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J Appl Physiol 97: 17-28, 2004; doi:10.1152/japplphysiol.01355.2003
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Influenza virus-induced sleep responses in mice with targeted disruptions in neuronal or inducible nitric oxide synthases

Lichao Chen, Deborah Duricka, Scott Nelson, Sanjib Mukherjee, Stewart G. Bohnet, Ping Taishi, Jeannine A. Majde, and James M. Krueger

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

Submitted 18 December 2003 ; accepted in final form 11 February 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Influenza viral infection induces increases in non-rapid eye movement sleep and decreases in rapid eye movement sleep in normal mice. An array of cytokines is produced during the infection, and some of them, such as IL-1{beta} and TNF-{alpha}, are well-defined somnogenic substances. It is suggested that nitric oxide (NO) may mediate the sleep-promoting effects of these cytokines. In this study, we use mice with targeted disruptions of either the neuronal NO synthase (nNOS) or the inducible NO synthase (iNOS) gene, commonly referred to as nNOS or iNOS knockouts (KOs), to investigate sleep changes after influenza viral challenge. We report that the magnitude of viral-induced non-rapid eye movement sleep responses in both nNOS KOs and iNOS KOs was less than that of their respective controls. In addition, the duration of rapid eye movement sleep in nNOS KO mice did not decrease compared with baseline values. All strains of mice had similar viral titers and cytokine gene expression profiles in the lungs. Virus was not isolated from the brains of any strain. However, gene expression in the brain stem differed between nNOS KOs and their controls: mRNA for the interferon-induced gene 2',5'-oligoadenylate synthase 1a was elevated in nNOS KOs relative to their controls at 15 h, and IL-1{beta} mRNA was elevated in nNOS KOs relative to their controls at 48 h. Our results suggest that NO synthesized by both nNOS and iNOS plays a role in virus-induced sleep changes and that nNOS may modulate cytokine expression in the brain.

virus; cytokine; brain

EXCESSIVE SLEEPINESS OFTEN accompanies influenza and other flulike viral infections. Previous studies in our laboratory (2, 19) and others (62) showed that inoculation of high doses of influenza virus via the intranasal route results in enhanced non-rapid eye movement sleep (NREMS) and suppressed rapid eye movement sleep (REMS), as well as a characteristic hypothermia. Proinflammatory cytokines are implicated in the sleep changes seen in response to viruses (12, 42). Yet the underlying mechanisms responsible for sleep changes in response to acute viral infections remain largely unknown.

One approach taken to understanding sleep changes was to study the genetic basis for the NREMS responses after influenza infection (63). One of the candidate genes identified was growth hormone releasing hormone receptor, and subsequently this gene was shown to be important for the influenza-induced increases in NREMS but not other acute-phase responses assessed (2). It is also well known that a cascade of cytokines, such as IL-1{alpha}, IL-1{beta}, TNF-{alpha}, IL-6, IFN-{alpha}/{beta}, and IFN-{gamma}, is secreted during influenza viral infections in mice (27). Notably, some of the cytokines, such as TNF-{alpha} and IL-1{beta}, as well as growth hormone releasing hormone, are well-defined sleep regulatory substances (50). However, the links between these humoral regulators and the sleep responses after influenza challenge are largely missing.

One possible downstream mediator that could fill this gap is nitric oxide (NO). NO is a gas that plays extraordinarily diverse physiological roles in living systems. NO is produced by three types of NO synthases (NOSs): neuronal (nNOS or NOS-1), inducible (iNOS or NOS-2), and endothelial (eNOS or NOS-3). Among them, nNOS and eNOS are constitutively expressed. Although iNOS is not normally expressed in adult animals, it has great potential to produce NO if induced in response to inflammation (47). Cytokines such as TNF-{alpha} and IFN-{gamma} are potent inducers of iNOS; they are also capable of increasing the expression of nNOS and eNOS (4, 10, 38). NO is produced in influenza-infected lungs by iNOS (1), but influenza virus infection of the lung is not known to induce NO synthesis by any NOS isoform in the brain.

NO is predominantly produced in the brain by nNOS under physiological conditions and is involved in the regulation of sleep and circadian rhythms. NOS inhibitors reduce sleep (32), whereas NO donors promote sleep (33). There is particularly strong evidence that NO plays an important role in REMS regulation. For example, the cholinergic projections from pedunculopontine tegmental nuclei and the laterodorsal tegmental nucleus to the medial pontine reticular formation are crucial in REMS generation. Local NOS inhibition within the medial pontine reticular formation reduces acetylcholine release and decreases the amount of REMS (45). The suprachiasmatic nucleus (SCN) in the anterior hypothalamus is the main biological clock that controls circadian rhythms. Both nNOS in neurons and eNOS in astrocytes are found in and around the SCN (8, 67). One role that NO may play is to synchronize environmental light with the biological clock in the SCN (51).

NO is important in the pathogenesis and control of viral infections (6, 54). For instance, mice that are deficient in the iNOS gene can survive a dose of influenza virus that causes death in the wild-type controls (34). In contrast, other studies indicate that nNOS may have a protective role during some viral infections (20, 39).

In this study, we use mice with targeted disruptions in the nNOS or iNOS genes, commonly referred to as nNOS or iNOS knockouts (KOs), to investigate sleep changes after influenza viral challenge. In addition, we have characterized the gene expression profile for IL-{beta}, TNF-{alpha}, and 2',5'-oligoadenylate synthase 1a (OAS), an IFN-induced enzyme, in mouse lung and brain stem following viral challenge.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals

Male mice, 2–3 mo of age (20–28 g), acquired from Jackson Laboratory (Bar Harbor, ME), were used and maintained in Association for Assessment and Accreditation of Laboratory Animal Care accredited animal facilities. The specific strains were nNOS KO mice (B6;129S4-Nos1tm1Plh), nNOS control mice (B6129SF2/J), iNOS KO mice (B6.129P2-Nos2tm1Lau), and iNOS control mice (C57BL/6J). The control strains used were those designated as optimal controls by Jackson Laboratory geneticists (see http://www.jaxmice.jax.org/info/index.html for more information) (9). The mice were quarantined for 2 wk. Then they were acclimated to our sleep recording room for another 2 wk. The mice then either underwent surgery to implant sleep recording electrodes, followed by recovery in an environmental chamber (maintained at 29 ± 1°C), or were directly put into environmental chambers for other experiments, as described below. All experiments commenced after the mice had adapted to the environmental chamber for at least 1 wk. Both the recording room and the environmental chambers were kept on a 12:12-h light-dark cycle (light onset at 0800, dark onset at 2000). All animal experiments were approved by the Washington State University Animal Care and Use Committee.

Virus Stock Preparation

The influenza strain employed was mouse-adapted A/PR/8/34, H1N1 (PR8), a strain that is highly lethal for mice. The PR8 was grown in specific pathogen-free chick embryos, and the resultant allantoic fluid was harvested under nonpyrogenic conditions by Specific Pathogen-Free Avian Supply (SPAFAS, North Franklin, CT), shipped on dry ice, and stored at –80°C. The virus was then washed and purified by sucrose gradient ultracentrifugation, diluted to a protein concentration of 200 µg/ml, aliquoted, snap frozen on dry ice, and stored at –80°C (2, 11). Samples were tested for both endotoxin (<0.125 endotoxin units/ml) and mycoplasma (negative for mycoplasma and acholeplasma species), as previously described (2). Viral titrations were performed in Madin-Darby canine kidney cells (MDCK; ATCC CCL-34, purchased from the American Type Culture Collection) and cultured in a 1:1 mixture of Dulbecco's MEM (Sigma) and Ham's F-12 (Sigma) with 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin/streptomycin. Tenfold dilutions of virus were incubated at 34°C for 72 h in the presence of residual trypsin from cell transfer. The median tissue culture infectious dose (TCID50) was defined as the dilution giving ~50% cell death (as determined by visual inspection) at 72 h. The starting titer of the purified PR8 virus was 1 x 108 TCID50/ml.

Tissue Viral Titrations

A group of mice (32 total, 8 for each strain) was housed under the same conditions as those used for sleep recording experiments (see below). On the experimental day, mice were infected intranasally as described below. Mice were then killed by cervical dislocation 24 h after viral challenge. Whole brains and lungs were then removed from the animals and placed on dry ice. Samples were then stored at –80°C until assay.

For lung tissue titration, individual lungs were weighed and macerated in Dounce glass homogenizers (15 ml, Kontes Glass, Vineland, NJ) on wet ice. The tissue homogenates were diluted in ice-cold PBS to make a 10% suspension (weight/volume). The lung suspensions were centrifuged for 10 min at 1,000 g to pellet cell debris and titrated in MDCK cells, as described above.

Several modifications were made for determining brain tissue viral titers. To improve sensitivity, the tissue homogenate was diluted to a concentration of 20% by adding less PBS and titrated by using twofold rather than 10-fold dilutions. Finally, to avoid a possible toxic effect of brain lipids on the MDCK cells, 3 h after cells were added, the tissue suspension was aspirated from those wells that contained the homogenates, and fresh medium was added. Cells were cultured for 5 days to improve the possibility of detecting any cytopathic effect.

Virus Inoculation

Purified PR8 virus stock was diluted 1:10 in Hanks' balanced salt solution containing Ca2+ and Mg2+ (Gibco BRL, Rockville, MD). Heat-inactivated virus was prepared by placing the diluted virus stock in a boiling water bath for 15 min before inoculation. The viral preparations were inoculated intranasally with a micropipette at a dose of 1 x 106 TCID50 (live or heat inactivated) in a volume of 50 µl/mouse (delivered to both nares) following light methoxyfluorane (Metofane, Schering-Plough Animal Health, Kenilworth, NJ) inhalation anesthesia. This dose of PR8 killed the mice between 4 and 5 days after inoculation.

Surgery

Mice were implanted with two stainless steel electromyogram (EMG) electrodes and two stainless-steel EEG electrodes (part E363/1/Spc., 15 mm length; Plastic One, Roanoke, VA) under ketamine-xylazine anesthesia (8.7 and 1.3 mg/kg, respectively, given intraperitoneally). EMG electrodes were placed into the dorsal neck muscles. EEG electrodes were placed over the frontal and parietal cortexes. A third EEG electrode was placed on the opposite side of the parietal cortex to serve as the ground. All electrodes were fixed on the skull with 3M p-10 resin bonded ceramic (3M Dental Products, St. Paul, MN). The leads from all electrodes were routed to a Teflon pedestal (Plastic One). The pedestal and leads were then attached to the skull with dental cement (Memphis Dental Manufacturing, Memphis, TN). After surgeries, an antibiotic (gentamycin, 1 mg/mouse) was administered subcutaneously to reduce the risk of infection.

Sleep Recording Procedure

A total of 64 mice were used (n = 16 for each strain). Baseline sleep was determined for each mouse for 48 h starting from dark onset (2000). The baseline values of sleep obtained from the mice used in this study were previously reported as part of a larger study of spontaneous sleep in these four strains of mice (9). After baseline recording, mice were inoculated intranasally with live influenza virus at 1100 the following day. Sleep recordings were resumed the same day at dark onset and continued for 4 days.

Recording and Analysis

After surgeries, animals were housed in individual Plexiglas shoebox cages placed in environmental chambers (Hot Pack 352600; Philadelphia, PA). The mice were kept on a 12:12-h light-dark cycle with light onset at 0800 and dark onset at 2000. The ambient temperature was kept at 29 ± 1°C. Water and food were provided ad libitum throughout the experiment. The mice were connected to the recording system and handled daily around dark onset for at least 4 days before the first recording day. A flexible tether connected the electrodes to an electronic swivel (SL6C, Plastic One). The leads from the swivel were routed to a Grass model 12C data-acquisition system. The EEG signals were amplified and filtered between 0.1 and 35 Hz. The EMG signals were amplified and filtered between 300 Hz and 100 kHz. The amplified signals were sampled at a rate of 128 Hz by an analog-to-digital converter, and the data were transferred to an IBM-compatible personal computer. The EEG data were analyzed by online fast Fourier transformation (FFT). The FFT results were averaged for every 10 s. The sleep data and FFT results were saved in 10-s intervals to a hard disk for offline analyses.

The sleep-wakefulness states were scored visually offline with our in-house software. The vigilance states were determined in 10-s epochs. NREMS was identified by high-voltage EEG delta (0.5–4 Hz) waves and decreased muscle tone. REMS was identified by the appearance of EEG theta activity (6–8 Hz) and the absence of neck muscle tone. Wakefulness was identified by low-voltage fast EEG activity and an increased level of muscle activity. In addition, the number of NREMS and REMS episodes and mean episode lengths were determined by using a computer program with the criterion that each episode lasted ≥30 s. For EEG power spectrum analyses, the EEG power density was calculated in 1-Hz intervals in the 0.5- to 25-Hz range during NREMS. The values obtained during baseline recordings in each 1-Hz frequency bin in each mouse were normalized to 100. Each corresponding 1-Hz bin posttreatment value was expressed as a relative percentage of the baseline.

Tissue Sample Collection For Gene Expression Analysis

Mice were lightly anesthetized with methoxyfluorane at either 2000 (n = 10 for each strain, total = 40) or 1100 (n = 10 for each strain, total = 40) and inoculated with live or heat-inactivated virus, as described above. Animals were killed by cervical dislocation at 15 h (1100) after challenge (the mice that were inoculated at 2000) and 48 h after challenge (the mice that were inoculated at 1100) for mRNA analysis. Thus all strains, whether challenged at 2000 or 1100, were killed at the same time of the day (1100), and baseline values were pooled from the two experiments. At the time of death, the lungs and brain stem were quickly dissected and placed in liquid nitrogen. Samples were stored at –80°C until processed.

Real-time Reverse Transcriptase PCR

Isolation of RNA, cDNA preparation, and real-time RT-PCR were performed as previously described (58). Briefly, the PCR reaction mixture (25 µl) contained 3 µl of diluted cDNA (6 ng), 12.5 µl of 2 x PLATINUM Quantitative PCR SuperMix-UDG (GIBCO BRL), 0.25 µl each of SYBRgreen (1:1,000 dilution) and fluorescence (1: 1,000 dilution), and 0.5 µl of the following forward and reverse primers at 10 µM: IL-1{beta}, sense 5'-caaccaacacgtgatattctccatg, antisense 5'-gatccacactctccagctgca; TNF-{alpha}, sense 5'-gggacagtgacctggactgt, antisense 5'-gctccagtgaattcggaaag; OAS 1a, sense 5'-ctttgatgtcctgggtcatgt, antisense 5'-gctccgtgaagcaggtagag; and cyclophilin A, sense 5'-aaatgctggaccaacacaaac, antisense 5'-ttgatgccttctttcaccttc. A standard curve and efficiency analysis of the primers were performed, and the primers' efficiencies were determined to exceed 90%. To activate uracil DNA glycosylase, the mixtures were subjected to one cycle at 50°C for 2 min and 95°C for 2 min. This amplification was followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 58–62°C for 15 s, and extension at 72°C for 15 s. Finally, starting at 55°C and a 0.5°C increase after every 10 s, a melting curve was carried out for 80 cycles. If multiple peaks were observed in melt curve analysis, the data were not used. The threshold cycle (Ct) was determined by using SYBRgreen fluorescence.

The real-time PCR reactions were performed in triplicate. Each Ct value was an average of values obtained from the samples. The changes ({Delta}) in Ct values were determined by subtracting the cyclophilin A Ct values from the target gene Ct values in control or experimental samples. The gene expression level was computed by using a comparative Ct method (User Bulletin no. 2, ABI PRISM 7700 sequence detection system, PE Applied Biosystems; and Ref. 58) by using the formula 2–({Delta}Ct for exp – {Delta}Ct for control).

Statistical Analysis

Data concerning the time spent in each vigilance state, the number and the average length of NREMS or REMS episodes during either the dark period or the light period after infection, were analyzed by one-way repeated ANOVA for each strain (5 of 64 mice were excluded from sleep analyses because of artifacts in EEG signals). Dunnett's test for multiple comparisons was used to compare posttreatment values to baseline values, if there was a significant treatment effect. It should be noted that the sample sizes decreased in the course of the experiment because of animal deaths after infection. Total duration of NREMS or REMS during either the dark period or the light period after the infection (data from animals that survived during the 4-day recording period) were compared between KOs and their respective controls by using the Student unpaired t-test. For EEG power spectrum analyses, EEG power values were summed into four frequency bands: delta (0.5–4.0 Hz), theta (4.5–8.0 Hz), alpha (8.5–12.0 Hz), and beta/sigma (12.5–25.0 Hz). Two-way repeated ANOVA were used for each strain, and the two factors were treatment (baseline vs. infection) and frequency (four frequency bands). The Student-Newman-Keuls (SNK) test was used if there were significant time-treatment interactions. Data concerning the EEG power spectrum were analyzed for the first 48 h of postinfection recording because there were no animal deaths at that time point and hence no missing data in the analyses. Moreover, the EEG power data derived from the first 48 h are most likely reflecting physiological changes after infection. The Cox Proportional Hazards test was used to analyze differences in death rates between strains of mice. Viral titers were analyzed by Student's unpaired t-test. The mRNA values in a particular statistic were always obtained from the same plate. Values derived from administration of heat-inactivated virus were regarded as baseline and normalized to 100. Gene expression levels were analyzed by two-way ANOVA, where strain (KOs vs. controls) and treatment (live vs. heat-inactivated influenza virus) were the two factors employed. The SNK test was used if there were significant strain-treatment interactions. A value of P < 0.05 was accepted as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Sleep Responses in Uninfected Mice

During spontaneous baseline sleep, the duration of NREMS (Table 1) in the nNOS KO and iNOS KO mice was not significantly different from those values obtained from corresponding control mice during either the dark or the light period. In addition, the duration of REMS during the dark period in both KO mouse strains was not significantly different from those values in corresponding control mice (Table 2). In contrast, the duration of REMS in the nNOS KO mice was significantly less than that in their controls during the light period, whereas the duration of REMS in the iNOS KO mice was significantly more than that seen in their controls during the light period (Table 2). These results are consistent with our previous report (9), although some more subtle differences were found when sleep data were analyzed in 2-h intervals (i.e., there was less NREMS in iNOS KO mice during the day-night transition compared with the values in iNOS control mice, and there was slightly more REMS at one 2-h time interval during the dark period).


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  		Table 1. Changes in NREMS parameters in nNOS KOs, iNOS KOs, and their controls

 

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  		Table 2. Changes of REMS parameters in nNOS KOs, iNOS KOs, and their control strains

 
Sleep Responses in Infected Mice

Sleep responses in infected nNOS control mice.   NREMS was elevated in nNOS control mice during both dark [F(4,47) = 24.7, P < 0.01] and light periods [F(4,47) = 12.4, P < 0.01] after influenza virus inoculation compared with the baseline values (Fig. 1). Significant NREMS enhancement started on the first recording day postinfection and continued during each dark or light period throughout the 4-day recording session (P < 0.05, Dunnett's test). During dark periods, these increases were due to increases in the number [F(4,47) = 4.5, P < 0.01] but not the average length [F(4,47) = 1.4, P = 0.2] of NREMS episodes (Table 1). On the other hand, neither the number [F(4,47) = 1.2, P = 0.3] nor the average length of NREMS [F(4,47) = 1.4, P = 0.3] episodes changed significantly during the light period (Table 1).

REMS duration in nNOS control mice after viral infection significantly decreased during both dark [F(4,47) = 7.0, P < 0.01] and light periods [F(4,47) = 22.0, P < 0.01] (Fig. 1). Significant REMS decreases occurred on postinfection recording day 4 during the dark period and all light period recording sessions (P < 0.05, Dunnett's test). These increases were due to a reduced number of REMS episodes during both the dark [F(4,47) = 10.2, P < 0.01] and the light periods [F(4,47) = 25.1, P < 0.01] (Table 2). The average length of REMS episodes did not change significantly during the dark [F(4,47) = 1.4, P = 0.3] or during the light periods [F(4,47) = 0.2, P = 0.9] (Table 2).



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  		Fig. 1. Sleep responses of neuronal nitric oxide synthase (nNOS) knockout (KO) mice ({bullet}) and nNOS control mice ({circ}) to influenza virus infection (first 48 h: n = 15 for KOs, n = 14 for controls; for 49–72 h: n = 10 for KOs, n = 12 for controls; for 73–96 h: n = 7 for KOs, n = 11 for controls). A: non-rapid eye movement sleep (NREMS). B: rapid eye movement sleep (REMS). Values are means ± SE.

 
Sleep responses in infected nNOS KO mice.   NREMS was elevated in nNOS KO mice during both dark [F(4,43) = 21.9, P < 0.01] and light periods [F(4,43) = 8.1, P < 0.01] after influenza virus inoculation (Fig. 1). Significant NREMS enhancement started on the first recording day postinfection and continued in each dark or light period throughout the 4-day recording session (P < 0.05, Dunnett's test). During the dark periods, these increases were due to changes in both the number [F(4,43)= 3.7, P < 0.05] and the average length [F(4,43) = 3.4, P < 0.05] of NREMS episodes (Table 1). During the light periods, these increases were mainly due to the occurrence of a larger number of NREMS episodes [F(4,43) = 4.0, P < 0.01] but not the average length of such episodes [F(4,43) = 0.5, P = 0.7] (Table 1).

There were no significant changes in REMS duration after influenza challenge in nNOS KO mice during either dark [F(4,43) = 1.2, P = 0.3] or light periods [F(4,43) = 1.6, P = 0.2] (Fig. 1). There were also no changes in the number of REMS episodes in the dark [F(4,43) = 1.1, P = 0.4] or the light [F(4,43) = 2.5, P = 0.06] period (Table 2). The average length of REMS episodes did not change during the dark period [F(4,43) = 1.6, P = 0.2] but did so during the light period [F(4,43) = 2.9, P < 0.05] (Table 2).

Sleep differences between infected nNOS KO mice and nNOS control mice.   The duration of NREMS after infection in nNOS control mice was higher than that in nNOS KO mice during both the dark and light periods (P < 0.05) (Table 1). The duration of REMS after infection in nNOS control mice was not significantly different from that in nNOS KO mice during both dark and light periods (P > 0.05) (Table 2). It is also notable that the sleep pattern in nNOS KO mice still had diurnal variations and that variation was absent in nNOS control mice (Fig. 1).

Sleep responses in infected iNOS control mice.   NREMS was elevated in iNOS control mice during both dark [F(4,52) = 69.8, P < 0.01] and light periods [F(4,52) = 26.8, P < 0.01] after influenza virus inoculation (Fig. 2). Significant NREMS enhancement started on the first postinfection recording day and continued during each dark or light period throughout the 4-day recording session (P < 0.05, Dunnett's test). During dark periods, these increases were due to increases in the number [F(4,52) = 13.7, P < 0.01] but not the average length [F(4,52) = 1.2, P = 0.3] of NREMS episodes (Table 1). During the light periods, these increases were due to the increased number [F(4,52) = 5.7, P < 0.01] of NREMS episodes but not changes in the average length [F(4,52) = 1.5, P = 0.2] of NREMS episodes (Table 1).

REMS duration in iNOS control mice after viral infection increased during the dark period [F(4,52) = 8.2, P < 0.01] (Table 2). These increases occurred only during the dark period on postinfection recording day 1 (P < 0.05, Dunnett's test) but not the dark periods on the following days. In contrast, REMS duration significantly decreased during light periods [F(4,52) = 19.2, P < 0.01] (Table 2). Significant REMS decreases during the light periods started on postinfection recording day 1 and continued until the end of the recording (P < 0.05, Dunnett's test). The changes of REMS during the dark periods were due to the changes in the number of REMS episodes [F(4,52) = 6.4, P < 0.01] and changes in the average length [F(4,52) = 2.8, P < 0.05] of REMS episodes (Table 2). During the light periods, the decreases of REMS were due to the changes in the number [F(4,52) = 21.3, P < 0.01] and the average length of REMS episodes [F(4,52) = 2.7, P < 0.05] (Table 2).

Sleep responses in infected iNOS KO mice.   NREMS was elevated in iNOS KO mice during both dark [F(4,54) = 44.6, P < 0.01] and light periods [F(4,54) = 22.9, P < 0.01] after influenza virus inoculation compared with baseline (Fig. 2). Significant NREMS enhancement started on the first recording day postinfection and continued in each dark or light period throughout the 4-day recording session (P < 0.05, Dunnett's test). During dark periods, these increases were due to increases in the number [F(4,54) = 21.1, P < 0.01] and the average length [F(4,54) = 3.4, P < 0.05] of NREMS episodes (Table 1). During the light periods, these increases were due to the increased number [F(4,54) = 4.7, P < 0.01] of NREMS episodes but not changes of the average length [F(4,54) = 0.9, P = 0.5] of NREMS episodes (Table 1).



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  		Fig. 2. Sleep response of inducible NOS (iNOS) KO mice ({bullet}) and iNOS control mice ({circ}) to influenza virus infection (baseline and first 72 h: n = 15 for each strain; 73–96 h: n = 13 for KOs, n = 11 for controls). A: non-rapid eye movement (NREM). B: rapid eye movement (REM). Values are means ± SE.

 
REMS duration in iNOS KO mice after viral infection increased during the dark periods [F(4,54) = 6.5, P < 0.01] (Table 2). These increases occurred during the dark period on postinfection recording day 1 (P < 0.05, Dunnett's test) but not the dark periods of the following days. In contrast, REMS duration significantly decreased during light periods [F(4,54) = 22.8, P < 0.01] (Table 2). Significant REMS decreases during the light periods started on postinfection recording day 1 and continued until the end of the recording (P < 0.05, Dunnett's test) (Table 2). The changes in REMS during the dark periods were due to the changes in the number of REMS episodes [F(4,54) = 6.4, P < 0.01] but not changes in the average length [F(4,54) = 0.7, P = 0.5] of REMS episodes (Table 2). During the light periods, the decreases in REMS were due to changes in the number of REMS episodes [F(4,54) = 31.9, P < 0.01]. The average length of REMS episodes did not change significantly during the light period [F(4,54) = 2.0, P = 0.1] (Table 2).

Sleep differences between iNOS KO mice and iNOS control mice after infection.   The duration of NREMS after infection in iNOS control mice was higher than that in iNOS KO mice during both dark and light periods (P < 0.05) (Fig. 2, Table 1). The duration of REMS after infection in iNOS control mice was not significantly different from that in iNOS KO mice during both dark and light periods (P > 0.05) (Table 2). Similar to the differences seen between nNOS KO mice and their controls, the diurnal sleep pattern in iNOS KO mice was still intact, whereas variation was attenuated in iNOS control mice (Fig. 2).

EEG Power Spectrum Changes During NREMS

Infected nNOS control mice and nNOS KO mice.   The EEG power during NREMS in nNOS control mice did not change significantly during the first 12 h of recording after infection [infection effect: F(1,13) = 1.5, P = 0.2] (Fig. 3). EEG power decreased in the next 36 h, starting at the highest frequency band and then gradually moved to lower frequency bands [13–24 h, infection and frequency interaction: F(3,39) = 3.3, P < 0.05; P < 0.05 for beta/sigma band, SNK test; 25–36 h, infection and frequency interaction: F(3,39) = 10.1, P < 0.01; P < 0.05 for alpha and beta/sigma band, SNK test; 37–48 h, infection and frequency interaction F(3,39) = 17.0, P < 0.01; P < 0.05 for all frequency bands except delta bands, SNK test] (Fig. 3).



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  		Fig. 3. Effects of influenza infection on the NREMS EEG power of nNOS KOs ({bullet}) and nNOS controls ({circ}). The baseline EEG power is normalized as 100. A: first 12 h. B: 13–24 h. C: 25–36 h. D: 37–48 h. Each data point is expressed as percentage of control ± SE.

 
The EEG power during NREMS in nNOS KO mice did not change significantly in the first 36 h of recording after infection [infection effect, first 12 h: F(1,14) = 0.15, P = 0.7; 13–24 h: F(1,14) = 2.5, P = 0.1; 25–36 h: F(1,14) = 3.3, P = 0.09]. During the 37- to 48-h period, EEG power decreased in higher frequency bands [infection and frequency interaction: F(3,42) = 3.3, P < 0.05; P < 0.05 for all frequency bands except delta bands, SNK test] (Fig. 3).

Infected iNOS control mice and iNOS KO mice.   The EEG power during NREMS in iNOS control mice did not change significantly during the first 12 h of recording after infection [infection effect: F(1,13) = 0.3, P = 0.6] (Fig. 4). EEG power decreased broadly in the second 12 h [13–24 h, infection effect: F(1,13) = 5.4, P < 0.05]. EEG power decreased at higher frequency bands at the next 24 h [25–36 h, infection and frequency interaction: F(3,39) = 15.1, P < 0.01; P < 0.05 for all frequency bands except delta bands, SNK test; 37–48 h, infection and frequency interaction: F(3,39) = 31.1, P < 0.01; P < 0.05 for all frequency bands except delta bands, SNK test] (Fig. 4).



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  		Fig. 4. Effects of influenza infection on the NREMS EEG power of iNOS KOs ({bullet}) and nNOS controls ({circ}). The baseline EEG power is normalized as 100. A: first 12 h. B: 13–24 h. C: 25–36 h. D: 37–48 h. Each data point is expressed as percentage of control ± SE.

 
The EEG power during NREMS in iNOS KO mice significantly decreased in the first 12 h after infection in the delta band [infection and frequency interaction: F(3,42) = 25.1, P < 0.01; P < 0.05 for delta band, SNK test] (Fig. 4). The EEG power further decreased in all frequency bands in the next 24 h [13–24 h, infection and frequency interaction: F(3,42) = 5.8, P < 0.01; P < 0.05 for all frequency bands, SNK test; 25–36 h, infection and frequency interaction: F(3,42) = 17.3, P < 0.01; P < 0.05 for all frequency bands, SNK test]. During the 37- to 48-h period, the EEG power in iNOS KO mice decreased significantly in the higher frequency bands [infection and frequency interaction: F(3,42) = 37.6, P < 0.01; P < 0.05 for all frequency bands except delta bands, SNK test] (Fig. 4).

Mortality

The Cox Proportional Hazards analyses revealed no significant differences in survival between nNOS KOs and nNOS controls or the iNOS KOs and iNOS controls (P > 0.05) (data not shown). However, iNOS KO mice survived significantly longer than nNOS KO mice (P < 0.05), whereas no difference was found between survival times of the nNOS control and iNOS control mice (P > 0.05).

Tissue Viral Titers

Lung viral titers were not significantly different between nNOS KO mice (titer = 102.88 ± 0.36 TCID50/mg tissue) and their controls (titer = 102.67 ± 0.23 TCID50/mg) (P > 0.05) or between iNOS KOs (titer = 102.88 ± 0.22 TCID50/mg) and their controls (titer = 102.79 ± 0.21 TCID50/mg) (P > 0.05) 24 h postinfection. There was also no significant difference in lung viral titers between nNOS KO and iNOS KO mice (P > 0.05) or between nNOS control and iNOS control mice (P > 0.05). Viable virus was not detected in the brain 24 h after infectious challenge in any mouse strain.

Lung mRNA Levels

Baseline IL-1{beta}, TNF-{alpha}, and OAS expressions in the lung did not differ between nNOS KO mice and nNOS control mice or between iNOS KO mice and iNOS control mice after inoculation with heat-inactivated virus (Fig. 5). After infection, however, influenza challenge greatly stimulated mRNA levels of all genes at both the 15- and 48-h postinfection time points in nNOS KO mice and their controls [virus effect, 15 h, IL-1{beta}: F(1,23) = 11.6, P < 0.01; TNF-{alpha}: F(1,24) = 39.3, P < 0.01; OAS: F(1,24) = 117.3, P < 0.01; 48 h, IL-1{beta}: F(1,23) = 38.2, P < 0.01; TNF-{alpha}: F(1,24) = 65.5, P < 0.01; OAS, 48 h: F(1,24) = 102.3, P < 0.01], as well as iNOS KO mice and their controls [virus effect, 15 h, IL-1{beta}: F(1,25) = 5.4, P < 0.05; TNF-{alpha}: F(1,25) = 12.2, P < 0.02; OAS: F(1,25) = 10.5, P < 0.01; 48 h, OAS: F(1,25) = 158.3, P < 0.01; F(1,25) = 45.7, P < 0.01; F(1,25)= 19.7, P < 0.01] (Fig. 5).



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  		Fig. 5. Effect of intranasal influenza administration on lung IL-1{beta}, TNF-{alpha}, and 2',5'-oligoadenylate synthase 1a (OAS) mRNAs in control mice (open bars) and KO mice (solid bars) 15 and 48 h after influenza infection. A: nNOS KOs and their controls. B: iNOS KOs and their controls. Baseline samples were collected after inoculation with heat-inactivated virus in control mice (normalized to 100). Data are expressed as relative fold increase in relation to baseline levels ± SE. Horizontal bar: P < 0.05 within group between baseline and postinfection values.

 
Brain Stem mRNA Levels

Baseline IL-{beta} and OAS expression in the brain stem was similar in both nNOS KO mice and their controls and iNOS KO mice and their controls after inoculation with heat-inactivated virus (Fig. 6). Influenza infection did not activate IL-1{beta} mRNA [virus effect, nNOS KOs and their controls: F(1,22) = 3.0, P = 0.1; iNOS KOs and their controls: F(1,25) = 0.08, P = 0.8] in the brain stem 15 h after infection. However, after 48 h, IL-1{beta} mRNA levels were elevated in all strains [virus effect: F(1,22) = 8.1, P < 0.01; F(1,23) = 7.5, P < 0.05]. In addition, a significant group effect was obtained for IL-1{beta} mRNA levels 48 h after infection [F(1,22) = 9.4, P < 0.01] in which nNOS KO mice had higher IL-1{beta} expression than that in nNOS control mice (Fig. 6). On the other hand, OAS mRNA was significantly increased in all strains at both 15- and 48-h postinfection time points [virus effect, 15 h: F(1,22) = 9.9, P < 0.01 for nNOS KOs and their controls; F(1,24) = 7.4, P < 0.05 for iNOS KOs and their controls; 48 h: F(1,23) = 41.6, P < 0.01 for nNOS KOs and their controls; F(1,23) = 38.6, P < 0.01 for iNOS KOs and their controls]. Moreover, OAS levels were also higher in nNOS KO mice than in nNOS control mice at 15 h postinfection [group effect: F(1,22) = 5.4, P < 0.05] (Fig. 6).



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  		Fig. 6. Effects of intranasal influenza administration on the brain stem IL-1{beta} and OAS mRNAs in control mice (open bars) and KO mice (solid bars) 15 and 48 h after influenza infection. A: nNOS KOs and their controls. B: iNOS KOs and their controls. Baseline samples were collected after inoculation with heat-inactivated virus in control mice (normalized to 100). Data are expressed as relative fold increase in relation to baseline levels ± SE. *P < 0.05 between groups (control and KO). Horizontal bars: P < 0.05 within group between baseline and postinfection values.

 

   DISCUSSION
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Current results show that influenza infection induced NREMS in both NOS KO strains and their respective controls, although the amplitude of these increases was smaller in the two NOS KO strains than in their respective controls. These results imply that both nNOS and iNOS play a role in NREMS changes after viral challenge.

Systemic cytokines act on the brain to induce nonspecific symptoms of sickness, such as decreased locomotor activity, decreased water and food intake, and sleep changes (12,35, 41). Circulating cytokines may affect these behavioral changes through several different mechanisms (41). Alternatively, localized tissue cytokines may act through activation of the vagus nerve (24). Stimulation of the vagus nerve can affect sleep and EEG synchronization (53). Electrical stimulation of the central end of the vagus nerve induces increases in the IL-1{beta} mRNA and protein levels in the hypothalamus (29). Sensory neurons of the vagus nerve express receptors for IL-1{beta} (17), and vagal paraganglia cells bind the IL-1 receptor antagonist (23). Subdiaphragmatic vagotomy attenuates sleep (24, 43, 52) and other central responses, such as fever (21), induced by systemic IL-1{beta} and systemic TNF-{alpha}. Vagotomy blocks the induction of IL-1{beta} in the brain stem in response to intraperitoneal IL-1{beta} injections (26). IL-1{beta} induces c-Fos immunoreactivity in primary afferent neurons of the vagus nerve (22). The vagus nerve projects to the nucleus tractus solitarius, which, in turn, projects to the paraventricular nucleus (PVN) of the hypothalamus (12, 35). Interestingly, many nNOS neurons locate to the same neuronal structure and can be activated during stress.

Chemosensory responses from the vagus nerve are not the only route by which cytokines signal the brain. For example, vagotomy blocks the IL-1{beta}-induced fever at low doses but not at high doses where circulating cytokines prevail (25). Another important pathway of cytokine signaling to the brain is through the induction of readily diffusible molecules such as NO and prostaglandins at cerebrovasculature and perivascular elements, which are the most prominent IL-1 receptor type 1 mRNA expression sites in the rodent brain (56, 64). IL-1{beta} induces perivascular iNOS gene expression and NO production (68). The combination of IL-1{beta}, TNF-{alpha}, and IFN-{gamma} increases iNOS mRNA in the PVN (66). In addition, many other cytokines are able to induce iNOS gene expression (31, 47). On the other hand, cytokines can also induce nNOS mRNA: bacterial lipopolysaccharide treatment in rats increases nNOS mRNA in the PVN and IFN-{gamma}, or IL-12 treatment increases nNOS gene expression in neurons (38, 40).

It is possible that NO produced by both iNOS and nNOS mediates the NREMS enhancement by somnogenic cytokines. When one source of NO production was removed, as in the case of nNOS KO or iNOS KO mice, the amplitude of NREMS enhancement is decreased. In line with this, intracerebroventricular (ICV) administration of NO donors increases NREMS (33). Systemic or ICV injection of NOS inhibitors at light onset reduces NREMS in rats within the first few hours (32, 49). However, this effect also depends on the time of the day. Endogenous NOS activity (and protein synthesis activity) varies substantially in the brain, particularly the hypothalamus, over 24 h (3). Injection of NOS inhibitors at dark onset increases NREMS (7).

Among cytokines that are induced by influenza infection, IL-1{beta} and TNF-{alpha} are the two best characterized NREMS-promoting substances. IL-1 and TNF-{alpha} are also key players in other aspects of sickness behavior (12). Studies of their sleep-enhancing mechanisms are still evolving. Two probable sites of action of cytokines are the brain stem and the anterior hypothalamus/basal forebrain. Injection of IL-1 and TNF-{alpha} into the locus coeruleus or dorsal Raphe nucleus enhances NREMS (13, 30). Another active site for these cytokines is the anterior hypothalamus/preoptic area (44, 59, 60).

Our virus isolation studies do not support a role for virus in the brain as a mechanism for inducing cytokine and sleep alterations. However, preliminary studies using extremely sensitive methods such as nested RT-PCR and real-time RT-PCR do suggest that virus is undergoing replication at a very low level in the brain stem and hypothalamus of wild-type mice infected with doses of PR8 similar to those used in this study. Whether there are differences in viral invasion in NOS KOs has not yet been determined. The exact role of brain virus in sleep regulation is under investigation.

With respect to REMS, both iNOS KO mice and the two wild-type control strains showed decreased REMS during the light period, whereas the nNOS KO mice did not show this response. Influenza infection elicits inflammatory stress (16). All forms of stress activate the hypothalamic-pituitary-adrenal axis, which results in systemic elevation of glucocorticoids (18). Indeed, influenza infection increases plasma concentrations of glucocorticoids (15, 16, 28). It is well known that glucocorticoids inhibit REMS (57). nNOS plays an important role in the stress response (55). Systemic or ICV injection of the NOS inhibitor L-NAME reduces ACTH release (36). Stress-induced glucocorticoid increases are attenuated in nNOS KO mice (5). Therefore, it is not surprising that nNOS KO mice failed to show a decrease in their REMS during the daytime, as did other strains. Another interesting observation is that iNOS KO mice and their controls increased their REMS during the first night after infection. Acute stress often leads to enhanced REMS. Interestingly, regardless of when the animal is exposed to the stress, the sleep changes always occur during the dark period (37). Although we also observed a slight increase of REMS during the first dark period after infection in nNOS KO mice and their controls, those changes were not significant. This difference may reflect a strain variation in the response to stress (48). In summary, our studies may support a role for nNOS in REMS changes after persistent activation of hypothalamic-pituitary-adrenal axis.

In line with the literature, we observed influenza-induced EEG power decreases at various frequency bands, especially at high-frequency bands. In addition, we observed that nNOS KO mice are relatively resistant to EEG power changes after infection compared with their controls. On the other hand, iNOS KO mice showed decreased EEG power faster and at lower frequency bands first. Influenza-infected mice have hypothermia (19, 62), and this low body temperature could reduce EEG power (14, 46). In addition, EEG power varies with cerebral blood flow (69). Neurons containing nNOS project to microvessels and are involved in cerebral blood flow regulation (61, 65). On the other hand, iNOS may also be involved in blood flow regulation when iNOS in perivascular microglial cells is induced. Further study on blood flow changes after influenza infection may clarify this issue.

All strains of mice showed similar increases in their lung cytokine expression in a time-dependent manner. There were no gene expression differences between KO strains and their respective controls. This, taken together with 24-h postinfection viral titer, suggests that KO mice and their controls had similar immune and inflammatory responses at this early stage of infection. Moreover, there is no significant difference with respect to the mortality rate among KO strains and their controls. Although others have shown reduced mortality in iNOS KOs, this protection was lost at higher doses of virus similar to those that we have employed (34). Although we have not examined pathological changes per se in the lungs of the NOS KOs, previous studies have shown that dramatic changes in sleep can occur in the absence of altered lung pathology (2). Thus the sleep differences we see between KO mice and their controls are most likely due to differences in the central nervous system, rather than different peripheral cytokine profiles. Indeed, cytokine gene expression is different at the brain stem level between nNOS KOs and their controls. Preliminary studies to date do not show changes in cortical cytokine expression induced by infection.

In conclusion, the results suggest that both nNOS and iNOS participate in influenza infection-induced sleep. The increase in NREMS seen in all mouse strains, except the nNOS KOs, may reflect enhanced immune defenses in the responsive strains, although no survival advantage was seen in these studies. NO produced by these enzymes may mediate the effects of proinflammatory cytokines on sleep, although other responses such as blood flow could also contribute to sleep changes seen in NOS KOs.


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This work was supported in part by National Institutes of Health Grants NS-31453 and HD-36520.


   ACKNOWLEDGMENTS
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 ABSTRACT
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The authors thank Dr. B. Slinker for help with statistical analyses, Dr. A. De for help with MDCK cell culture, and R. Brown for excellent technical assistance and animal care.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Krueger, Dept. of VCAPP, Washington State Univ., 204 Wegner Hall, 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. Section 1734 solely to indicate this fact.


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L. Kapas, S. G. Bohnet, T. R. Traynor, J. A. Majde, E. Szentirmai, P. Magrath, P. Taishi, and J. M. Krueger
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