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Am J Physiol Regul Integr Comp Physiol 279: R404-R413, 2000;
0363-6119/00 $5.00
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Vol. 279, Issue 2, R404-R413, August 2000

Nuclear factor-kappa B inhibitor peptide inhibits spontaneous and interleukin-1beta -induced sleep

Takeshi Kubota, Tetsuya Kushikata, Jidong Fang, and James M. Krueger

Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University College of Veterinary Medicine, Pullman, Washington 99164-6520


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear factor-kappa B (NF-kappa B) is a transcription factor that when activated promotes production of several sleep-promoting substances such as interleukin-1beta (IL-1beta ), tumor necrosis factor-alpha , and nerve growth factor. Therefore, we hypothesized that inhibition of NF-kappa B activation would attenuate sleep. A NF-kappa B cell-permeable inhibitor peptide (IP) was injected intracerebroventricularly (5 and 50 µg for rats, 100 µg for rabbits). On a separate day, time-matched control injections of a cell-permeable inactive control peptide were done in the same animals. The 50-µg dose of IP in rats and the 100-µg dose in rabbits significantly inhibited non-rapid eye movement sleep and rapid eye movement sleep if administered during the light period. Moreover, pretreatment of rabbits with 100 µg of the IP 12 h before intracerebroventricular injection of IL-1beta (10 ng) significantly attenuated IL-1beta -induced sleep and febrile responses. The current data support the hypothesis that a brain cytokine network is involved in sleep regulation and that NF-kappa B is a crucial factor in physiological sleep regulation.

electroencephalogram; power density; rapid eye movement sleep; rabbits; rats


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE NUCLEAR FACTOR-kappa B (NF-kappa B) family includes the proteins p50, p52, p65, c-Rel, and Rel B. They are capable of forming heterodimers and as such complex with kappa B DNA sequence motifs and thereby affect transcription (1, 38). NF-kappa B exists in an inactive form in the cytosol combined to its inhibitory subunit Ikappa B. Nuclear translocation of NF-kappa B occurs after phosphorylation of Ikappa B in response to various stimuli, including cytokines, nerve growth factor (NGF), bacterial and viral products, excitatory neurotransmitters, oxidative stress, etc. Large proteins such as NF-kappa B require active nuclear import, and this is dependent on a short peptide signal composed mainly of basic amino acids; p50 has such a nuclear localization sequence. Within the central nervous system (CNS), NF-kappa B has a role in inflammatory responses, neuronal plasticity, development, and some pathological processes (reviewed in Ref. 38).

Sleep is regulated in part via an extensive biochemical network involving changes in expression of several sleep regulatory substances (reviewed in Refs. 20, 22). Several of these sleep regulatory substances are either upregulated in response to NF-kappa B activation or themselves induce NF-kappa B activation. For example, several somnogenic substances activate NF-kappa B; the list includes interleukin-1 (IL-1; 7, 14, 34, 53), tumor necrosis factor (TNF; 7, 51, 53), NGF (6, 32, 51), interferon-alpha (7), epidermal growth factor (37, 53), acidic fibroblast growth factor (5), insulin (3), and insulin-like growth factor (37, 53; sleep effects reviewed in Refs. 19, 20, 23, 36). Furthermore, NF-kappa B is involved in the expression of IL-2 (17), cyclooxygenase-2 (COX-2; 18, 34), inducible nitric oxide synthase (NOS-2; reviewed in Refs. 12, 48), IL-1, TNF (reviewed in Refs. 1, 38), NGF (14-16), and the adenosine A1 receptor (35); all of these substances are part of the biochemical network involved in sleep regulation. Some substances, such as IL-4, IL-10 (9, 10, 50), and glucocorticoids (reviewed in Ref. 2), directly or indirectly inhibit NF-kappa B activation, and they inhibit sleep (reviewed in Ref. 23). Collectively, such considerations led us previously to demonstrate that sleep deprivation promotes NF-kappa B activation in murine cerebral cortex and that cortical NF-kappa B activation has a diurnal rhythm, with higher levels of activation occurring during daylight hours (the sleep period in mice) than during the dark period (8). Thus the activation of NF-kappa B correlated with higher sleep propensity. However, direct evidence linking NF-kappa B activation to sleep has heretofore been lacking.

Previously, a cell-permeable NF-kappa B inhibitor peptide (IP) bearing the nuclear localization sequence of the NF-kappa B p50 subunit required for the nuclear uptake of NF-kappa B was described (30). The IP also contains a cell-permeable hydrophobic region of the signal peptide of a Kaposi fibroblast growth factor as a membrane-translocating carrier, and thereby it can bring the nuclear localization sequence into cells. It is thought that the IP inhibits NF-kappa B translocation by competing with NF-kappa B complexes for the cellular machinery responsible for nuclear translocation of NF-kappa B (30). We hypothesized that inhibition of NF-kappa B activation by the IP would inhibit sleep. We report herein that the NF-kappa B cell-permeable IP inhibits spontanenous sleep in rats and rabbits and IL-1beta -induced sleep in rabbits.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Agents

A NF-kappa B cell-permeable IP (amino acid sequence: AAVALLPAVLLALLAPVQRKRQKLMP; mol wt 2781.5) and a NF-kappa B cell-permeable inactive control peptide (CP) (amino acid sequence: AAVALLPAVLLALLAPVQRNGQKLMP; mol wt 2668.3) were purchased from Calbiochem (San Diego, CA). The inactive CP has the substitutions (underlined) of Asn for Lys and Gly for Arg in the nuclear localization sequence region of the IP peptide. In the murine endothelial LE-II cell line, lipopolysaccharide-induced nuclear translocation of the NF-kappa B complexes is inhibited by IP but not by CP. The maximum cellular uptake of IP was observed at 37°C between 30 min and 1 h, and the maximum inhibitory effect was observed at the concentration of 18 µM (30). In our studies, IP and CP were dissolved in pyrogen-free isotonic saline (PFS; Abbott). The concentrations of these peptides were 5 µg/4 µl and 50 µg/4 µl for rats and 100 µg/25 µl for rabbits. Recombinant human IL-1beta was purchased from R&D Systems (Minneapolis, MN). It was dissolved in PFS at a concentration of 10 ng/25 µl. The peptides were stored under sterile conditions at -80°C until the experiment.

Animals

Twenty-three male Sprague-Dawley rats (360-420 g) and 22 male New Zealand White rabbits (4.0-5.5 kg) were surgically implanted with electroencephalographic (EEG) electrodes, a brain thermistor, a lateral intracerebroventricular (icv) cannula, and electromyographic (EMG) electrodes (only in rats) under ketamine-xylazine anesthesia as previously described (25). Briefly, the guide cannula was placed in the left lateral ventricle for icv injection. A calibrated 30-kOmega thermistor (model 44008; Omega Engineering, Stamford, CT) was implanted on the dura mater over the parietal cortex to measure brain temperature (Tbr). The leads from the electrodes and the thermistor were routed to a Teflon pedestal. The pedestal, guide cannula, and leads were attached to the skull with dental acrylic (Duz-All; Coralite Dental Products, Skokie, IL). In rats, the patency of the guide cannula was verified by a drinking response induced by icv injecion of 40 ng angiotensin II (25). After a 1-wk (for rats) or a 2-wk (for rabbits) recovery period, the animals were placed in experimental chambers (Hot Pack 352600, Philadelphia, PA). The animals were kept on a 12:12-h light-dark cycle (lights on at 0800 for rats or 0600 for rabbits) at 22 ± 1°C (for rats) or 21 ± 1°C (for rabbits) ambient temperature. Water and food were ad libitum throughout the experiment. Rats and rabbits were habituated to the recording procedure for at least 3 days and 1 day, respectively.

Recording and Analysis

A flexible tether connecting the EEG and EMG electrodes and the thermistor led to an electronic swivel (SL6C, Plastics One). In rabbits, body movements were detected by ultrasonic detectors (Biochemical Instrumentation, University of Tennessee). The leads from the swivel and movement detectors were routed to Grass model 7D polygraphs in an adjacent room. The EEG was filtered below 0.1 Hz and above 35 Hz. The amplified signals were digitized at the frequency of 128 Hz for the EEG and at 2 Hz for Tbr and motor activity. Tbr data were saved on a computer in 10-s intervals. Online Fourier analysis of the EEG was performed. The vigilance states of wakefulness, non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) were visually determined off-line in 10-s epochs by using criteria previously reported (25-27, 46, 47). In brief, wakefulness was characterized by fast low-amplitude EEG waves, gradually increasing Tbr, and a high incidence of gross body movements. NREMS was associated with slow high-amplitude EEG waves, slowly decreasing Tbr, and lack of body movements. In contrast, REMS was characterized by fast low-amplitude EEG waves, appearance of theta activity in the EEG, rapidly increasing Tbr at REMS onset, and a lack of body movement. The average of EEG power density in the delta frequency band (0.5-4.0 Hz) during NREMS, which is called EEG slow-wave activity (SWA), was calculated. The average power of SWA throughout the entire 23-h control-recording period in each animal was normalized to 100%. Then all SWA data were expressed as a percent of the control value. In rabbits, power spectrum analysis during NREMS was performed for the 0.5-25 Hz frequency range. The average power in each 1-Hz frequency bandwidth during NREMS of control recordings was normalized to 100%, and then all EEG power data during the treatment-recording period was converted to a percent of these values. The average amount of time spent in each vigilance state, SWA, and Tbr were calculated for 3-h intervals and used for statistical analysis.

Experimental Protocols

Experiment 1: effects of IP and CP on spontaneous sleep in rabbits. A total of seven rabbits was used. Each rabbit received two icv injections of 25 µl PFS 15 min apart on the control day. On the next day they were injected with 100 µg of CP or IP in a volume of 25 µl PFS followed by 25 µl PFS (15-min interval). On another day they received 100 µg of CP or IP (each rabbit received a different drug from that administered on the first experimental day) followed by 25 µl PFS (15-min interval). All injections were performed between 0830 and 0915. After injections, all rabbits were recorded from for 23 h.

Experiment 2: effects of IP on IL-1beta -induced-sleep in rabbits (pretreatment 15 min before IL-1beta administration in the light period). A total of seven rabbits was used. Each rabbit received two icv injections of 25 µl of PFS 15 min apart on the control day. On the next day they were injected with 25 µl of PFS or 100 µg of IP in a volume of 25 µl PFS followed 15 min later by 10 ng IL-1beta in a volume of 25 µl PFS. Seven days later they received 100 µg of IP or 25 µl of PFS (each rabbit received a different drug from that of the previous administration) followed 15 min later by 10 ng IL-1beta in a volume of 25 µl PFS. All injections were performed between 0830 and 0915. After injections, all rabbits were recorded from for 23 h.

Experiment 3: effects of IP or CP on IL-1beta -induced sleep in rabbits (pretreatment 12 h before IL-1beta administration at dark onset). A total of eight rabbits was used. On the control day each rabbit received icv injection of 25 µl PFS just before the light onset (0530-0600), and they received the second icv injection of 25 µl PFS just before the dark onset (1730-1800). On the next day they were injected with 100 µg of CP or IP in a volume of 25 µl PFS just before the light onset, and they also received 10 ng of IL-1beta in a volume of 25 µl PFS just before the dark onset. Seven days later they received 100 µg of CP or IP (each rabbit received a different drug from that previously injected) just before the light onset and 10 ng of IL-1beta in a volume of 25 µl PFS just before the dark onset. After injections all rabbits were recorded from for 23 h.

Experiment 4: effects of IP and CP on spontaneous sleep in rats. A total of 23 rats was used. On the control day all rats received an icv injection of 4 µl PFS to obtain the control values. On the next day these rats received 5 µg of CP or IP (n = 7) or 50 µg of CP or IP (n = 8) in a volume of 4 µl PFS. On the second experimental day each rat received an equal dose of CP or IP. These injections took place between 0730 and 0800. One-half of the rats were injected icv with IP and then CP, whereas the rest of them received CP and then IP. After injections, EEG, EMG, and Tbr were recorded for the next 23 h. Furthermore, in an additional eight rats the same experiment was performed, except that injections were done just before dark onset (1930-2000).

Statistical Analysis

All analyses were performed with two-way analysis of variance (ANOVA) for repeated measures across the entire recording period and 3-h time blocks followed by Student-Newman-Keuls (SNK) test. For power spectrum analysis data the actual EEG power density values were summed in four frequency bands [delta (0.5-4.0 Hz), theta (4.5-8.0 Hz), alpha (8.5-12.0 Hz), beta (12.0-25.0 Hz)] wave activities, and then one-way ANOVA for repeated measures was performed for these four frequency bands. A significant level of P < 0.05 was accepted.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1: Effects of IP and CP on Spontaneous Sleep in Rabbits

The 100-µg dose of IP significantly inhibited NREMS [ANOVA for 23-h postinjection period, treatment effect: F(2,12) = 7.60, P < 0.01; with time-treatment interaction: F(14,84) = 4.42, P < 0.0001; SNK test: control vs. IP, q(3,12) = 5.25, P < 0.05 and CP vs. IP q(2,12) = 4.09, P < 0.05 (Table 1)]. This inhibitory effect began 10 h postinjection and continued to 21 h postinjection (Fig. 1). After the 100 µg of IP, REMS was also inhibited [ANOVA for 23-h postinjection period, treatment effect: F(2,12) = 10.86, P < 0.005; SNK test: control vs. IP, q(2,12) = 5.06, P < 0.05 and CP vs. IP, q(3,12) = 6.19, P < 0.05 (Fig. 1 and Table 1)]. During the first 3 h postinjection the IP enhanced NREMS above values obtained after control [SNK test: control (1-3 h) vs. IP (1-3 h), q(6,84) = 5.32, P < 0.05] but not after CP (Fig. 1). Similarly, although a transient increase in EEG SWA was observed during the first 3 h after injection of the IP compared with the control [ANOVA, time-treatment interaction: F(14,84) = 2.84, P < 0.005; SNK test: control (1-3 h) vs. IP (1-3 h), q(3,84) = 4.95, P < 0.05], the IP did not significantly affect EEG SWA compared with results obtained after CP injections during this period. EEG SWA occurring 10-21 h after the IP tended to decrease; the effect was in parallel with the suppression of NREMS during the same period, but this effect did not reach significance (Fig. 1). Power density in the EEG during the first 6 h postinjection (when NREMS was increased after IP) tended to increase in the 0.5- to 5-Hz frequency band and decrease in the 9- to 16-Hz band (Fig. 2). Although the increases in the delta frequency band did not reach significance, the decreases in the alpha frequency band were significant compared with corresponding values in control and CP groups [ANOVA, treatment effects: F(2,12) = 6.33, P < 0.05; SNK test: control vs. IP, q(2,12) = 4.181, P < 0.05 and CP vs. IP, q(3,12) = 4.51, P < 0.005] (Fig. 2A). In contrast, EEG power densities during NREMS in the dark period (10-21 h) postinjection in frequency bands lower than 15 Hz were inhibited after 100 µg of the IP, whereas power densities in frequency bands between 16 and 20 Hz increased compared with results obtained after the control or CP injection (Fig. 2B). The decrease in the delta frequency band after the IP was significant compared with corresponding values in the control and CP groups [ANOVA, treatment effect: F(2,12) = 7.23, P < 0.01; SNK test: control vs. IP, q(3,12) = 4.89, P < 0.05 and CP vs. IP, q(2,12) = 4.36, P < 0.05]. The IP slightly increased Tbr during the initial 9-h postinjection period compared with the other two groups [ANOVA, time-treatment interaction: F(14,84) = 10.09, P < 0.0001].

                              
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  		Table 1.   Effects of NF-kappa B cell-permeable inhibitor peptide on spontaneous and IL-1beta -induced sleep in rabbits



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  		Fig. 1.   Effects of intracerebroventricular (icv) injection of the nuclear factor-kappa B (NF-kappa B) cell-permeable inhibitor peptide (IP; 100 µg) and the NF-kappa B cell-permeable inactive control peptide (CP; 100 µg) on non-rapid eye movement sleep (NREMS), rapid eye movement sleep (REMS), electroencephalographic (EEG) slow-wave activity (SWA), and brain temperature (Tbr) in rabbits. All data are expressed as means ± SE and are averaged over 2-h intervals. , vehicle treatment; open circle , CP treatment; black-down-triangle , IP treatment. The IP significantly inhibited NREMS and REMS with a several-hour time delay.



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  		Fig. 2.   Power spectrum analysis of the EEG during NREMS in experiment 1. The percent change in each power density frequency band from time- and sleep state-matched values obtained during vehicle recordings pyrogen-free isotonic saline [(PFS) + PFS] are shown. open circle , CP treatment; black-down-triangle , IP treatment. A: power spectrum analysis in NREMS during the initial 6 h after injection. B: power spectrum analysis in NREMS during the dark period.

Experiment 2: Effects of IP on IL-1beta -Induced Sleep in Rabbits (Pretreatment 15 min Before IL-1beta Administration in the Light Period)

IL-1beta significantly increased NREMS; however, pretreatment with IP did not inhibit IL-1beta -induced NREMS (Table 1) [ANOVA for 23-h postinjection period, treatment effects: F(2,12) = 3.89, P < 0.05; with time-treatment interaction: F(14,84) = 3.61, P < 0.0005; SNK test: control vs. PFS + IL-1beta , q(3,12) = 3.84, P < 0.05]. Although IL-1beta significantly inhibited REMS, pretreatment with the IP did not affect the REMS inhibition induced by IL-1beta [ANOVA for 23-h postinjection period, treatment effects: F(2,12) = 8.54, P < 0.005; with time-treatment interaction: F(14,84) = 1.99, P < 0.05; SNK test: control vs. PFS + IL-1beta , q(2,12) = 4.03, P < 0.05 and control vs. IP + IL-1beta , q(3,12) = 5.68, P < 0.05] (Table 1). The effect of IL-1beta on EEG SWA was time dependent. IL-1beta significantly increased EEG SWA during the initial 3-h postinjection period; however, nonsignificant reductions in EEG SWA were observed beginning 10 h postinjection. Therefore, IL-1beta reduced the total value of EEG SWA during the 23-h postinjection period, but it did not reach significance [ANOVA for 23 h: F(2,12) = 2.92, P = 0.0929 (Table 1)]. These effects were not affected by pretreatment with the IP. IL-1beta significantly increased Tbr; however, the IP did not inhibit this effect [ANOVA for 23-h postinjection period, treatment effects: F(2,10) = 23.41, P < 0.0005; with time-treatment interaction: F(14,70) = 3.40, P < 0.0005; SNK test: control vs. PFS + IL-1beta , q(3,10) = 9.33, P < 0.05 and control vs. IP + IL-1beta , q(2,10) = 6.90, P < 0.05 (Table 1)].

Experiment 3: Effects of IP and CP on IL-1beta -Induced Sleep in Rabbits (Pretreatment 12 h Before IL-1beta Administration at Dark Onset)

IL-1beta administration plus pretreatment with the CP markedly increased NREMS (about 3 h of extra NREMS occurred compared with the base line). This effect was partly blocked by pretreatment with the IP [ANOVA for 23-h treatment effects: F(2,14) = 25.44, P < 0.0001; with time-treatment interaction: F(14,98) = 4.42, P < 0.0001; SNK test: control vs. CP + IL-1beta , q(3,14) = 10.08, P < 0.05; control vs. IP + IL-1beta , q(2,14) = 6.89, P < 0.05; and CP + IL-1beta vs. IP + IL-1beta , q(2,14) = 6.43, P < 0.05 (Table 1, Fig. 3)]. IL-1beta in combination with pretreatment with the CP significantly suppressed REMS (there was about 20 min less of REMS compared with saline control). Similarly, IL-1beta in combination with pretreatment with the IP also induced REMS inhibition; this REMS inhibition was not significantly changed from that observed after IL-1beta and the CP [ANOVA for 23-h postinjection treatment effects: F(2,14) = 5.86, P < 0.05; with time-treatment interaction: F(14, 98) = 2.70, P < 0.005; SNK test: control vs. CP + IL-1beta , q(3,14) = 4.66, P < 0.05 and control vs. IP + IL-1beta , q(2,14) = 3.48, P < 0.05 (Fig. 3, Table 1)]. Time-dependent changes in EEG SWA were also observed after IL-1beta treatment. IL-1beta plus CP pretreatment significantly increased EEG SWA during the initial 6 h; however, EEG SWA was significantly lower during the 15- to 23-h postinjection period (Fig. 3). As a consequence, EEG SWA was suppressed in the CP + IL-1beta group during the entire 23-h period (Table 1). In contrast, there was no significant differences in EEG SWA between the control and IP + IL-1beta group [ANOVA for 23-h postinjection period, treatment effects: F(2,14) = 4.28, P < 0.05; with time-treatment interaction: F(14,98) = 6.26, P < 0.0001; SNK test: control vs. CP + IL-1beta , q(3,14) = 4.14, P < 0.05]. The power spectrum analysis in NREMS during the initial 8 h of the dark period revealed that relative delta power (0.5-4 Hz) in the CP + IL-1beta -treated group significantly increased; this effect was significantly inhibited by the IP [ANOVA, treatment effects: F(2,14) = 7.47, P < 0.01; SNK test: control vs. CP + IL-1beta , q(3,14) = 5.15, P < 0.05 and CP + IP vs. IP + IL-1beta , q(2,14) = 4.17, P < 0.05] (Fig. 4A). In the initial 8 h of the light period (12 h after IL-1beta and 24 h after IP or CP injections), there was a significant reduction of power in the all-frequency bands in the CP + IL-1beta and IP + IL-1beta groups (Fig. 4B). The reduction of the relative power in this frequency band in the IP + IL-1beta group was less than that in the CP + IL-1beta group (Fig. 4B); however, this effect did not reach significance between the two groups. IL-1beta significantly increased Tbr after the pretreatment with the CP or the IP compared with the control; however, the IP partly blocked IL-1beta -induced febrile responses [ANOVA for 23-h postinjection period, treatment effects: F(2,12) = 18.22, P < 0.0005; with time-treatment interaction: F(14,84) = 8.88, P < 0.0001; SNK test: control vs. CP + IL-1beta , q(3,12) = 8.45, P < 0.05; control vs. IP + IL-1beta , q(2,12) = 5.28, P < 0.05; and CP + IL-1beta vs. IP + IL-1beta , q(2,12) = 3.17, P < 0.05 (Fig. 3, Table 1)].


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  		Fig. 3.   Effect of pretreatment with the IP 12 h prior to icv injection of interleukin-1beta (IL-1beta ) on NREMS, REMS, EEG SWA, and Tbr in rabbits. , PFS + PFS treatment; open circle , CP + IL-1beta ; black-down-triangle , IP + IL-1beta treatment. Pretreatment with the IP significantly attenuated IL-1beta -induced increases in NREMS and Tbr; however, the IP pretreatment did not affect IL-1-induced REMS responses.



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  		Fig. 4.   Power spectrum analysis of the EEG during NREMS in experiment 3. open circle , CP + IL-1beta treatment; black-down-triangle , IP + IL-1beta treatment. Pretreatment with the CP or the IP was performed just before the light onset, and IL-1beta injection was performed 12 h later just before the dark onset. A: power spectrum analysis during initial 8 h after injection. B: power spectrum analysis during initial 8 h after the light onset.

Experiment 4: Effects of IP and CP on Spontaneous Sleep in Rats

Intracerebroventricular administration of 5 µg of the IP had no effect on NREMS, REMS time, SWA, and Tbr in rats. Although 50 µg of the CP slightly increased NREMS compared with the control after light onset administration, 50 µg of the IP decreased the total amount of time spent in NREMS compared with the control or the CP group [ANOVA for 23-h postinjection period, treatment effects: F(2,14) = 17.52, P < 0.0005; SNK test: control vs. CP, q(2,14) = 4.03, P < 0.05; control vs. IP, q(2,14) = 4.34, P < 0.05; and CP vs. IP, q(3,14) = 8.37, P < 0.05 (Table 2)]. The 50-µg dose of IP given at the light onset also suppressed REMS compared with the control but not compared with results obtained after the CP [ANOVA for 23-h postinjection period, treatment effects: F(2,14) = 6.38, P < 0.05; SNK test: control vs. IP, q(3,14) = 5.02, P < 0.05]. Neither SWA nor Tbr was affected by the 50-µg dose. CP administration at dark onset did not affect any sleep parameters (Table 2).

                              
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  		Table 2.   NF-kappa B cell-permeable inhibitor peptide attenuates spontaneous sleep in rats


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major finding of the present study is that the IP inhibits spontaneous NREMS in rats and rabbits. The inhibitory actions of the IP on NREMS are similar to those we previously reported for IL-4 (27) and IL-10 (26) in rabbits in that the inhibitory effects began 8-10 h postinjection and were observed only during the dark period after administration in the light period. The reason for this delay remains unknown, but there are several possibilities. For example, the time for the diffusion of the IP from cerebrospinal fluid to effective sites could be long. The NREMS inhibitory action of the IP could result from the suppression of the production of new sleep-promoting substances such as IL-1beta and TNF-alpha . The mRNA levels of IL-1beta and TNF-alpha are already high during the initial few daylight hours (4, 45); therefore, IL-1beta and TNF-alpha levels in the CNS were likely high at the time of the IP injection during the light period. Because bolus injections of IL-1beta or TNF-alpha enhance sleep for 6-12 h, high levels of endogenous IL-1beta or TNF-alpha may also maintain sleep for several hours, the IP-induced inhibition only becoming manifest as newly produced IL-1beta or TNF-alpha , etc. is reduced.

Another result of this study is that the IP inhibited IL-1beta -induced sleep in rabbits. We performed this experiment because the IL-1 type I receptor signals via NF-kappa B (13, 40). Pretreatment of rabbits with the IP 15 min before IL-1beta administration was without effect. However, there was a significant inhibition of IL-1beta -induced NREMS if rabbits were pretreated 12 h prior to IL-1beta administration. The reasons for this time delay are likely similar to those described above for the actions of the IP on spontaneous sleep.

EEG delta-wave amplitudes are thought to reflect the intensity of NREMS. For example, EEG supranormal slow waves occur during the deep sleep after sleep deprivation (46). In the current study the IP inhibited spontaneous EEG SWA in the 0.5- to 4-Hz frequency band during the dark period in rabbits. Moreover, the IP attenuated IL-1beta -induced augmentation in EEG SWA (0.5-4 Hz) during the dark hours. These findings provide support for the idea that NF-kappa B is involved in physiological sleep regulation. Regardless, changes in EEG SWA did not always correspond to those of NREMS in the current study. This suggests that the mechanism of maintaining NREMS is different from that of generating EEG SWA. Many previous studies support this notion. For example, electrolytic lesions of the preoptic area of the hypothalamus induce long-term reduction of EEG SWA, whereas NREMS returns close to normal values after 8 days postlesion (44). Rats allowed to eat only during the daylight hours shift their diurnal rhythm of NREMS, becoming daytime active and nighttime sleep, but do not shift the diurnal rhythm of changes in EEG SWA (42). Moreover, benzodiazepines enhance NREMS but decrease EEG SWA, whereas GABAA receptor agonists enhance NREMS with an increase in EEG SWA. (29, reviewed in Ref. 28).

In the present study, we showed that the IP also inhibits REMS in rats and rabbits; however, it neither antagonized nor augmented the inhibition of REMS induced by IL-1beta . Interestingly, the onset of IP-induced inhibition of REMS is faster than that of NREMS, occurring within 4-6 h postinjection. These data are also consistent with the REMS inhibitory actions of IL-4 (27) and IL-10 (26) and support the involvement of NF-kappa B in the REMS inhibitory mechanisms of IL-4 and IL-10. It is thought that sleep regulatory mechanisms of REMS are different from those of NREMS. A possible mechanism of IL-4 and IL-10 could be due to the inhibition of NOS-2 (26, 27). A brain stem NOergic mechanism is implicated in REMS regulation. Microinjection of Nomega -nitro-L-arginine, a NOS inhibitor, into the pedunculopontine tegmental area reduces REMS in cats (11). NO is thought to provide a negative feedback signal via NF-kappa B; NO inhibits transcription of the NOS-2 gene by interfering with binding of NF-kappa B to target DNA sites (39). Another possible mechanism of REMS suppression could be due to a suppression of NGF expression. NGF promotes REMS in rabbits (47) and cats (52). NGF is important in the development and maintenance of cholinergic basal forebrain neurons; these neurons are involved in REMS regulation (reviewed in Ref. 49). Collectively, it is reasonable to assume that the IP inhibition of REMS results from several processes.

The IP did not increase Tbr in rats but slightly increased Tbr in rabbits; the cause of this mild febrile response in rabbits is unknown. This effect was transient and occurred before IP-induced inhibition of NREMS occurred. It is possible, though not certain, that in the process of manufacturing the IP small amounts of contaminants such as endotoxin were introduced. Regardless, the IP attenuated IL-1beta -induced febrile responses. A possible inhibitory mechanism for this effect involves prostaglandins (PG). IL-1beta increases PGE2 synthesis via induction of COX-2 (34). NF-kappa B is involved in the production of COX-2 mRNA (18, 34). Therefore, it is possible that the IP inhibited IL-1beta -induced COX-2 gene expression and thereby attenuate the febrile response of IL-1beta .

Perspectives

Current results indicate that the inhibition of DNA transcription, a subcellular event, results in an inhibition of sleep, a multicellular event. The mechanism by which this occurs remains unknown, although the results are consistent with our hypothesis concerning sleep mechanisms (reviewed in Refs. 21, 24). We had described the involvement of growth factors, including several NF-kappa B-sensitive substances such as IL-1, TNF, NGF, the adenosine A1 receptor, COX-2, NOS-2, fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, IL-4, IL-10, and IL-2, in NREMS regulation (reviewed in Ref. 23). These factors are organized in parallel redundant interacting systems, each affecting each other and collectively regulating sleep. We posited that within small groups of highly interconnected neurons, the intense use of neurons leads to the production of one or more of the above-mentioned growth factors; this has been demonstrated for IL-1, NGF, and neurotrophin-2 (NT-2; 33, 41, 43). The growth factors, in turn, via autocrine and paracrine actions induced altered input-output relationships for the affected neurons (e.g., Ref. 31). We proposed that these altered input-output relationships are sleep at the local neuronal group level. Thus if NF-kappa B transcription is inhibited by the IP, new neuronal use-induced production of these growth factors would be curtailed, as would the shift to altered input-output relationships and hence sleep. That some sleep persists in IP-treated animals can be explained in part by the fact that some somnogenic growth factors, e.g., NT-2, are NF-kappa B independent. It also seems unlikely that a single bolus injection of the IP would lead to the inhibition of all kappa B binding sites. Finally, the IP may not inhibit nuclear translocation of all the NF-kappa B/Rel family heterodimers.

Results are also consistent with our idea of sleep function. Many of the NF-kappa B-sensitive growth factors also play a role in synaptic plasticity and efficacy. Because the microcircuitry of the brain remains dynamic throughout adulthood and is determined to a large degree by its use and disuse, some mechanism is needed to maintain synapses responsible for innate and acquired memories. For example, many of the synapses involved in those processes, such as mating behavior, respiratory responses to high carbon dioxide levels, or for recall of memories formed years before, are seldom used, yet are maintained. These synapses, like others, need stimulation to remain efficacious. Growth factors, induced by neuronal use via their ability to alter expression of genes involved in synapse formation, provide the structural basis for synapses and as a result alter the microcircuitry as a function of neural use. The manner by which they keep seldom-used synapses functional is that they also have secondary actions affecting membrane potentials of nearby cells and thereby alter the input-output relationships of those cells (e.g., Ref. 31). Thus synapses that were not activated by an initial environmental stimulus are secondarily activated after a time delay due to growth factor production and diffusion times (see Krueger and Obál for a model of these events, reviewed in Refs. 21, 24). Such actions shift activity patterns within neural groups and divorce the output of such affected groups from immediate reference to the environment. Thus the shift in activity serves to preserve synapses not directly activated by environmental cues, and the desynchrony between environmental input and neural group output provides a basis for the reduced responsiveness to the environment associated with sleep. Current results suggest that NF-kappa B could play a role in this cascade of events.

In conclusion, current data are consistent with the hypothesis that the cytokine network in the CNS is involved with sleep regulation. NF-kappa B is a critical transcriptional factor regulating the CNS cytokine network, and it likely plays an important role in physiological sleep regulation.


    ACKNOWLEDGEMENTS

We thank Richard A. Brown for expertise in animal care.


    FOOTNOTES

This work was supported in part by grants from the National Institutes of Health (NS-25378, NS-31453, and HD-36520).

Address for reprint requests and other correspondence: J. M. Krueger, Dept. of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State Univ., College of Veterinary Medicine, PO Box 646520. Pullman, WA 99164-6520 (E-mail: krueger{at}vetmed.wsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 22 December 1999; accepted in final form 25 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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