Day- and nighttime injection of a nitric oxide synthase inhibitor elicits opposite sleep responses in rats

Ana C. Ribeiro and Levente Kapás

Department of Biological Sciences, Fordham University, Bronx, NY 10458 R-00605-2004.R2 Running Title: Nitric oxide and sleep Corresponding Author: Levente Kapás, M.D. Department of Biological Sciences Fordham University 441 E. Fordham Rd. Bronx, NY 10458 tel: (718) 817-3891 fax: (718) 817-3645 e-mail: kapas@fordham.edu

Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 28, 2005). doi:10.1152/ajpregu.00605.2004

Copyright © 2005 by the American Physiological Society.

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Abstract

Previous studies suggest that nitric oxide (NO) may play a role in sleep regulation,

particularly in the homeostatic process. The present studies were undertaken to compare the

sleep effects of injecting a NO synthase (NOS) inhibitor when homeostatic sleep pressure is

naturally highest (light onset) or when it is at its nadir (dark onset), in rats. Sleep,

electroencephalogram (EEG) delta-wave activity during non-rapid-eye-movement sleep

(NREMS), also known as slow-wave activity (SWA), and brain temperature responses to three

doses of the NOS inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME), (5, 50 and 100 mg/kg)

injected intraperitoneally at light or dark onset were examined in rats (n = 6 to 8). The effects of

5 mg/kg L-NAME were determined in both normal and vagotomized (VX) rats. Light onset

administration of 50 mg/kg L-NAME decreased NREMS amounts and suppressed SWA and

increased rapid-eye-movement sleep (REMS) amounts. At dark onset, L-NAME injection also

dose-dependently suppressed SWA, however, unlike light onset injections, both NREMS and

REMS amounts were increased after all three doses. Sleep responses to 5 mg/kg L-NAME were

not different in control and VX rats, suggesting that the sleep effects of L-NAME are not

mediated through the activation of sensory vagal mechanisms. The present findings suggest that

timing of the injection is a major determinant of the sleep responses observed after systemic L-

NAME injection, in rats.

Key words: Nω-nitro-L-arginine methyl ester, slow-wave activity, homeostatic regulation,

vagotomy, suprachiasmatic nucleus, thermoregulation, EEG power spectrum.

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Introduction

Sleep regulation relies on the interplay between the homeostatic and circadian processes

(5). The homeostatic component manifests itself as an increasing sleep pressure due to prior

waking periods, while the circadian component is an oscillating process that permits sleep to

occur only during low sleep threshold periods. Normally, increased homeostatic pressure

coincides with periods of decreased sleep threshold, thus allowing sleep to occur. In nocturnal

animals, such as rats, this occurs at the beginning of the light period. A growing number of

studies suggest that nitric oxide (NO)-ergic mechanisms play a role in sleep regulation,

particularly in the homeostatic component.

The two-process model of sleep regulation postulates that the homeostatic process relies

on a neuronal mechanism, the activity of which increases during wakefulness and is dissipated

during sleep (5). There is evidence that the activity of NO-generating mechanisms in the brain

stem, thalamus and cortex fits this pattern. For example, NO is released in an activity-dependent

manner from neurons in brainstem areas involved in sleep regulation (29). In rats, cortical (6)

and thalamic (49) NO levels are the highest during wakefulness and brain NO synthase (NOS)

activity exhibits diurnal variation peaking during the active (dark) period (3). Also,

administration of NO donor molecules or the NO precursor L-arginine during the dark phase of

the cycle mimics the effects of prolonged wakefulness, i.e., dose-dependently increases non-

rapid-eye-movement sleep (NREMS) in rats (26). On the other hand, NOS inhibitors such as

Nω-nitro-L-arginine methyl ester (L-NAME) (24, 35, 36, 40) and 7-nitro indazole (7-NI) (7, 17)

suppress sleep when administered at light onset in rats.

The most direct evidence, to date, implicating NO-ergic neurotransmission in

homeostatic regulation of sleep is that rebound increases in sleep after sleep deprivation (SD) are

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shorter in duration and lower in amplitude in NOS inhibitor-treated rats then sleep rebounds in

control animals (40). Another approach to study the role of NO homeostatic components of

sleep regulation is to test the effects of a NOS inhibitor when a) the activity of the homeostatic

mechanism is spontaneously at its highest and b) when the activity of homeostatic mechanisms is

the lowest. In rats, homeostatic sleep pressure is the highest at the end of the active period and

lowest at the end of the rest period. The aim of the present study was to determine the effects of

L-NAME on sleep after light and dark onset administration in rats. The results show that

systemic injection of L-NAME suppresses NREMS after light onset injection, but promotes

NREMS and rapid-eye-movement sleep (REMS) when injected at dark onset. Our findings are

in line with the hypothesis that NO-ergic mechanisms are involved in homeostatic sleep-

promoting mechanisms and also suggests that, in addition, they may play a role in arousal

mechanisms.

Methods

Animals

Using combined ketamine (87 mg/kg) and xylazine (13 mg/kg) anesthesia, male Sprague-

Dawley rats (250-350 g) were implanted with cortical electroencephalographic (EEG) electrodes,

nuchal electromyographic (EMG) electrodes and a calibrated brain thermistor. The EEG

electrodes were anchored over the frontal and parietal cortices and the thermistor was placed on

the dura over the parietal cortex. Following the surgery, the animals were kept in sound

attenuated individual sleep-recording cages for habituation to the experimental conditions. After

a 1-wk recovery period, the animals were connected to the recording cables and injected daily for

seven days with isotonic NaCl solution intraperitoneally (ip) 5-15 min before light (for

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Experiment I) or dark onset (for Experiment II). Animals were kept on a 12:12 h (light onset at

0400 h) and an ambient temperature of 24 ± 1 °C, for at least two weeks prior to surgeries,

during the recovery and habituation periods and throughout the experimental procedure. Water

and food were always available ad libitum. The experiments are consistent with the “Guiding

Principles for Research Involving Animals and Human Beings” issued by the American

Physiological Society. The experimental protocol was approved by the Institutional Animal

Care and Use Committee of Fordham University.

Experimental Protocol

Experiment I: The effect of light onset injection of L-NAME on sleep, SWA and Tbr.

On the control day, animals received 2 ml/kg isotonic NaCl ip and sleep was recorded for

23 h starting at 0400 h. The next day, the animals received 5 mg/kg (n = 7), 50 mg/kg (n = 6) or

100 mg/kg (n = 6) L-NAME ip (Sigma, St. Louis, MO) dissolved in isotonic NaCl (2 ml/kg). L-

NAME is an irreversible inhibitor of all three isoforms of NOS; in rats, single bolus injection of

L-NAME suppresses brain NOS activities by 50% for at least 14 h (14). The injections were

performed 5-15 min prior to light onset. Sleep on an additional, recovery, day was also recorded

for the 50 mg/kg and 100 mg/kg doses; the animals received saline ip at light onset.

Experiment II: The effect of dark onset injection of L-NAME on sleep, SWA and Tbr.

The experimental design was similar to that of Experiment I with the exception that all

injections were performed 5-15 min before dark onset (1600 h). Three doses of L-NAME were

tested, 5 mg/kg, 50 mg/kg (n = 6) and 100 mg/kg (n = 6). For 50 mg/kg L-NAME a recovery

day was also recorded, when the animals received isotonic NaCl before dark onset. The 5 mg/kg

dose was tested on two groups of rats. One group (n = 7) was subjected to bilateral

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subdiaphragmal vagotomy (VX), the other (n = 8) consisted of sham-operated rats. Three to 4

weeks after the surgeries the animals were implanted with EEG, EMG and Tbr, and habituated to

the recording conditions. After the experiments, the completeness of the vagotomies was

confirmed using the 2-deoxy-D-glucose/neutral red test (9).

Recordings

EEG, EMG and brain temperature (Tbr) were recorded by computer. EMG activity

served the sole purpose of aiding in determining the vigilance states of the animals and was not

further quantified. The EEG was filtered below 0.1 Hz and above 40 Hz (Biopac Systems,

Branco, CA). The amplified signals were digitized at the frequency of 128 Hz for EEG and

EMG and 2 Hz for Tbr. Single Tbr samples were saved on the hard disc in 10-s intervals.

Average Tbr was calculated in 3-h time blocks. The vigilance states were determined off-line in

10-s epochs. Time spent in NREMS and REMS was calculated in 3-h time blocks. Also, the

number of NREMS and REMS episodes was recorded and the average episode durations were

computed. On-line fast Fourier transformation (FFT) analysis of the EEG was also performed in

10-s intervals on 2-s segments of the EEG in 0.5 Hz bands of the 0.5-20 Hz frequency range.

The EEG power density values were summed in four frequency bands for each 10-s epoch. The

spectral data were paired with the vigilance states and EEG power was computed in each of the

four bands separately for each vigilance state. Hourly average delta- (0.5 - 4 Hz), theta- (4.5 - 8

Hz), alpha- (8.5 - 12 Hz) and beta-wave (12.5 - 20 Hz) activities (μV2) were then calculated for

NREMS, REMS and wakefulness epochs. On the baseline day, average power densities were

computed across the entire 23 h for each rat to obtain a reference value for each animal. Power

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densities in 3-h blocks on the baseline day and the test days were then expressed as a percentage

of that reference value.

Statistical Analysis

Statistical comparisons were made between the baseline days and the test days and also

between baseline and recovery days. Analysis of variance (ANOVA) was done separately for

the dark and light periods. Two-way ANOVA for repeated measures was performed on data

averaged in 3-h time blocks for sleep amounts and episode numbers and on 1-h time blocks for

Tbr. Samples sizes for sleep episode duration, SWA and theta-wave activity during REMS were

not perfectly matched between the baseline and test days. The reason for this is that in some

animals there were a few instances when REMS and/or NREMS were absent for an entire 3-h

period. For these time blocks, EEG analysis could not be performed resulting in missing data

points which prevented us from performing statistical tests for repeated measures. Therefore,

two-way ANOVA for non-repeated measures was performed on data averaged in 3-h time blocks

for sleep episode durations, SWA and theta-wave activity during REMS. When ANOVA

indicated significant treatment effects, Student-Newman-Keuls test was performed a posteriori.

ANOVA was also performed on 12-h averages across the light and dark periods between

baseline and experimental days for NREMS, REMS and SWA. Two-way ANOVA was

performed to compare the NREMS, REMS and SWA effects of L-NAME in VX and control

rats. We report only the results of the treatment effects from the ANOVAs in the Tables 1-3 and

in the text.

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Results

Light onset administration of L-NAME (see Table 1 and 2 for statistical results).

Confirming our previous findings (24, 40), systemic injection of L-NAME at light onset

suppressed NREMS in rats. The lowest dose tested, 5 mg/kg L-NAME, did not elicit any

changes in NREMS. REMS amounts, however, were increased in the dark period, 13-23 h after

L-NAME injection (Fig. 1); these increases were not accompanied by significant changes in the

number or average duration of REMS epochs (Fig. 2). Injection of 50 mg/kg L-NAME

decreased NREMS amounts by 52.1 ± 8.6 min in the 12-h period following the injection (Fig. 3).

REMS amount was unchanged during the light period, but the average duration of REMS

episodes decreased (Fig. 1 and 2). In the subsequent dark period, REMS episode durations

significantly increased and there was a strong tendency towards increased number of REMS

episodes; these changes together resulted in elevated REMS amounts, this increase, however,

did not reach the level of statistical significance (Fig. 1). In the 12-h period following 100 mg/kg

L-NAME treatment, NREMS amounts were slightly below baseline levels although not

significantly. REMS amounts did not deviate from baseline during the light period, however, in

h 12-23, REMS was increased by 31.8 ± 9.8 min, ~122% above baseline (Fig. 3).

Delta-wave activity of the EEG during NREMS, which is also called slow-wave activity

(SWA) and is often regarded as a measure of NREMS intensity (5), showed dose-dependent

decreases in response to L-NAME. While there was no change in SWA following the injection

of 5 mg/kg L-NAME, both 50 and 100 mg/kg L-NAME elicited decreases in SWA that lasted

across the test and recovery days (Fig. 1). Across the two days, SWA was suppressed by 16 ±

4.4% and 23.9 ± 3.4% by 50 and 100 mg/kg L-NAME, respectively. EEG theta-wave activity

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during REMS was suppressed by all three doses of L-NAME; these effects were still evident

during the recovery day of the 100 mg/kg dose (Fig. 1). In addition, FFT analysis of all four

wave-bands across all three vigilance states after 50 mg/kg L-NAME revealed significant

increases in alpha activity during REMS and wakefulness and increases in the beta activity

during wakefulness (Fig. 4). Light onset administration of L-NAME did not have significant

effect on brain temperature (Fig. 1).

Dark onset administration of L-NAME (see Table 2 and 3 for statistical results).

Dark onset administration of L-NAME induced dose-dependent increases in both

NREMS and REMS (Fig. 5). In response to 5 mg/kg L-NAME, NREMS and REMS were

increased by 22.1 ± 3.6 min and 23.7 ± 3.5 min, respectively, during the first 12-h period in

sham-operated rats (Fig. 3). The increase in REMS was due to the increased numbers and

average durations of REMS episodes (Fig. 6). Vagotomy itself did not have significant effects

on baseline sleep amounts and SWA (control vs. VX, two-way ANOVA, treatment effects;

NREMS: F(1,26) = 0.09, n.s., REMS: F(1,26) = 0.01, n.s., SWA: F(1,16) = 0.01, n.s.).

Furthermore, NREMS and REMS responses to 5 mg/kg L-NAME in sham-operated rats were

not statistically different from those in VX rats (Tukey test; REMS: p = 0.642, n.s.; NREMS:

p = 0.531, n.s.).

After the injection of 50 mg/kg L-NAME, NREMS amounts were increased by

56.5 ± 9.1 min in h 1-12, then returned to baseline for the rest of the recordings (Fig. 3 and 5).

Changes in REMS showed a biphasic pattern. In the dark phase after the treatment, REMS

increased by ~250% compared to baseline. In the subsequent light period, REMS returned to

baseline levels, but during the next dark period, h 25-36, a second significant increase in REMS

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of ~ 90% took place. The first phase of REMS increase was due to increased numbers of REMS

episodes and the second phase was due to a combination of increased episode numbers and

durations (Fig. 6). The highest dose of L-NAME, 100 mg/kg, also increased NREMS and

REMS amounts in the 12-h dark period by 60.9 ± 12.2 and 43.0 ± 16.3 min, respectively (Fig. 3).

Both the number and average duration of REMS episodes increased significantly in the dark

phase whereas the number and average durations of NREMS episodes did not change from

baseline (Fig. 6).

SWA was dose-dependently suppressed after dark onset injection of L-NAME (Fig. 3

and 5). Following 5 mg/kg L-NAME, SWA was 10.9 ± 1.9% below baseline levels in h 12-23

(Fig. 3). Fifty mg/kg L-NAME suppressed SWA by 22.5 ± 1.9% on the test day and by 15.0 ±

5.2% on the recovery day (Fig. 5). After 100 mg/kg L-NAME, SWA was reduced by 26.6 ±

1.8% across the 23-h recording period. Theta-wave activity of the EEG was suppressed during

the light phase after each L-NAME treatment (Fig. 5). In addition, FFT analysis of all four

wave-bands across all three vigilance states after 50 mg/kg L-NAME revealed significant

decreases in theta activity during NREMS and increases in alpha and beta activities in REMS

and wakefulness (Fig. 4). Dark onset injection of 50 mg/kg L-NAME elicited slight but

statistically significant decreases in brain temperature in the first 12 h after the treatment (Fig. 5).

Discussion

Confirming previous findings, both NREMS amounts and SWA were decreased in

response to systemic L-NAME treatment during the rest period in rats (24, 36, 40). Consistent

also with our prior observations, the SWA responses to L-NAME were long lasting (40);

following 50 and 100 mg/kg L-NAME SWA remained below baseline for at least 36-48 h.

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Administration of L-NAME at dark onset dose-dependently suppressed SWA, however, contrary

to the light onset experiments both NREMS and REMS amounts were increased.

During the light period, the activity of the homeostatic sleep-promoting mechanisms is

the highest in rats. Our finding that L-NAME decreased NREMS only when injected at light

onset is consistent with the hypothesis that NO-ergic mechanisms play a role in sleep regulation

by modulating the homeostatic process. Further support for this hypothesis comes from studies

showing that systemic injection of L-NAME suppresses both rebound sleep amounts and

intensity seen after SD (40) and cortical (6) and thalamic (49) release of NO are higher during

wakefulness than in NREMS. Moreover, NOS activity is highest during behaviorally active

periods (3), adding support to the involvement of NO-ergic mechanisms in homeostatic sleep

regulation.

Sleep increases in response to dark onset injection of L-NAME cannot be explained by

the putative role of NO in homeostatic sleep-promoting mechanisms. NO has also been shown

to be a crucial signaling molecule in the suprachiasmatic nucleus (SCN). Glutamate elevates NO

levels in the SCN, which, in turn, activate soluble guanylyl cyclase, increasing cyclic guanosine

monophosphate levels. Intra-SCN microinjection or in vitro application of glutamate (12, 33) or

NO donors (12) to SCN slices elicits phase shifts similar to those produced by in vivo light

pulses. In contrast, cerebral injections of chemicals that block this signaling cascade prevent

light-induced phase shifts (2, 12, 48). Phase resetting by the light/glutamate/NO pathway is only

active during the dark period (19), which is the phase when L-NAME strongly enhances

NREMS and REMS. The actions of NO in the SCN suggest that NO-ergic mechanisms may

also be part of the circadian process of sleep regulation. We hypothesize that during the dark

period, when homeostatic sleep pressure is low, sleep responses to L-NAME are due to its

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effects on the light/glutamate/NO pathway in the SCN. In fact, preliminary studies have shown

that microinjection of NOS inhibitors into the medial preoptic region (41) or anterior

diencephalic area (31) increased sleep, particularly REMS; while bilateral microinjections of NO

donor into the SCN suppressed REMS (42).

The overall action of L-NAME on sleep is likely the net of the effects on the homeostatic

and circadian processes. Our results suggest that the activation of those

NO-ergic mechanisms involved in the homeostatic and in the circadian component of sleep

regulation elicits opposite effects. We hypothesize that during the light period,

L-NAME interferes with the sleep-promoting homeostatic effects of NO-ergic mechanisms. In

vitro studies indicate that the SCN during this time is not sensitive to NO (19). During the dark

period, however, when homeostatic mechanisms are the least active but the SCN is sensitive to

L-NAME, the target for L-NAME is likely the SCN and increased sleep is due to the actions of

L-NAME on the circadian process. The site of sleep-suppressing action of L-NAME is not

known although various brain stem structures may possibly play a role. Evidence for the brain

stem site of action of NO arose from microinjection experiments where pontine administration of

NOS inhibitors decreased NREMS and REMS amounts in rats (21, 25) and in cats (10), and

injection of NG-nitro-L-arginine into the medial pontine reticular formation decreased REMS in

cats (30). Furthermore, injection of NO donors (10, 21) or the NO precursor L-arginine (21) into

the pedunculopontine tegmental nucleus elicited increases in NREMS and REMS. In addition,

injection of NOS inhibitors such as L-NAME (35, 36) or 7-NI (7, 36) into the dorsal raphe

nucleus resulted in decreased amounts of NREMS or REMS. L-NAME facilitated REMS when

it was given before dark or light onset. Dark onset injection of L-NAME enhanced REMS

immediately while light onset increased REMS after a 12-h delay, i.e., during the next dark

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phase. Systemic injection of L-NAME likely has an overall REMS-enhancing effect. It is

possible, however, that this effect is not manifested during the light periods because REMS is

already high.

Irrespective of the timing of the treatment, SWA was always suppressed in response to L-

NAME. The changes in EEG power spectrum, however, were not restricted to the delta wave

activity during NREMS, theta activities during REMS were also suppressed during the light

periods after each treatment. Theta activities during REMS show diurnal variation and it is

likely that theta suppression was confined to the light phases because during the dark it was

already at its daily nadir. Similar suppressions in theta activities, restricted mainly to the light

phase, were reported in rats in response to systemic injections of 7-NI, an inhibitor of neuronal

NOS (15). We hypothesize that this suppression in EEG power may reflect a generalized

inhibition of EEG delta- and theta-wave generating mechanisms, which is, at least in part,

independent of sleep. Confirming our previous findings (40), SWA and REMS theta

suppressions lasted for at least 48 h and some of the sleep effects were also evident as late as 36

– 40 h after the injection. These long lasting effects are likely due to the fact that L-NAME is an

irreversible inhibitor of NOS (14); for example, systemic bolus injection of L-NAME suppresses

NOS activity in the forebrain for at least 24 h (22). We have also shown that brain NOS activity

is decreased in response to an ip bolus injection of 50 mg/kg L-NAME in the hypothalamus,

brain stem and cerebellum by 30 – 75% in rats (4).

L-NAME elicits vasoconstriction and, as a result, systemic L-NAME treatment results in

increased blood pressure. Blood pressure changes may also trigger sleep responses through

vagal afferents (38). We studied the effects of 5 mg/kg L-NAME in vagotomized rats to

determine if an intact vagus is required for the sleep effects of L-NAME. The lowest dose was

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chosen for the VX experiment because, on the one hand, this dose still exhibits significant sleep-

promoting actions and, on the other hand, within the dose range we tested, brain NOS activities

are the least affected by this dose (22) while it still eliciting vasoconstriction and elevated blood

pressure (39). Our results that VX did not prevent the REMS-promoting and SWA-suppressing

effects of L-NAME suggest that these effects are not likely to be triggered by vagal sensory

pathways. L-NAME also elicits vasoconstriction in cerebral blood vessels (43), and both

reduced (47) and unchanged (18) cerebral blood flow (CBF) have been reported after systemic

injection of NO synthase inhibitors. It is unlikely, however, that reduced CBF would be

responsible for the sleep effects, especially for both wake- and sleep-promoting effects, of L-

NAME administration, since reduced CBF per se does not result in changes in sleep or EEG

activity. For example, in rats, systemic administration of 10 mg/kg indomethacin reduces CBF

by 30-50% (32) but does not affect sleep (37). Similarly, a decrease in CBF induced by

indomethacin or hyperventilation does not diminish EEG slow-wave activity in humans (28).

NO has been implicated in thermoregulatory mechanisms. Inhibitors of NOS suppress

febrile responses to lipopolysaccharide (13, 45) and interleukin 1 (34, 44) [but see also (1) and

(20)] and inhibit stress-induced hyperthermia (11) and cold-induced thermogenesis (23). In the

present study, peripheral injection of 50 mg/kg L-NAME at dark onset elicited a 0.2-0.3 oC drop

in brain temperature. This confirms our (24, 40) and others’ (11, 34, 46) observation that

systemic injection of L-NAME in rats causes minimal decreases in body temperature and is in

line with the notion that NO-producing mechanisms may contribute to thermogenesis.

Our present study has its limitations. First, L-NAME is a non-isoform specific inhibitor

of NOS, therefore the relative contribution of inhibiting neuronal, endothelial and inducible NOS

to the observed effects on sleep and EEG cannot be determined. Both inducible and neuronal

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NOS have been implicated in sleep regulation and their involvement in REMS-generating

mechanisms appears to be different (8). Second, systemic injection of L-NAME suppresses the

activity of both peripheral and brain NOS activities. Finally, systemic injection L-NAME likely

inhibits NOS activities in all brain regions that express NOS, therefore the relative importance of

specific regions and neuronal circuits in the effects of L-NAME is not clear.

The present studies reveal that the effect of L-NAME on sleep depends on the time of the

administration. There has been some controversy in the literature regarding the effects of L-

NAME on sleep. While the majority of the experiments reported thus far have shown that

systemic administration of NOS inhibitors such as L-NAME (24, 27, 35, 36, 40), 7-NI (7, 17) or

3 bromo-7-NI (15) suppress sleep, in contrasting reports, increased NREMS and/or REMS

amounts were found in response to systemic injection of the NOS inhibitors L-NAME (7) and L-

NMMA (16). In the latter studies, the NOS inhibitors were administered at dark onset or during

the dark period. These studies further support the present observation that the effects of L-

NAME on sleep depend on the timing of the injections, presumably by modulating

predominantly the circadian or homeostatic components of sleep regulation.

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23

Table 1: The effects of light onset injection of Nω-nitro-L-arginine methyl ester

(L-NAME) on sleep: statistical results

h 1-12 h 13-23 h 24-36 h 37-47

5 mg/kg NREMS F(1,6) = 0.0 n.s.

F(1,6) = 0.5 n.s.

# NREMS F(1,6) = 0.0 n.s.

F(1,6) = 3.9 n.s.

NREMS duration

F(1,48) = 0.1 n.s.

F(1,47) = 4.0 n.s.

REMS F(1,6) = 0.0 n.s.

F(1,6) = 7.2 p = 0.037

# REMS F(1,6) = 3.5 n.s.

F(1,6) = 1.3 n.s.

REMS duration

F(1,48) = 2.5 n.s.

F(1,40) = 0.2 n.s.

50 mg/kg NREMS F(1,5) = 30.8 p = 0.003

F(1,4) = 0.0 n.s.

F(1,5) = 7.0 p = 0.045

F(1,4) = 0.2 n.s.

# NREMS F(1,5) = 3.1 n.s.

F(1,4) = 2.5 n.s.

F(1,5) = 0.0 n.s.

F(1,4) = 0.2 n.s.

NREMS duration

F(1,39) = 1.6 n.s.

F(1,38) = 1.3 n.s.

F(1,40) = 4.4 p = 0.043

F(1,35) = 0.5 n.s.

REMS F(1,5) = 0.0 n.s.

F(1,4) = 5.2 n.s.

F(1,5) = 2.9 n.s.

F(1,4) = 0.0 n.s.

# REMS F(1,5) = 0.2 n.s.

F(1,4) = 1.6 n.s.

F(1,5) = 7.9 p = 0.037

F(1,4) = 4.5 n.s.

REMS duration

F(1,36) = 11.5p = 0.002

F(1,33) = 17.7 p < 0.001

F(1,39) = 1.3 n.s.

F(1,30) = 4.2 p = 0.049

100 mg/kg NREMS F(1,6) = 5.2 n.s.

F(1,6) = 0.2 n.s.

F(1,5) = 11.3 p = 0.020

F(1,5) = 0.0 n.s.

# NREMS F(1,6) = 5.2 n.s.

F(1,6) = 0.7 n.s.

F(1,5) = 3.7 n.s.

F(1,5) = 5.7 n.s.

NREMS duration

F(1,47) = 10.0p = 0.003

F(1,43) = 5.0 p = 0.031

F(1,40) = 42.4 p < 0.001

F(1,34) = 0.2 n.s.

REMS F(1,6) = 0.0 n.s.

F(1,6) = 8.7 p = 0.026

F(1,5) = 2.1 n.s.

F(1,5) = 2.3 n.s.

# REMS F(1,6) = 3.2 n.s.

F(1,6) = 3.0 n.s.

F(1,5) = 5.4 n.s.

F(1,5) = 2.4 n.s.

REMS duration

F(1,47) = 0.0 n.s.

F(1,42) = 7.3 p = 0.010

F(1,40) = 9.5 p = 0.004

F(1,28) = 4.1 n.s.

NREMS, non-rapid-eye-movement sleep; REMS, rapid-eye-movement sleep.

24

Two-way analysis of variance (ANOVA) for repeated measures was performed for NREMS,

REMS and episode number (# NREMS and # REMS), between the baseline and treatment (L-

NAME) days and between baseline and recovery days. Two-way ANOVA was performed for

episode duration (NREMS and REMS duration).

ANOVA across the specified hours was performed on 1-h time blocks for Tbr and on 3-h time

blocks for sleep. The F-values for the treatment effects are indicated.

n.s.: non significant difference between control and test condition (p ≥ 0.05). The actual p values are shown if they fell in the range 0.05 > p ≥ 0.001; when p < 0.001, it is

indicated as such. Statistically significant differences (p < 0.05) are emphasized in bold.

25

Table 2: The effects of injection of L-NAME on electroencephalogram (EEG) and brain

temperature (Tbr): statistical results

Light Onset

h 1-12 h 13-23 h 24-36 h 37-47

5 mg/kg SWA F(1,48) = 1.5 n.s.

F(1,48) = 1.8 n.s.

REMS theta

F(1,48) = 19.2p < 0.001

F(1,32) = 0.7 n.s.

TbrF(1,4) = 0.7

n.s. F(1,4) = 2.0

n.s.

50 mg/kg SWA F(1,40) = 28.0 p < 0.001

F(1,36) = 19.7 p < 0.001

F(1,40) = 26.8 p < 0.001

F(1,36) = 6.0 p = 0.020

REMS theta

F(1,40) = 28.7p < 0.001

F(1,30) = 0.0 n.s.

F(1,40) = 0.1 n.s.

F(1,34) = 0.2 n.s.

TbrF(1,3) = 0.6

n.s. F(1,3) = 0.0

n.s. F(1,3) = 0.1

n.s. F(1,3) = 0.6

n.s.

100 mg/kg SWA F(1,48) = 67.5p < 0.001

F(1,48) = 117.8p < 0.001

F(1,40) = 55.6 p < 0.001

F(1,40) = 32.4 p < 0.001

REMS theta

F(1,48) = 34.0p < 0.001

F(1,38) = 0.8 n.s.

F(1,40) = 21.1 p < 0.001

F(1,28) = 2.0 n.s.

TbrF(1,3) = 0.2

n.s. F(1,3) = 0.1

n.s. F(1,3) = 0.0

n.s. F(1,3) = 0.5

n.s.

Dark Onset h 1-12 h 13-23 h 24-36 h 37-47

5 mg/kg SWA F(1,40) = 12.4p < 0.001

F(1,40) = 12.5 p < 0.001

REMS theta

F(1,28) = 3.3 n.s.

F(1,32) = 6.4 p = 0.017

TbrF(1,6) = 1.2

n.s. F(1,6) = 3.1

n.s.

26

50 mg/kg SWA F(1,40) = 157.4p < 0.001

F(1,40) = 67.2 p < 0.001

F(1,40) = 9.3 p = 0.004

F(1,40) = 28.1 p < 0.001

REMS theta

F(1,36) = 1.0 n.s.

F(1,40) = 20.0 p < 0.001

F(1,32) = 2.0 n.s.

F(1,38) = 14.3 p < 0.001

TbrF(1,4) = 17.7

p = 0.014 F(1,4) = 6.0

n.s. F(1,4) = 1.3

n.s. F(1,4) = 22.4

p = 0.009

100 mg/kg SWA F(1,30) = 143.7p < 0.001

F(1,24) = 127.2p < 0.001

REMS theta

F(1,30) = 1.0 n.s.

F(1,24) = 25.8 p < 0.001

TbrF(1,3) = 5.9

n.s. F(1,3) = 11.2

p = 0.044

5 mg/kg VX SWA F(1,32) = 16.9

p < 0.001 F(1,32) = 8.8

p = 0.006

REMS theta

F(1,30) = 1.3 n.s.

F(1,32) = 7.6 p = 0.010

TbrF(1,5) = 0.2

n.s. F(1,5) = 0.3

n.s.

Two-way ANOVA for repeated measures was performed for Tbr, between the baseline and

treatment (L-NAME) days and between baseline and recovery days. Two-way ANOVA was

performed for SWA and REMS theta.

ANOVA across the specified hours was performed on 1-h time blocks for Tbr and on 3-h time

blocks for EEG. The F-values for the treatment effects are indicated.

p < 0.05: significant difference between control and test condition.

n.s.: non significant difference between control and test condition (p ≥ 0.05). The actual p values are shown if they fell in the range 0.05 > p ≥ 0.001; when p < 0.001, it is

indicated as such. Statistically significant differences (p < 0.05) are emphasized in bold.

SWA, delta-wave activity during NREMS; REMS theta, theta-wave activity during REMS; VX,

vagotomized rats.

27

Table 3: The effects of dark onset injection of L-NAME on sleep: statistical results *

h 1-12 h 13-23 h 24-36 h 37-47

5 mg/kg NREMS F(1,7) = 32.4 p < 0.001

F(1,7) = 0.0 n.s.

# NREMS F(1,7) = 3.6 n.s.

F(1,7) = 2.3 n.s.

NREMS duration

F(1,56) = 0.3 n.s.

F(1,56) = 4.7 p = 0.034

REMS F(1,7) = 40.6 p < 0.001

F(1,7) = 17.6 p = 0.004

# REMS F(1,7) = 31.1 p < 0.001

F(1,7) = 9.2 p = 0.019

REMS duration

F(1,54) = 4.3 p = 0.042

F(1,56) = 0.2 n.s.

50 mg/kg NREMS F(1,5) = 31.9 p = 0.002

F(1,5) = 5.2 n.s.

F(1,5) = 2.7 n.s.

F(1,5) = 0.0 n.s.

# NREMS F(1,5) = 0.3 n.s.

F(1,5) = 2.7 n.s.

F(1,5) = 0.7 n.s.

F(1,5) = 3.9 n.s.

NREMS duration

F(1,38) = 0.2 n.s.

F(1,38) = 0.3 n.s.

F(1,40) = 4.6 p = 0.038

F(1,36) = 2.4 n.s.

REMS F(1,5) = 86.0 p < 0.001

F(1,5) = 2.2 n.s.

F(1,5) = 26.1 p = 0.004

F(1,5) = 3.5 n.s.

# REMS F(1,5) = 59.6 p < 0.001

F(1,5) = 0.1 n.s.

F(1,5) = 7.9 p = 0.038

F(1,5) = 5.7 n.s.

REMS duration

F(1,38) = 0.6 n.s.

F(1,38) = 0.5 n.s.

F(1,40) = 9.6 p = 0.004

F(1,36) = 10.2p = 0.003

100 mg/kg NREMS F(1,4) = 20.6 p = 0.011

F(1,4) = 0.2 n.s.

# NREMS F(1,4) = 1.0 n.s.

F(1,4) = 4.2 n.s.

NREMS duration

F(1,29) = 0.4 n.s.

F(1,32) = 8.5 p = 0.007

REMS F(1,4) = 18.9 p = 0.012

F(1,4) = 0.3 n.s.

# REMS F(1,4) = 14.0 p = 0.020

F(1,4) = 0.3 n.s.

REMS duration

F(1,27) = 11.1p = 0.003

F(1,31) = 1.4 n.s.

28

5 mg/kg VX NREMS F(1,6) = 5.7

n.s. F(1,6) = 5.1

n.s.

# NREMS F(1,6) = 0.1 n.s.

F(1,6) = 2.1 n.s.

NREMS duration

F(1,48) = 8.0 p = 0.007

F(1,48) = 8.7 p = 0.005

REMS F(1,6) = 34.0 p < 0.001

F(1,6) = 4.3 n.s.

# REMS F(1,6) = 61.0 p < 0.001

F(1,6) = 10.9 p = 0.016

REMS duration

F(1,47) = 11.4p = 0.002

F(1,48) = 5.2 p = 0.028

*See footnotes for Table 1.

29

Figure Legends

Fig. 1. The effects of light onset administration of three doses of Nω-nitro-L-arginine methyl

ester (L-NAME) on sleep, electroencephalogram (EEG) slow-wave activity (SWA),

theta-wave activity during rapid-eye-movement sleep (REMS) and brain temperature

(Tbr) in rats. Horizontal solid bars denote the dark phases of the day. Open symbols:

baseline day. Solid symbols: L-NAME day. The data were averaged in 3-h time

blocks. Error bars: standard error. Horizontal dotted lines: significant treatment effect

by ANOVA (p < 0.05). Asterisks: significant difference between baseline and

experimental treatments by Student-Newman-Keuls test (p < 0.05).

Fig. 2. The effects of light onset administration of L-NAME on the number and average

duration of NREMS and REMS episodes (see legend to Fig. 1 for details).

Fig. 3. Changes in sleep amounts and SWA in response to L-NAME administration at light

and dark onset. Values represent change from baseline levels. The first three groups of

bars on each panel show changes during the first 12-h period after L-NAME injection;

the second three groups of bars shows changes in the subsequent 12-h period (i.e. h 13-

23 after injection). Within each group, fine dashed bars represent the effects of 5

mg/kg, medium dashed bars 50 mg/kg, coarse bars are 100 mg/kg. Non-dashed bars

show the effects of 5 mg/kg L-NAME in VX rats. Dark background on the columns

refers to the dark period, whereas white background refers to the light periods. Error

30

bars: standard error. Asterisks represent significant differences between control and

treatment conditions (p < 0.05; Tukey test).

Fig. 4. The effects of dark and light onset injection of 50 mg/kg L-NAME on EEG power

spectrum in rats. The effects are expressed as percent change from baseline ± SE

(baseline: 100%). Upper row: delta-wave activity, second row from top: theta-wave

activity, third row: alpha-wave activity, bottom row: beta-wave activity; W:

wakefulness. EEG power spectra were averaged in 12-h time blocks. I: first 12-h (h 1-

12), II: second 12-h (h 13-24), III: third 12-h (h 25-36), and IV: fourth 12-h (h 37-48).

Asterisks represent significant differences between control and treatment conditions (p

< 0.05; paired t-test).

Fig. 5. The effects of dark onset administration of L-NAME on sleep, EEG and Tbr of normal

and vagotomized (VX) rats (see legend to Fig. 1 for details).

Fig. 6. The effects of dark onset administration of L-NAME on the number and average

duration of NREMS and REMS episodes (see legend to Fig. 1 for details).

31

31

Figure 1.

32

32

Figure 2.

33

33

Figure 3.

34

34

Figure 4.

35

35

Figure 5.

36

Figure 6.