of age, 5 male ob/ob (36.7 ± 1.3 g) and 5 age matched wt mice (25.0 ± 1.2 g) were individually housed in cages with free access to a running wheel. Animals were maintained on a 12:12 L:D cycle (lights on 06:00) with food and water available ad libitum. After 2 weeks of acclimation to the recording environment and running wheels, activity patterns were recorded using Chronobiology Kit software (Stanford Software Systems, Stanford, CA). Data from the second week of recording, when animals were 9-10 weeks of age, were selected for analysis. The animal weights at the beginning of wheel running recordings were ob/ob (43.7.2±1.8 grams) and wt (26.9±1.1 grams). For each day of recording, activity counts were accumulated for consecutive 6-minute intervals, as well as for the 12-hr light phase, 12-hr dark phase and overall 24-hr period using ClockLab software (Actimetrics, Evanston, IL). Activity counts in each of these intervals were averaged over the 7 days of recording in each ob/ob and wt mouse in order to reduce variability within the samples. Statistical Analysis Repeated-measures analysis of variance (ANOVA) was used in instances where genotype (wt vs. ob/ob), time (12-hr light vs. 12-hr dark phases, or 2-hr intervals) and genotype x time effects were examined. The ANOVA was applied to analyze data from baseline and sleep deprivation recordings, as well as locomotor activity profiles. In a few instances, t-tests were utilized to make simple between and within genotype comparisons. Significance levels were set at p<.05 for all comparisons. Statistical analyses were performed using Statistica (StatSoft, Inc., Tulsa, OK).

9 9 Results: Sleep time is increased in ob/ob vs. wt mice: Over the 24-hr baseline period, TST differed between genotypes (genotype main effect, F (1,15) = 12.2, P<.01), with ob/ob mice sleeping more ( min) than wt mice (Figure 1). The increased TST in ob/ob mice was time dependent (genotype x time interaction, F (1,15) = 14.6, P<.01) due to an elevation during the 12-hr dark phase (P<.001) and a non-significant decrease during the 12-hr light phase. With respect to individual sleep stages, the amount of 24-hr NREM sleep time was increased in ob/ob mice (genotype main effect, F (1,15) = 13.9, P<.01). This effect was time dependent (genotype x time interaction, F (1,15) = 13.7, P<.01) as reflected by increased NREM sleep only in the 12-hr dark phase (P<.001) (Figure 1). Wt and ob/ob mice had similar amounts of 24-hr REM sleep (F (1,15) = 1.0, P>.05) (Figure 1). However, a genotype x time interaction (F (1,15) = 7.7, P<.01) showed that in ob/ob mice, REM sleep was significantly reduced during the 12-hr light phase (P<.05) and non-significantly elevated during the 12-hr dark phase, compared to wt mice. In addition, Figure 1 shows specific 2-hr intervals across the L:D cycle during which wt and ob/ob differed in wake (genotype main effect, F (1,15) = 12.2, P<.01; genotype x time interaction, F (11,165) = 4.1, P<.001), NREM sleep (genotype main effect, F (1,15) = 13.9, P, <.01; genotype x time interaction, F (11,165) = 4.1, P<.001) and REM sleep (genotype x time interaction, F (11,165) = 3.2, P<.001) time. Quantitative analysis of NREM EEG waveforms We analyzed EEG activity during NREM sleep by determining the average power density in the delta (1-4 Hz), theta (4-8 Hz) and sigma (11-15 Hz) frequency bands under baseline sleep conditions (Table 1). There were no genotype differences in delta (F (1,15) = 0.4, P>.05), theta (F (1,15) = 1.9, P>.05) or sigma (F (1,15) = 0.4, P>.05) power. There was an overall time effect in

10 10 which delta power (time main effect, F (1,15) = 8.5, P<.01) and theta power (time main effect, F (1,15) = 31.5, P<.001) were elevated during the 12-hr dark compared to 12-hr light phase. Sleep consolidation is disrupted in ob/ob vs. wt mice: In both wt and ob/ob mice, NREM (time main effect, F (1,15) = 267.0, P<.001) and REM (time main effect, F (1,15) = 146.3, P<.001) sleep were concentrated to the light phase and wakefulness (time main effect, F (1,15) = 296.9, P<.001) to the dark phase, indicating a general overall intact diurnal sleep-wake distribution under entrained conditions (Figure 1). However, when the genotypes were compared on sleep-wake distribution by determining the light:dark ratio (amount of sleep occurring in the 12-hr light phase as a percentage of 24-hr sleep time), 69.4% of total NREM sleep occurred in the light phase in wt mice compared to 60.9% for ob/ob mice (unpaired t-test, t (15) =4.4, P<.001). Similarly, a higher percentage of total REM sleep occurred during the light phase in wt (79.6%) compared to ob/ob (70.2%) mice (unpaired t-test, t (15) = 2.7, P<.05). In ob/ob mice, individual sleep-wake cycles were less consolidated, as indicated by a high level of sleep fragmentation (Table 2). Ob/ob mice had more arousals from sleep (genotype main effect, F (1,15) = 18.9, P<.001; genotype x time interaction, F (1,15) = 4.4, P<.05) and stage shifts (genotype main effect, F (1,15) = 22.6, P<.001) compared to wt mice. In addition, the number of individual wake bouts (genotype main effect, F (1,15) = 14.9, P<.01) and NREM bouts (genotype main effect, F (1,15) = 127.8, P<.001) was higher in ob/ob mice. On the other hand, the average duration of individual wake bouts (genotype main effect, F (1,15) = 8.2, P<.01; genotype x time interaction, F (1,15) = 7.0, P<.05) and NREM bouts (genotype main effect, F (1,15) =9.5, P<.01) was shorter in ob/ob mice (Table 2). These data indicate that ob/ob mice fell asleep more frequently but were unable to maintain individual sleep episodes for the same duration as wt mice, reflective of alterations in sleep architecture in ob/ob mice. There were no genotype

11 11 differences in REM bout number (F (1,15) = 0.01, P>.05) or REM bout duration (F (1,15) = 0.91, P>.05). The diurnal pattern of homeostatic sleep drive was determined by normalizing NREM delta (1-4 Hz) power for each baseline 2-hr interval as a percentage of the 24-hr baseline NREM delta power (Figure 2). In wt mice, NREM delta power was elevated at light onset, when sleep pressure is normally the highest, and showed the expected decline across this phase as sleep time accumulated. NREM delta power showed a typical elevation during the dark phase in association with high levels of wakefulness and an increased pressure for sleep. Ob/ob mice produced an attenuated NREM delta power rhythm (genotype x time interaction, F (11,143) = 2.4, P<.01) with respect to the peak at light onset, the gradual decline across the light phase and the increase during the dark phase compared to wt mice (Figure 2). Body temperature is lower in ob/ob mice: Ob/ob mice had a lower average 24-hr body temperature in wake (genotype main effect, F (1,13) = 47.6, P<.001), NREM sleep (genotype main effect, F (1,13) = 19.0, P <.001) and REM sleep (genotype main effect, F (1,13) = 16.6, P<.01) compared to wt mice (Table 3). Both genotypes exhibited a higher body temperature during the 12-hr dark phase vs. 12-hr light phase during wakefulness (time main effect, F (1,13) = 159.9, P<.001), NREM sleep (time main effect, F (1,13) = 63.8, P<.001) and REM sleep (time main effect, F (1,13) = 24.3, P<.001), indicating an intact diurnal rhythm under entrained conditions. In addition, we compared genotypes on the magnitude of the decrease in body temperature during NREM and REM sleep compared to wakefulness. During NREM sleep, body temperature was significantly lower than in wakefulness in both wt (paired t-test, t (13) = 2.8, P<.01) and ob/ob (paired t-test, t (13) = 2.2, P<.05) mice (Table 3). However, this effect was smaller in ob/ob (-0.51 ± 0.06 C) compared to wt (-1.0 ± 0.1 C) mice (unpaired t-test, t (13) = 3.2, P<.01). Similarly, body temperature during REM sleep was lower than during wakefulness in wt

12 12 (paired t-test, t (13) = 3.3, P<.01) and ob/ob (paired t-test, t (13) = 3.1, P<.01) mice (Table 3), and this difference was smaller in ob/ob (-0.73 ± 0.07 C) compared to wt (-1.19 ± 0.16 C) mice (unpaired t-test, t (13) = 2.9, P<.01). Recovery from 6-hr sleep deprivation: The recovery response following sleep deprivation was first assessed separately in each genotype by comparing sleep during the 18-hr recovery period (18:00 to 12:00) to sleep during the previous day's corresponding baseline period. The recovery period began at dark onset and consisted of the 12-hr dark phase and the 6-hr light phase, as indicated in Figure 3. In wt mice, NREM sleep time was significantly elevated during the first 6 hours of the recovery period, as well as during hours 9 and 10 of recovery (condition x time interaction, F (8,40) = 11.5, P<.001), (Figure 3). A decrease in NREM sleep time was noted in the last 2 hours of the light phase of the recovery period commonly referred to as a negative rebound. Similarly, REM sleep was increased during the first 6 hours of the recovery period and remained non-significantly elevated during the following 4 hours (condition x time interaction, F (8,40) = 4.5, P<.001), (Figure 3). NREM delta power was significantly increased during the first 2 hours of recovery (paired t-test, t (5) = 5.7, P <.001), indicating an increase in the intensity of NREM sleep (Figure 3). In ob/ob mice, the rebound in both NREM (condition x time interaction, F (8,56) = 5.0, P<.001) and REM sleep (condition x time interaction, F (8,56) = 2.2, P<.05) was restricted to the first 4 hours of the recovery period and to the last 2 hours of the dark phase of the recovery period (Figure 3). NREM delta power was significantly increased in the first 2 hours of recovery sleep (paired t- test, t (7) = 6.1, P <.001). Because there are differences in baseline sleep between ob/ob and wt mice in the dark phase, we chose to make genotype comparisons based on the magnitude of their rebound response relative to baseline, rather than the absolute values of recovery sleep. Compared to baseline levels, ob/ob had a significantly smaller rebound in NREM (ob/ob, ± 3.4 vs. wt,

13 ± 15.4 %, unpaired t-test, t (12) = 2.9, P<.05) and REM (ob/ob, ± 10.4 vs. wt, ± 59.1 %, unpaired t-test, t (12) = 2.6, P<.05) sleep during the 12-hr dark phase compared to wt mice. We examined changes in NREM sleep bout number and duration between the first 12 hrs of recovery sleep and the corresponding baseline period (Table 4). NREM recovery sleep in wt mice was primarily due to an increase in NREM bout number, whereas in ob/ob mice, the recovery was due to an increase in average NREM bout duration. We did not perform a bout analysis for REM sleep because the low amount of REM sleep in wt mice during the dark phase baseline recording precluded an accurate analysis of bout duration. Amount and circadian rhythm of wheel running activity is altered in ob/ob mice The number of running wheel revolutions was significantly decreased in ob/ob mice over the 24-hr period (genotype main effect, F (1,8) =150.7, p<.001) (Figure 4). A genotype x time interaction occurred (F (1,8) =250.7, p<.001), such that ob/ob mice exhibited more wheel running activity during the 12-hr light phase and less activity during the 12-hr dark phase compared to wt mice (Figure 4). Therefore, a dramatic genotype difference existed in the percentage of 24-hr activity expended during the dark phase (ob/ob, 58.2 ± 4.5 vs. wt, 96.5 ± 0.8 %, unpaired t-test, t (8) = 8.3, p<.001), and the light phase (ob/ob, 41.8 ± 4.5 vs. wt, 3.5 ± 0.8 %, unpaired t-test, t (8) = p<.001).

14 14 Discussion: The purpose of this study was to examine the effect of leptin deficiency on sleep architecture and homeostasis using the ob/ob mouse, a genetic model of impaired leptin production that results in severe obesity and accompanying symptoms of metabolic dysregulation (18, 49, 56). A number of alterations in baseline sleep patterns were detected in ob/ob mice compared to age-matched controls. Sleep fragmentation was notably elevated in ob/ob mice, including an increased number of arousals from sleep, frequent stage shifts, increased sleep bout numbers and decreased sleep bout durations (Table 2). Therefore, ob/ob mice awakened and fell asleep more frequently than wt mice and were unable to sustain individual sleep-wake bouts for a normal length of time. In addition, ob/ob mice had significantly elevated baseline NREM sleep time in the 12-hr dark phase, resulting in an overall 24-hr increase in total sleep time compared to wt mice (Figure 1). While the total amount of 24- hr REM sleep was similar between genotypes, ob/ob mice had significantly less REM sleep in the light and a non-significant increase in the dark phase. There is little data available to define a physiologically meaningful amount of sleep in rodents, and in fact, there is still debate about how much nightly sleep is important in humans. According to a recent National Sleep Foundation poll, the average amount of nightly sleep in humans has decreased by 1-2 hours within the past few decades (1), and these data have served as a catalyst to begin investigating the health consequences of chronic partial sleep restriction (43-45). Animal studies are only beginning to implement comparable paradigms and results from these studies will provide necessary to begin making interpretations about construct validity of effects sizes in sleep time. One interpretation is that the increased sleep time in ob/ob mice is more likely a compensatory response to increased sleep fragmentation or an effect of hormonal alterations, as discussed below, rather than a direct cause of their metabolic phenotype.

15 15 The amount of slow wave activity in the EEG during NREM sleep, called NREM delta (1-4 Hz) power, is used as a quantitative measure of sleep intensity and homeostatic sleep drive (7). In wt animals under baseline conditions, NREM delta power reached a peak at light onset, decreased across the light phase as sleep drive dissipated and was elevated during the dark phase in association with accumulating sleep drive (Figure 2). Even though ob/ob mice had normal levels of absolute NREM delta power (Table 1), they exhibited less diurnal variation in normalized NREM delta power across the 24-hrs (Figure 2), consistent with the possibility that they were unable to accumulate and dissipate sleep drive normally. It may be that this attenuated diurnal rhythm resulted from ob/ob mice sleeping more in the dark phase, therefore, dissipating sleep drive more evenly throughout the 24-hr period. Similarly, the increase in NREM sleep fragmentation during the light phase could have prevented the normal dissipation of sleep drive. It may also be that ob/ob mice have a more fundamental deficit in their circadian regulatory system, as discussed in later paragraphs. In response to sleep deprivation, both wt and ob/ob mice generated a compensatory increase in NREM and REM sleep time, as well as NREM delta power (Figure 3), indicating the basic mechanisms involved in generating the sleep homeostatic response were intact in ob/ob mice. It should be noted that the NREM and REM rebound in ob/ob mice was shorter in duration and smaller in magnitude compared to wt animals. Because ob/ob mice already had higher levels of baseline sleep time during the dark phase compared to wt mice, they may have been unable to generate the same magnitude of recovery response, referred to as a ceiling effect. Nonetheless, in the current sleep deprivation paradigm, ob/ob mice did not make up for lost sleep time to the same extent as wt mice. Another genotype difference emerged for NREM recovery sleep in that wt mice primarily increased NREM bout number, whereas ob/ob mice clearly increased NREM bout duration, another indication that ob/ob mice have an intact homeostatic response to sleep deprivation. The lack of increase in NREM bout duration during recovery in wt mice is probably

16 16 due to large variability in this measure during baseline sleep, since sleep time is low during the active phase in wt mice. Our data are consistent with findings in rats harboring a mutation in the leptin receptor, the obese Zucker rat (9, 26). In these animals, baseline NREM sleep time was increased and the diurnal sleep rhythm was flattened, although these results should be interpreted cautiously because lean Zucker rats were not used as controls (9). A later study demonstrated that during a brief 6 hr recording period the obese Zucker rats had normal sleep amounts, but an increase in sleep fragmentation compared to lean control rats (26). Despite some methodological limitations, these data parallel our findings in the ob/ob mouse and provide another example of altered sleepwake patterns in the context of impaired leptin signaling. The data from our study indicate that leptin deficiency, or some physiological consequence of leptin deficiency, has a negative impact on many aspects of sleep regulation in rodents. An important function of hormonal signals of metabolic status, such as leptin, may be to coordinate long-term energy homeostasis with multiple physiological processes, including the regulation of sleep architecture and sleep amount. More generally, an important function of such peripheral signals may be to modulate sleep in order to put the animal in an optimal state for anabolic or catabolic processes to be carried out. In support of this hypothesis is the finding that when food availability is restricted to the light phase, rats readjust their sleep-wake cycle so that they are awake more in the light and sleep more in the dark phase, therefore, maintaining temporal alignment between metabolic processes and sleep-wake state (36). Previous studies have shown that changes in energy balance influence sleep-wake patterns. Studies utilizing high-fat feeding and food restriction paradigms have shown that changes in energy consumption affect sleep-wake patterns (10, 20, 27). Also, hormones that are secreted in response to acute changes in energy status, such as cholecystokinin, ghrelin and insulin have demonstrated effects on sleep-wake patterns, particularly NREM sleep time and

17 17 NREM sleep intensity (29). Hyperinsulinemia is a component of the metabolic phenotype in ob/ob mice. Considering that ob/ob mice are both hyperphagic and hyperinsulinemic, their increased sleep time may represent increased postprandial sleep. An interesting future experiment will be to test the effects of insulin sensitizing agents on sleep-wake patters in ob/ob mice. The metabolic deficits in ob/ob mice include alterations in hypothalamic neurotransmitters involved in the control of feeding behavior and energy expenditure, which have also been shown to be involved in the control of sleep-wake states, such as histamine (38, 41) and orexin/hypocretin (24, 47, 52). The concentrations of hypothalamic histamine and the expression level of prepro-orexin mrna are reduced in ob/ob mice (19, 53), and the administration of leptin can increase histamine turnover in mice and pre-pro orexin mrna levels in rats (54, 55). Interestingly, orexin knockout (28, 51) and histidine decarboxylase deficient mice (31) have increased levels of sleep fragmentation. Therefore, one mechanism by which leptin is linked to sleep-wake pathways could involve interactions with histamine and orexin neurons, particularly since leptin receptors have been identified in regions of the hypothalamus where histamine and orexin/hypocretin are synthesized (16, 40). An interesting future experiment will be to determine whether leptin replacement is able to correct the sleep-wake deficits in ob/ob mice. The brainstem contains cholinergic and serotonergic nuclei important for the balance of sleep-wake states and integration with respiratory and metabolic function. Ob/ob mice have decreased levels of M2 muscarinic receptor protein in the brain and brainstem(12). Injections of the anticholinesterase agent, neostigmine, into the pontine reticular nucleus yields differential effects in wt and ob/ob mice on sleep time and respiratory control (12). Because the serotonergic system has been implicated in the control of feeding and energy expenditure, the raphé nucleus may represent another important brain region where sleep-wake and metabolic regulatory signals

18 18 intersect. Leptin receptor immunoreactivity has been identified in the dorsal raphé nucleus, and ob/ob mice have decreased levels of serotonin transporter mrna in dorsal raphé neurons (8). In combination, these findings present a number of candidate anatomical sites that may have important roles in coordinating sleep and metabolic activity. Recent studies have shown that adipose cells release a number of adipokines, including tumor necrosis factor (TNF) alpha and interleukin (IL)-6, and levels of these are high in obesity (22). There is substantial evidence that TNF alpha and possibly IL-6, as well as other cytokines, influence sleep-wake patterns (29). Therefore, it is possible that the changes in sleep in ob/ob mice are a response to an inflammatory component of obesity. There are few data on the circadian rhythms of ob/ob mice with regard to behavioral, physiological or molecular parameters (17, 37). In the current study, we found that the diurnal distribution of NREM sleep was slightly attenuated, but that the rhythm of REM sleep and NREM delta power are notably altered in ob/ob mice (Figures 1 and 2). Ob/ob mice had reduced activity levels and a pronounced flattening of the locomotor activity rhythm (Figure 4). Taken together, these results indicate that in ob/ob mice the output rhythmic signals to a variety of physiological systems are attenuated with respect to the amplitude of the signals. While the circadian system provides an important signal for the propensity and organization of sleep-wake states (7), recent data have emerged to suggest that circadian processes are also critically involved in energy homeostasis. For example, mice harboring a mutation in the circadian clock gene, Clock, develop obesity, hyperphagia, symptoms of the metabolic syndrome and alterations in the regulation of hypothalamic neuropeptides involved in metabolic signaling (50). In addition, environmentally induced disruption of the normal circadian alignment to the L:D cycle, as occurs in shift workers, has been associated with increased body mass index, insulin resistance and cardiovascular disease (11, 21). Interestingly, in an in vitro preparation, leptin administration had direct effects on circadian pacemaker cells,

19 19 suprachiasmatic nuclei (SCN), and induced phase shifts in the oscillation of these neurons (34). Future studies will be needed to determine whether ob/ob mice have a primary deficit in the function of the circadian pacemaker in the SCN and/or in central and peripheral circadian clock gene regulation. It will also be important to determine whether various rhythms in behavioral (i.e., feeding, activity), physiological (i.e., circulating hormones, hypothalamic neuropeptides, body temperature) and molecular (i.e., circadian clock genes) systems are misaligned in ob/ob mice. In the current study, we found that ob/ob mice have a lower body temperature in all sleep-wake states across all periods of the light-dark cycle compared to wt animals. While ob/ob mice did show the expected reduction in body temperature in NREM and REM sleep compared to wakefulness, the magnitude of the decrease was smaller than in wt mice. Because of the close interaction between body temperature and sleep-wake regulation, it is possible that thermoregulatory deficits in ob/ob mice (48) contribute, at least in part, to the disruption of their sleep-wake organization. In addition, ob/ob mice exhibit reduced baseline ventilation and an impaired hypercapnic ventilatory response (HCVR) during both wakefulness and sleep(23, 30). The respiratory changes during sleep in ob/ob mice are not likely to result from sleep apnea since there is no indication of upper airway closure in these mice (30). Therefore, the most notable feature of sleep in the ob/ob mice, increased sleep fragmentation, could result from the inability to regulate blood gas levels and the subsequent arousal responses to relieve CO 2 retention. These findings suggest that the ob/ob mouse represents a valuable animal model for studying the relationship between obesity, respiratory control and sleep. Summary Clinical and experimental studies have shown that chronic sleep loss is associated with impaired metabolic function. One question still to be examined in genetic animal models is whether sleep disruption (i.e. altered sleep architecture, diurnal rhythmicity or sleep amount)

20 20 precedes or is a response to the onset of metabolic disruption. It would not be surprising that sleep and metabolic processes act reciprocally on each other in the context of disease states such as obesity, diabetes and the metabolic syndrome. There are a number of ways in which leptin deficiency could lead to alterations in sleep patterns ranging from direct interactions with sleeprelated nuclei to indirect pathways via changes in hormone levels or circadian regulation. Animal models of leptin dysregulation, such as ob/ob and db/db (leptin receptor mutation) mice will be valuable in clarifying the mechanisms and pathways by which leptin is involved in complex behaviors, such as sleep and feeding.

21 21 Acknowledgements: This work was supported by grants from the National Institutes of Health (AG011412, AG018200, HL and HL075029).

22 22 Figure legends: Figure 1. Baseline sleep-wake patterns. Comparison of baseline sleep-wake states (percentage of recording time) in age-matched wild type (wt, filled circles and bars) and ob/ob (open circles and bars) mice. (Left panels) Distribution of wake, non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep in 2-hr intervals across a 12:12 L:D cycle. Values represent group means ± s.e.m. Recordings were initiated at light onset (zeitgeber time, ZT0) and the dark phase is indicated by the shaded background. (Right panels) Amounts of baseline sleep-wake states were averaged over the 12-hr light, 12-hr dark and 24-hr intervals for each genotype. In both panels, ANOVA revealed 24-hr and time of day differences in sleep amounts between wt and ob/ob mice. *p<.05; **p<.01, ***p<.001. Figure 2. Baseline sleep homeostasis: NREM delta power. Distribution of sleep homeostatic drive across the 12:12 L:D cycle in wild type (wt, filled circles) and ob/ob (open circles) mice. The average power density in the delta (1-4 Hz) frequency range was determined for NREM sleep in 2-hr intervals. These values were normalized and graphed as a percentage of 24-hr NREM delta power. Recordings were initiated at light onset (zeitgeber time, ZT0) and the dark phase is indicated by the shaded background. ANOVA testing revealed a number of time points at which wt and ob/ob mice differed. *p<.05; **p<.01, ***p<.001. Figure 3: Recovery from sleep deprivation in wild type (wt) and ob/ob mice. Animals were sleep deprived the last 6 hrs of the light phase, indicated by "SD" on the x-axis. The recovery phase, noted as "Recovery" on the x-axis began at dark onset (zeitgeber time, ZT12) and continued throughout the 12-hr dark period (indicated by the shaded background) and the 6-hr light phase. (Left panels) Wt baseline (filled circles) and recovery (open circles) patterns for NREM sleep, REM sleep and absolute NREM delta power graphed in 2 hr intervals. ANOVA was used to

23 23 make comparisons between baseline and recovery conditions. (Right panels) The same comparisons and analyses were made for baseline (filled circles) and recovery (open circles) patterns in ob/ob mice. *p<.05; **p<.01, ***p<.001. Figure 4: Diurnal pattern of wheel running activity. Wild type (wt, filled circles and bars) and ob/ob (open circles and bars) mice at 9 weeks of age. Wheel running counts were collected and averaged (mean ± s.e.m.) over a 7-day recording period, in order to reduce within group variability. (Top panel): The number of wheel rotations per 6-minute interval were plotted across a 12:12 L:D cycle to compare the circadian rhythm of activity between genotypes. Recordings began at light onset (zeitgeber time, ZT0) and the dark phase is indicated on the x-axis by the shaded horizontal bar. (Middle panel): The number of wheel rotations was accumulated over 12- hr light, 12-hr dark and 24-hr intervals. ANOVA was used to make genotype and time comparisons. *p<.05; **p<.01, ***p<.001.

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27 27 Table 1. NREM EEG in wild type and ob/ob mice wt Delta (1-4 Hz) Theta (4-8 Hz) Sigma (11-15 Hz) Light (40.3) (46.9) 61.0 (11.7) Dark (56.9) (50.6) 60.7 (11.9) 24-hrs (44.0) (47.5) 61.0 (11.7) ob/ob Light (52.6) (57.1) 69.8 (13.6) Dark (55.7) (60.8) 75.3 (14.2) 24-hrs (53.1) (57.5) 71.8 (13.8) Genotype (G) Time (T) ** *** GXT EEG average power density (uv 2 /Hz) for delta, theta and sigma frequency (Hz) bands during non-rapid eye movement (NREM) baseline sleep. Values were calculated for the 12-hr light (Light), 12-hr dark (Dark) and total 24-hr period in wild type (wt, N=9) and ob/ob (N=8) mice. ANOVA was used to identify main effects and interactions for Genotype and Time factors in each frequency range (** P<.01, *** P<.001).

28 28 Table 2. Baseline sleep/wake fragmentation wt ob/ob wt ob/ob Arousals Light Dark 24-hrs 75.8 (4.5) 33.4 (4.4) (7.2) 87.9 (4.2) 61.9 (2.4) *** (4.5) *** SS Light Dark 24-hrs (113.7) (17.6) (25.2) (25.8) (18.1) (38.8) *** #W Light Dark 24-hrs # NREM Light Dark 24-hrs 74.1 (5.2) 51.2 (16.2) (9.5) (5.9) 77.1 (8.1) (11.6) (10.3) 84.0 (7.4) (15.2) ** (10.2) (8.1) 324.9(15.2) *** Dur W Light Dark 24-hrs Dur NREM Light Dark 24-hrs 3.4 (0.3) 11.6 (1.9) 6.5 (0.9) 3.0 (0.1) 2.6 (0.2) 2.8 (0.2) 2.3 (0.2) 5.5 (0.5) ** 3.6 (0.3) ** 2.22 (0.2) 1.9 (0.1) 2.1 (0.1) ** # REM Light Dark 24-hrs 42.3 (2.0) 11.3 (1.4) 53.7 (2.2) 27.0 (4.0) 17.3 (1.9) 54.3 (5.6) Dur REM Light Dark 24-hrs 1.3 (0.04) 1.3 (0.1) 1.3 (0.04) 1.2 (0.04) 1.1 (0.1) 1.2 (0.04) Comparison of sleep fragmentation between wild type (wt) and ob/ob mice across the 12-hr light, 12-hr dark and total 24-hr recording period. Measurements include the number (#) of arousals, stage shifts (SS), wake (W) bouts, non-rapid eye movement (NREM) sleep bouts and rapid eye movement (REM) sleep bouts, as well as the average duration (Dur), in minutes, of invididual W, NREM and REM sleep bouts. All values represent group means ± s.e.m. ANOVA was used to make genotype and time comparisons. ** p<.01, *** p<.001.

29 29 Table 3. Body temperature in wild type and ob/ob mice wt Wake NREM REM Light (0.22) (0.25) (0.26) Dark (0.25) (0.29) (0.32) 24-hrs (0.24) (0.26) (0.27) ob/ob Light (0.14) (0.19) (0.19) Dark (0.17) (0.19) (0.20) 24-hrs (0.15) (0.18) (0.18) Genotype (G) *** *** ** Time (T) *** *** *** G X T Body temperature ( C) during the baseline sleep-wake recording was averaged over the 12-hr light (Light), 12-hr dark (Dark) and total 24-hr period in wild type (wt, N=7) and ob/ob (N=8) mice. Genotype and time comparisons were made for wake, non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM) using ANOVA. (**P<.01, *** P<.001).

30 30 Table 4. NREM bout number and duration: recovery from sleep deprivation wt Bout # Base Recov Bout Dur Base Recov ob/ob Bout # Base Recov Bout Dur Base Recov ZT 12 ZT 14 ZT 16 ZT 18 ZT 20 ZT (1.5) 16.7(2.9) * 2.9(0.9) 3.2(0.6) 19.9(3.6) 21.6(3.1) 8.3(2.7) 15.5(3.0) * 2.6(0.3) 3.2(0.4) 21.6(2.5) 20.4(2.7) 9.0(3.7) 15.7(1.4) 2.2(0.5) 3.2(0.4) 24.9(2.6) 22.1(4.1) 20.5(3.3) 17.8(2.8) 3.1(0.4) 3.9(0.7) 32.0(2.4) 30.6(3.1) 16.7(1.7) 16.3(1.7) 2.5(0.3) 3.7(0.7) 23.5(2.6) 24.5(3.2) 8.5(1.8) 6.5(2.3) 3.5(0.3) 3.2(0.7) 16.5(2.2) 22.8(2.5) * 1.4(0.1) 2.8(0.4) ** 1.7(0.1) 2.6(0.2) ** 2.3(0.3) 2.5(0.6) 2.2(0.2) 2.3(0.2) 1.9(0.1) 2.2(0.3) 2.1(0.2) 2.1(0.2) The number of NREM bouts (Bout #) and average duration of NREM bouts (Bout Dur, minutes) were determined in wild type (wt, N=6) and ob/ob (N=8) mice for the first 12 hours of recovery sleep (Recov) and the corresponding baseline (Base) period. Data were divided into 2-hr intervals starting at dark onset (zeitgeber time, ZT12). Statistical comparisons between baseline and recovery were made separately at each time interval using paired t-tests (*p<.05, **p<.01, p=.07).

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