Interleukin-1β has a Role in Cerebral Cortical State-Dependent Electroencephalographic Slow-Wave Activity

Transcription

1 MS 273.qxp 1/14/ :55 AM Page 177 Interleukin-1β has a Role in Cerebral Cortical State-Dependent Electroencephalographic Slow-Wave Activity Tadanobu Yasuda, PhD, MD 1 ; Hitoshi Yoshida, PhD, MD 2 ; Fabio Garcia-Garcia, PhD 1 ; Daniel Kay, HS 1 ; James M. Krueger, PhD 1 1 Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, Washington; 2 Department of Anesthesiology, University of Hirosaki School of Medicine, Hirosaki, Japan Study Objectives: To investigate the hypothesis that interleukin (IL)-1β is involved in mediating localized electroencephalogram synchronization. Design: We evaluated bilateral cortical electroencephalograms after unilateral local application of IL-1β onto the somatosensory cortex of rats. Furthermore, we investigated the effects of unilateral application of an IL-1β inhibitor, the IL-1 soluble receptor, on spontaneous sleep and sleep rebound after sleep deprivation. Setting: University research laboratory. Interventions: N/A. Participants: Rats Measurements and Results: Neither dose of IL-1β or the IL-1 soluble receptor affected the duration of non-rapid eye movement sleep or rapid eye movement sleep. Unilateral application of IL-1β induced state- and frequency-dependent electroencephalogram asymmetries. During non-rapid eye movement sleep, but not during other states, electroencephalographic slowwave activity was greater on the side that received IL-1β (10- and 50-ng doses). Electroencephalographic power in the higher frequencies was not affected by IL-1β in any state. Unilateral application of the IL-1 soluble receptor (0.1, 1.0 and 5.0 µg) had no effect on the spontaneous sleep electroencephalogram. In contrast, unilateral application of the IL-1 soluble receptor (5.0 µg) attenuated sleep deprivation-enhanced electroencephalographic slow-wave power ipsilaterally during non-rapid eye movement sleep. Conclusions: Results suggest that IL-1β can induce state-dependent localized increases of electroencephalographic delta wave power, suggesting an enhancement of sleep intensity within the cortex. Key Words: Sleep, EEG, FFT, cytokine, brain, TNF and sleep deprivation Citation: Yasuda T; Yoshida H; Garcia FG et al. Interleukin-1β has a Role in Cerebral Cortical State-Dependent Electroencephalographic Slow- Wave Activity. SLEEP 2005;28(2) INTRODUCTION SLEEP IS USUALLY REGARDED AS A WHOLE-BRAIN PHE- NOMENON IMPOSED UPON THE BRAIN BY SLEEP-REGU- LATORY CIRCUITS. The basal forebrain-hypothalamus is one such circuit, and many sleep-regulatory substances act in the basal forebrain-hypothalamus to promote non-rapid eye movement (NREM) sleep. 1 For instance, interleukin (IL)-1β increases firing rates of sleep-active neurons and inhibits wake-active neurons in the basal forebrain-hypothalamus, 2 and IL-1β increases intracellular calcium levels in cultured hypothalamic neurons. 3 However, another view regards sleep as an intrinsic emergent property of groups of highly interconnected neurons (called neuronal groups). In this view, sleep is driven by activity-induced production of sleep-regulatory substances that act locally to induce functional state shifts within neuronal groups. Sleep-regulatory circuits play a similar role, in either view, to coordinate and aggregate neuronal activity into an organism state. However, the neuronal group functional state theory provides a mechanism to target sleep to neuronal groups dependent upon their prior activity. Thus, the new view posits that production of sleep-regulatory substances such as IL-1 is the mechanism by which the brain keeps track of prior activity Disclosure Statement This is not an industry supported study. Drs. Yasuda, Yoshida, Garcia-Garcia, Kay, and Krueger have indicated no financial conflicts of interest. Submitted for publication August 2004 Accepted for publication September 2004 Address correspondence to: James M. Krueger, PhD, Washington State University, College of Veterinary Medicine, Department of VCAPP, PO Box , Pullman, WA ; Tel: (509) ; Fax: (509) ; Krueger@vetmed.wsu.edu SLEEP, Vol. 28, No. 2, and thereby provides a mechanistic explanation of sleep homeostasis. In contrast, the classic literature regarding the sleep-regulatory circuit does not adequately address this issue. 1 IL-1β is a well-characterized sleep-regulatory substance. Administration of exogenous IL-1β increases NREM sleep in the species thus far tested, including rabbits 4, rats, 5,6 mice, 7 and cats. 8 In contrast, inhibition of endogenous IL-1β activity by administration of an IL-1 receptor fragment, an IL-1 soluble receptor (IL-1SR), the IL-1 receptor antagonist, or anti-il-1 antibodies attenuates spontaneous sleep and sleep rebound induced by sleep deprivation. 1 Brain levels of IL-1β mrna, cerebrospinal fluid levels of IL-1, and plasma levels of IL-1β vary with the sleep-wake cycle, with highest levels correlating with high sleep propensity. 9,10 Brain levels of IL-1β mrna also increase after sleep deprivation. 11,12 IL-1β promotes sleep by acting on several known sleep-regulatory circuits such as the locus coeruleus, 13 the dorsal raphe, 14 and the basal forebrainhypothalamus. 15 Tumor necrosis factor-α (TNFα) is another well-characterized sleep-regulating substance. Previously we reported that unilateral microinjection of TNFα onto the somatosensory cortex of rats induces state- and frequency-dependent electroencephalogram (EEG) asymmetries. 16 In contrast, unilateral injection of the TNFα soluble receptor (a competitive inhibitor of TNFα) attenuates EEG slow-wave activity (SWA) during NREM sleep after sleep deprivation. Because the activities of TNF and IL-1 are related, 17 we hypothesized that unilateral local application of IL-1β would induce EEG asymmetries. We thus injected IL-1β onto the somatosensory cortex of rats unilaterally. Further, we investigated the effects of an IL-1β inhibitor, an IL-1SR, on spontaneous sleep and sleep rebound after sleep deprivation.

2 MS 273.qxp 1/14/ :55 AM Page 178 MATERIALS AND METHODS Agents Rat recombinant IL-1β and recombinant human IL-1SR were purchased from R&D Systems, Inc. (Minneapolis, Minn). These agents were dissolved in pyrogen-free saline (PFS; Abbott Laboratories, North Chicago, Ill) and stored at -80 C until used in the experiments. IL-1β and IL-1SR were dissolved at concentrations of 1, 10, and 50 ng in a volume of 2 µl or 0.1, 1, and 5 µg in a volume of 2 µl, respectively. Animals Male Sprague-Dawley rats weighing 300 to 400 g were obtained from Taconic Farm, Inc. (Germantown, NY). The use of rats in these experiments was in accordance with Washington State University guidelines and was approved by the Animal Care and Use Committee. The rats were kept on a 12-hour:12-hour light-dark cycle (lights on at 9:00 AM) at 23 C ± 2 C ambient temperature. Water and food were available ad libitum throughout the experiment. Surgeries were performed under ketamine-xylazine (87 and 13 mg/kg, respectively) anesthesia. The rats were provided with a stainless-steel EEG electrode (model E363/20, Plastics One Inc, Roanoke, Virg) over the somatosensory cortex on both sides of the brain and with guide cannulas for injection whose tips were under the electrodes between the surface of the somatosensory cortex and the dura on each side of the brain. The stereotaxic coordinates for the electrodes were 2.5 mm posterior to the bregma and ± 5.5 mm bilaterally to the midline. Another EEG electrode used as the common reference was implanted 10 mm posterior from the bregma on the midline over the cerebellum. A stainless-steel electromyogram (EMG) electrode (model E363/78, Plastics One, Inc.) was placed in the dorsal neck muscles. Insulated leads from the electrodes were routed to a miniature plug and attached to the skull with dental cement. The reference electrode and cage were grounded. The position of guide cannula was verified as previously described. 16 Briefly, 2 µl of 20% lidocaine was injected, and a reduction in EEG power in all frequency bands verified cannula placement. Further, after experiments were over, in some rats 1% hematoxylin QS was injected through the cannula. The dye diffused across the surface of the cortex unilaterally. It did not reach the lateral ventricle. Recording and Analysis The rats were given at least 10 days to recover from surgery and then placed in the recording chambers for 3 days of acclimation. The rats were allowed relatively unrestricted movement inside the recording cages. A flexible tether connecting the EEG and EMG electrodes led to an electronic swivel, and cables from the swivel were routed to 3 amplifiers separately, 2 for EEG and 1 for EMG. The amplified signals were digitized at the frequency of 128 Hz. The EEG was filtered below 0.1 Hz and above 35 Hz. The EEG signals from each side of the brain were subjected to on-line fast Fourier transformation, as previously published. 18,19 The vigilance states of wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep were determined off line in 10-second epochs using criteria previously published. 18 The average amount of time spent in each vigilance state was calculated for 3-hour intervals. The EEG power densities were calculated using our original program. 18,19 The EEG power (µv 2 ) for each rat in each 1-Hz frequency bandwidth for each side of the brain was determined under baseline conditions (saline injection) and under experimental conditions. These values were used for the calculations illustrated in Figures 3 and 4. In addition, EEG SWA during NREM sleep was calculated as previously described. 18 Thus, the average power of EEG SWA throughout the entire 23-hours control-recording period was normalized to 100% for each animal. Then all EEG SWA data were expressed as a percentage of that control value. These EEG SWA values are shown in Figures 2 and 4. Experimental Protocol Experiment I: Effects of Unilateral Administration of IL-1β on EEG Activity Twenty-four rats were used for the experiment. All rats were injected unilaterally with 2 µl of the vehicle, PFS, on a control day. The next day, 1 of the following doses of IL-1β was injected onto the same side: 1, 10, and 50 ng in 2 µl of PFS (n = 8; injection side; right n = 4, left n = 4). These injections took place between 2:50 and 3:10 PM. After injections, EEG and EMG were recorded for the next 23 hours. Experiment II: Effects of Unilateral Administration of the IL-1SR on EEG Activity of Spontaneous Sleep Eighteen rats were used for the experiment. On the control day, all rats were injected unilaterally with 2 µl of the vehicle, PFS. The next day, 1 of the following doses of the IL-1 SR was injected onto the same side: 0.1, 1, and 5 µg in 2 µl of PFS (n = 6; injection side; right n = 3, left n = 3). These injections took place between 8:50 and 9:10 AM. After injections, EEG and EMG were recorded for the next 23 hours. Experiment III: Effects of Unilateral Administration of IL-1SR on EEG Activity of Sleep Rebound After Sleep Deprivation Six rats were used for the experiment. On the control day, all rats were injected unilaterally with 2 µl of the vehicle, PFS, between 8:50 and 9:10 AM (injection side; right n = 3, left n = 3). After injections, the rats were kept inside the recording cages without any restriction for 6 hours. EEG and EMG were recorded from 3:00 PM for the next 23 hours. Two days later, all rats were injected onto the same side with 5 µg of the IL-1SR in 2 µl of PFS between 8:50 and 9:10 AM. Then, a sleep deprivation took place during the initial 6 hours of the light period after the injections. Sleep deprivation was by gentle handling. The 23 hours of recordings of EEG and EMG were started after the 6 hours of sleep deprivation. In a previous study, 16 we showed that unilateral injection of PFS prior to the beginning of sleep deprivation had no effect on any of the sleep or EEG parameters. Statistical Analysis All data are presented as mean ± SEM. Two-way analysis of variance (ANOVA) for repeated measures followed by the Student-Newman-Keuls test were used to analyze data. A significance level of P <.05 was accepted. SLEEP, Vol. 28, No. 2,

3 MS 273.qxp 1/14/ :56 AM Page 179 RESULTS Experiment I: Effects of Unilateral Administration of IL-1β on EEG Activity Unilateral injection of IL-1β onto the somatosensory cortex did not affect the duration of NREM or REM sleep, irrespective of dose (Figure 1). Further, the number of and the average duration of NREM or REM sleep episodes were not affected (Table 1). Unilateral injection of the lowest dose (1 ng) of IL-1β had no effect on EEG SWA during NREM sleep on the side receiving IL- 1β (Figure 2). In contrast, unilateral injection of the 10-ng dose of IL-1β induced a significant increase in EEG SWA during NREM sleep on the side receiving IL-1β (ANOVA, treatment effect: F 1,7 = 9.046, P=.02; with time-treatment interaction: F 7,49 =6.700, P <.001) but not on the contralateral side. This effect persisted during the first 9 hours after injection after the 10-ng IL-1β dose (Figure 2). Similarly, unilateral injection of the 50-ng dose of IL-1β increased EEG SWA during NREM sleep during the first 6 hours after injection (ANOVA, time-treatment interaction: F 7,49 = 2.272, P <.05) (Figure 2). The 10-ng IL-1β dose enhanced EEG power unilaterally within the 0.5- to 6-Hz frequency band (ANOVA, treatment effect: F 1,7 =7.830, P =.027; with frequency-treatment interaction: F 24,168 = 6.91, P<.001) in NREM sleep during first 3 hours after IL-1β injection. This effect was not observed during REM sleep or wakefulness (Figure 3). Similarly, the 50-ng IL-1β enhanced EEG power in the 0.5- to 7-Hz frequency band unilaterally (ANOVA, treatment effect: F 1,7 =13.884, P=.007; with frequen- Figure 1 Effects of unilateral injection of interleukin (IL)-1β onto the surface of the somatosensory cortex on non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS). Data are from the rats used in Experiment I. One of the following doses of IL-1β 1 ng (n = 8), 10 ng (n = 8), and 50 ng (n=8) was injected. Open and closed circles represent vehicle control and IL-1β treatments, respectively. All data shown are averages obtained from 3-hour time blocks ± SEM. Hatched bars show dark hours. Table 1 Effects of unilateral injection of interleukin-1β onto the surface of the somatosensory cortex on sleep. Condition NREM sleep REM sleep Dose, ng n Time in NREM Number Duration of Time in REM Number Duration of sleep, min of episodes episodes, min sleep, min of episodes episodes, min ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.06 Values are mean ± SEM. Data are expressed as the time spent in non-rapid eye movement sleep (NREM sleep) or rapid eye movement sleep (REM sleep), the number and length of the episodes during the 23-hour recording period. The number and length of the episodes were determined using a computer program with the criterion that each episode lasted at least 30 seconds. No significant differences between the saline group and the interleukin-1β group were observed. SLEEP, Vol. 28, No. 2,

4 MS 273.qxp 1/14/ :56 AM Page 180 cy-treatment interaction: F 24,168 = , P <.001). The 50-ng IL-1β dose also did not affect the EEG power spectrum during REM sleep or wakefulness (Figure 3). Experiment II: Effects of Unilateral Administration of the IL-1SR on EEG Activity During Spontaneous Sleep Neither dose of the IL-1SR affected the duration of spontaneous NREM or REM sleep. The number of, and average duration of, NREM or REM sleep episodes were not affected (Table 2). Further, power-spectrum analysis showed that unilateral injection of the IL-1SR onto the somatosensory cortex did not alter EEG power in any frequency bands in any vigilance state (data not shown). Experiment III: Effects of Unilateral Administration of the IL-1SR on EEG Activity During Sleep Rebound After Sleep Deprivation Unilateral injection of PFS had no effect on any of the parameters recorded (data not shown). Sleep deprivation induced significant increases in NREM and REM sleep duration (Table 2), and these effects continued for 9 hours. Sleep loss also enhanced EEG SWA during NREM sleep on both sides of the brain. These data are consistent with multiple prior studies. However, there was a time-dependent difference in EEG SWA between the side receiving the IL-1SR and the noninjection side during the 23-hour recording period (ANOVA, timetreatment interaction: F 7,35 = 6.019, P <.001). These effects resulted from a significant reduction in EEG SWA during NREM Figure 2 Effects of unilateral injection of interleukin (IL)-1β onto the surface of the somatosensory cortex on electroencephalographic (EEG) slow-wave activity (SWA) during non-rapid eye movement (NREM) sleep. Data are from the rats used in Experiment I. One of the following doses of IL-1β 1 ng (n = 8), 10 ng (n = 8), and 50 ng (n = 8) was injected. Open and closed circles represent vehicle control and IL-1β treatments, respectively. All data shown are averages obtained from 3-hour time blocks ± SEM. Percent SWA values are EEG delta-wave amplitudes. Average power throughout the 23-hour controlrecording period was normalized to 100%, and then all SWA data were converted to relative percentage values. Hatched bars show dark hours. *indicates significant difference P <.05. SLEEP, Vol. 28, No. 2,

5 MS 273.qxp 1/14/ :56 AM Page 181 sleep on the side receiving the IL-1SR for the first 6 recording hours (Student-Newman-Keuls test). About 9 hours after the end of sleep deprivation, EEG SWA decreased to below control level on both sides (Figure 4). During first 3 hours after sleep deprivation, the IL-1SR reduced EEG power in the 0.5- to 5-Hz frequency band unilaterally (ANOVA, frequency-treatment interaction: F 24,120 = , P <.001) during NREM sleep but not during REM sleep or wakefulness. Although EEG theta activity on both sides of the brain increased during REM sleep and wakefulness, this effect was not significantly altered by the IL-1SR (Figure 4). DISCUSSION The major findings of this study are that unilateral application of IL-1β onto the somatosensory cortex of rats induced state- and frequency-dependent EEG asymmetries and that unilateral application of an IL-1SR onto the somatosensory cortex attenuated sleep deprivation-enhanced EEG slow-wave power ipsilaterally during NREM sleep. These results suggest that IL-1 can act locally, whether exogenous IL-1 is microinjected or whether IL- 1 is allowed to accumulate endogenously during sleep loss, to affect EEG power and sleep intensity. The idea that local events are responsible for localized EEG SWA is supported by many experimental findings. Cortical islands, isolated from thalamic input, wax and wane through states of high-amplitude slow waves. 20,21 More recently, it was shown that a slow component ( Hz) of the EEG is of cortical origin. 22 Further, a cellular model of EEG SWA is consistent with the idea that EEG SWA is localized to the corticothalamic circuits involved. 23 We have developed preliminary data indicating that the application of IL-1β onto the somatosensory cortex activates Fos immunoreactivity within the somatosensory cortex and the reticular nucleus of the thalamus. 24,25 The reticular nucleus of the thalamus receives input from the cortex and is involved in sleep regulation via its output to other thalamic nuclei. 26 Evoked response potentials of auditory cortical columns oscillate between 2 functional states as defined by the amplitude of the evoked response potential; one state most often occurs during sleep while the other occurs during wakefulness. 27,28 These cortical column functional states manifest independence from each other and from organism state, in that sometimes the functional state usually associated with sleep occurs during wakefulness. Further, the probability of a functional state occurring is dependent upon prior sleep-wake activity. 28 Collectively, such data suggest that local regulatory events control functional state expression within groups of highly interconnected neurons. Sleep-regulating substances constitute Process S in the 2- process model of sleep regulation. 29 EEG SWA (EEG power between 0.5 and 4 Hz) during NREM sleep is enhanced after sleep deprivation in animals and humans, 33 and arousal thresholds correlate with EEG SWA. 34 Furthermore, several Figure 3 Effects of unilateral injection of interleukin (IL)-1β onto the surface of the somatosensory cortex on electroencephalogram (EEG) power spectrum during each vigilance state. Data are from the rats used in Experiment I. One of the following doses of IL-1β 1 ng (n = 8), 10 ng (n = 8), and 50 ng (n = 8) was injected. Analyzed data are from 0 to 3 hours after injection, the period of maximum EEG slow-wave activity (SWA) effects (see Figure 2). EEG power-spectrum analyses during each vigilance state were performed for the 0.5- to 25-Hz frequency range. The EEG power (µv 2 ) for each rat in each 1-Hz frequency bandwidth was determined on a saline baseline day and on the IL-1β-treatment day for both sides of the brain. The values obtained on baseline day for each side were subtracted from values obtained from the same side obtained on the experimental day after no injection (open circles) or IL-1β treatment days (closed circles). *indicates significant differences, P <.05. NREMS refers to non-rapid eye movement sleep; REMS, rapid eye movement sleep. SLEEP, Vol. 28, No. 2,

6 MS 273.qxp 1/14/ :56 AM Page 182 sleep-regulating substances, eg, IL-1β, enhance EEG SWA during NREM sleep. 1 SWA is, therefore, thought to reflect sleep intensity during NREM sleep and is used to model Process S. 29 Several prior observations support the notion that unilateral localized disproportionate stimulation of part of the brain induces a higher intensity of sleep in the stimulated area and, thus, asymmetries in the EEG SWA. Prolonged stimulation of the right hand of human subjects increases EEG SWA during subsequent sleep in the left cortex. 35 Similar results have been obtained from rats after unilateral facial-whisker removal and sleep deprivation; the side ipsilateral to the cut whiskers thus received disproportionate sensory stimulation, and it exhibited enhanced EEG SWA in subsequent sleep. 36 Furthermore, enhanced EEG SWA during sleep following a specific motor learning task is localized unilaterally to associative cortical areas corresponding to Brodmann areas 7 and 40 cortex and, thus, interpreted as a manifestation of local sleep. 37 Finally, some marine mammals, such as dolphins, never have high-amplitude EEG slow-wave NREM sleep simultaneously in both cerebral hemispheres and, thus, clearly demonstrate state-dependent EEG asymmetries. 38 Whether such effects are due in part to IL-1β remains unknown. We have proposed that local functional state changes occur as the result of the activity-dependent production of sleep-regulating substances such as IL-1β. 39 Such sleep-regulating substances act as autocrines, juxtacrines, and paracrines to alter input-output activity of nearby neurons within the neuronal group where they are made. Thus the input-output relationships of a neuronal group would be different in the presence of certain sleep-regulating substances, and, thus, a different functional state would occur. Within the fever literature, it is well known that IL-1β can induce altered input-output relationships within the hypothalamus and, hence, a different functional state (fever). 40 Current results suggest that IL- 1β can also initiate input-output changes within the cortex. There is only limited evidence that IL-1β is produced as a result of neuronal activity. IL-1β production is enhanced in brain during seizure-like activity, 41 and this is sleep dependent. 42 Brain IL-1β mrna levels have a diurnal rhythm with highest levels occurring at the onset of daylight hours in rats, suggesting an accumulation of IL-1β mrna occurring during the prior nighttime active period. 9,43 Brain IL-1β mrna levels also increase during sleep deprivation. 11,44 Finally, as mentioned above, IL-1β applied to the cortex induces Fos-immunoreactivity activation in reticular thalamic neurons. Juxtapositioned next to these Fos-positive cells are glia expressing IL-1β-immunoreactivity, 24 thereby suggesting that the neuronal activity driving the Fos expression may also be responsible for the expression of IL-1β in nearby cells. Such findings are exciting, since glia may play a role in EEG SWA. 45 Although systemic IL-1β can induce IL-1β mrna brain production via afferent neuronal input, 46 the issue of localized activity-dependent production of IL-1β in sleep needs further clarification. There are some differences between the effects of TNFα and IL-1β applied to the somatosensory cortex. For instance, after higher doses of TNFα, EEG power has been shown to be enhanced across a broader frequency range, and these increases extended into REM sleep and wakefulness (similar effects occur after prolonged sleep loss but not after shorter periods of sleep deprivation). 16 In contrast, after IL-1β, the higher-dose-induced increases in EEG SWA remained state specific, occurring only during NREM sleep (Figure 3). The reasons for such differences remain unknown. IL-1β and TNFα induce each other, and one might thus anticipate similar actions by both molecules. However, in mice lacking the type I IL-1 receptor, their sleep deficit occurs during the first few nighttime hours. 7 In contrast, in mice lacking the TNF 55 kd receptor, the sleep deficit occurs mostly during the daytime. 47 Further, IL-1-enhanced sleep is inhibited by the soluble TNF receptor, and, conversely, TNF-enhanced sleep is inhibited by the IL-1 soluble receptor. 17 However, the time courses of these inhibitory effects are different, suggesting unique disturbances by each of the soluble receptors. 17 In a pilot study, we tested another member of the sleep molecular network by injecting the adenosine agonist CGS Neither a 50- nor a 100-ng dose of this material affected the EEG under our recording conditions. We also tested prostaglandin D 2 (1, 10, and 100 nmole), and it also failed to affect any of the parameters measured. In yet another pilot study, we injected 2 µl of 3 M KCl unilaterally to induce spreading depression; no EEG asymmetries under our recording conditions were detected. Such results suggest that current data represent some degree of speci- Table 2 Effects of unilateral injection of interleukin-1soluble receptor onto the surface of the somatosensory cortex on spontaneous sleep and recovery from 6-hour sleep deprivation Condition NREM sleep REM sleep n Time in NREM Number Duration of Time in REM Number Duration of sleep, min of episodes episodes, min sleep, min of episodes episodes, min Saline ± ± ± ± ± ± 0.16 IL-1SR 0.1µg ± ± ± ± ± ± 0.07 Saline ± ± ± ± ± ± 0.10 IL-1SR 1µg ± ± ± ± ± ± 0.08 Saline ± ± ± ± ± ± 0.14 IL-1SR 5µg ± ± ± ± ± ± 0.13 Spontaneous sleep ± ± ± ± ± ± 0.10 Recovery from ± 30.1* ± ± ± 12.7* 73.3 ± 6.4* 1.72 ± 0.09 sleep deprivation Values are mean ± SEM. Data are expressed as the time spent in non-rapid eye movement sleep (NREM sleep) or rapid eye movement sleep (REM sleep), the number and length of the episodes during the 23-hour recording period. The number and length of the episodes were determined using a computer program with the criterion that each episode lasted at least 30 seconds. *P <.05 versus control. No significant differences between the saline group and the IL-1 soluble receptor (IL-1SR) group during spontaneous sleep were observed. Sleep deprivation significantly enhanced total amount of NREM sleep and REM sleep and number of REM sleep episodes. SLEEP, Vol. 28, No. 2,

7 MS 273.qxp 1/14/ :56 AM Page 183 ficity in that, thus far, only IL-1β and TNFα have induced EEG asymmetries after unilateral application to the cortex. IL-1β is a cerebral vasodilator, and, as such, it could affect EEG power. However, this seems unlikely to account for the current results. The other substances injected, the adenosine agonist (GS-21680), prostaglandin D 2, and KCl affect cerebral blood flow but had no effect on EEG power. There is an inverse correlation between delta and spindle activity and regional cerebral blood flow 48 ; thus, if IL-1 acted as a vasodilator, this action should decrease rather than increase delta activity. Further, in our conditions, IL-1β only affected delta activity, not spindle activity (12-15 Hz), and the effects we observed were state dependent. Finally, the IL-1SR, a vasoconstrictor, did not affect spontaneous EEG delta-wave activity. That the IL-1SR unilaterally inhibited sleep loss-induced increases in EEG SWA on the side injected (Figure 4), strongly implicates IL-1β in the mechanisms responsible for sleep lossenhanced EEG activity. However, the IL-1SR failed to alter EEG SWA during spontaneous sleep (Table 2). We do not know the reasons for this failure, although if the IL-1SR is given intracerebroventricularly in doses that inhibit the duration of NREM sleep, it does not affect EEG SWA during NREM sleep. 49 It is possible that, in order to affect spontaneous EEG SWA, the IL-1SR would have to be given at nighttime because it is during those hours that mice lacking a functional IL-1 receptor sleep less 7 ; in the current study, the IL-1SR was given during daytime hours. It is also possible that a different, or more extensive, set of IL-1 receptors are activated during NREM sleep after sleep loss than during spontaneous NREM sleep and that access to those receptors involved in spontaneous sleep regulation by the microinjected IL-1SR is more difficult. Further, the state of the sleep molecular network is likely very different during spontaneous sleep than during sleep following sleep deprivation due to the large number of feedback molecules induced by the molecular dynamics occurring during sleep loss; one might anticipate that the effects of a single feedback molecule, such as the IL-1SR, may be different under the 2 conditions. Regardless, currently we do not understand why the IL-1SR failed to affect spontaneous unilateral EEG SWA. In summary, current results provide strong evidence that IL- 1β is involved in the local processes responsible for EEG SWA. They also suggest that IL-1β is responsible, in part, for the sleep deprivation-induced EEG SWA. Results are also consistent with the notion that sleep begins as a localized process. ACKNOWLEDGEMENTS This work was supported in part by NIH Grant numbers NS and NS REFERENCES 1. Obal F Jr, Krueger JM. Biochemical regulation of non-rapid-eyemovement sleep. Front Biosci 2003;8: Alam N, McGinty D, Bashir T, Kumar S, Imeri L, Opp MR. Interleukin-1 modulates state-dependent discharge activity in preoptic area and basal forebrain neurons: role in sleep regulation. Eur J Neurosci 2004;20: De A, Churchill L, Obal F Jr, Simasko SM, Krueger JM. GHRH and IL1beta increase cytoplasmic calcium levels in cultured hypothalamic GABAergic neurons. Brain Res 2002;949: Krueger JM, Walter J, Dinarello CA, Wolff SM, Chedid L. Sleeppromoting effects of endogenous pyrogen (interleukin-1). Am J Physiol 1984;246:R Figure 4 Effects of unilateral injection of interleukin (IL)-1 soluble receptor (SR) onto the surface of the somatosensory cortex on sleep rebound after sleep deprivation. Data are from the rats used in Experiment III. The IL-1SR 5 µg dose was injected (n = 6). A: EEG SWA (% baseline) during each vigilance state. The power of delta wave (0.5-4 Hz) in each 3-hour time block of baseline recording was normalized to 100%, and then all power of delta wave of treatment recording were converted to a percentage of these values. B: Power-spectrum analyses of data obtained in hours 0 to 3 after sleep deprivation. Open and closed circles represent noninjection side and injection side, respectively. Hatched bars indicate dark hours. All data shown are averages of + /- S.E.M. *indicates significant differences, P <.05. SLEEP, Vol. 28, No. 2,

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