Nitric oxide production in the basal forebrain is required for recovery sleep

Article (PDF Available)inJournal of Neurochemistry 99(2):483-98 · November 2006with29 Reads
DOI: 10.1111/j.1471-4159.2006.04077.x · Source: PubMed
Abstract
Sleep homeostasis is the process by which recovery sleep is generated by prolonged wakefulness. The molecular mechanisms underlying this important phenomenon are poorly understood. Here, we assessed the role of the intercellular gaseous signaling agent NO in sleep homeostasis. We measured the concentration of nitrite and nitrate, indicative of NO production, in the basal forebrain (BF) of rats during sleep deprivation (SD), and found the level increased by 100 +/- 51%. To test whether an increase in NO production might play a causal role in recovery sleep, we administered compounds into the BF that increase or decrease concentrations of NO. Infusion of either a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or a NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), completely abolished non-rapid eye movement (NREM) recovery sleep. Infusion of a NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2diolate (DETA/NO), produced an increase in NREM that closely resembled NREM recovery after prolonged wakefulness. The effects of inhibition of NO synthesis and the pharmacological induction of sleep were effective only in the BF area. Indicators of energy metabolism, adenosine, lactate and pyruvate increased during prolonged wakefulness and DETA/NO infusion, whereas L-NAME infusion during SD prevented the increases. We conclude that an increase in NO production in the BF is a causal event in the induction of recovery sleep.
Nitric oxide production in the basal forebrain is required for
recovery sleep
A. V. Kalinchuk,* Y. Lu, D. Stenberg,* P. A. Rosenberg and T. Porkka-Heiskanen*
*Department of Physiology, Institute of Biomedicine, University of Helsinki, Helsinki, Finland
Department of Neurology, Children’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
Abstract
Sleep homeostasis is the process by which recovery sleep is
generated by prolonged wakefulness. The molecular mecha-
nisms underlying this important phenomenon are poorly
understood. Here, we assessed the role of the intercellular
gaseous signaling agent NO in sleep homeostasis. We
measured the concentration of nitrite and nitrate, indicative of
NO production, in the basal forebrain (BF) of rats during sleep
deprivation (SD), and found the level increased by 100 ± 51%.
To test whether an increase in NO production might play a
causal role in recovery sleep, we administered compounds into
the BF that increase or decrease concentrations of NO. Infu-
sion of either a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-
tetramethylimidazoline-1-oxyl-3-oxide, or a NO synthase
inhibitor, N
x
-nitro-
L
-arginine methyl ester (L-NAME), com-
pletely abolished non-rapid eye movement (NREM) recovery
sleep. Infusion of a NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-
ammonioethyl)amino]diazen-1-ium-1,2diolate (DETA/NO),
produced an increase in NREM that closely resembled NREM
recovery after prolonged wakefulness. The effects of inhibition
of NO synthesis and the pharmacological induction of sleep
were effective only in the BF area. Indicators of energy meta-
bolism, adenosine, lactate and pyruvate increased during
prolonged wakefulness and DETA/NO infusion, whereas
L-NAME infusion during SD prevented the increases. We
conclude that an increase in NO production in the BF is a
causal event in the induction of recovery sleep.
Keywords: adenosine, basal forebrain, nitric oxide, recovery
sleep, sleep deprivation.
J. Neurochem. (2006) 99, 483–498.
Sleep loss, induced by prolonged wakefulness, produces a
decline in cognitive and motor performance (Dinges et al.
1997), mood disturbances, memory deficits (Chee and Choo
2004) and affects immune function (Bryant et al. 2004).
These effects are restored by recovery sleep, which is
characterized by prolongation and intensification of both the
non-rapid eye movement (NREM) and rapid eye movement
(REM) components of sleep. Although the two-process
model of sleep regulation (Borbely 1982) accurately des-
cribes the expected duration of recovery sleep, the molecular
mechanisms that underlie this regulation remain less clear.
Endogenous sleep factors substances that accumulate in the
brain during prolonged wakefulness have been suggested
to be mediators of homeostatic sleep regulation (Borbely and
Tobler 1989). One potential sleep factor is the inhibitory
neuromodulator adenosine (Benington and Heller 1995);
during prolonged wakefulness extracellular adenosine con-
centration increases in the basal forebrain (BF) and induces
sleep (Porkka-Heiskanen et al. 1997). As adenosine is an
indicator of disturbed energy balance (Dunwiddie and
Masino 2001), we hypothesized that during sleep deprivation
(SD) continuous activity of the waking-promoting cells in the
cholinergic region of the BF (Detari et al. 1984; Szymusiak
and McGinty 1986; Szymusiak and McGinty 1989) leads to
unfavourable changes in energy demand/supply ratio and
consequent adenosine release in this area. Supporting our
Received January 9, 2006; revised manuscript received June 14, 2006;
accepted June 16, 2006.
Address correspondence and reprint requests to T. Porkka-Heiskanen,
Institute of Biomedicine, PO Box 63 (Haartmaninkatu 8), University of
Helsinki, Helsinki 00014, Finland. E-mail: porkka@cc.helsinki.fi
Abbreviations used: ac, anterior commissure; aCSF, artificial
cerebrospinal fluid; BF, basal forebrain; cPTIO, 2-(4-carboxyphenyl)-
4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DETA/NO, (Z)-1-
[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate;
D-NAME, N
x
-nitro-
D
-arginine methyl ester; EEG, electroencephalo-
gram; EMG, electromyogram; HDB, horizontal limb of the diagonal
band of Broca; LDT/PPT, laterodorsal/pedunculopontine tegmental
nuclei; L-NAME, N
x
-nitro-
L
-arginine methyl ester; MCPO, magnocel-
lular preoptic area; nBF, outside the BF; NOS, nitric oxide synthase;
NO
x
,NO
2
+NO
3
; NREM, non-rapid eye movement; ox, optic
chiasm; REM, rapid eye movement; SD, sleep deprivation.
Journal of Neurochemistry, 2006, 99, 483–498 doi:10.1111/j.1471-4159.2006.04077.x
Ó 2006 The Authors
Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498 483
hypothesis, experimentally induced local energy depletion in
the BF increased extracellular adenosine concentration and
concurrently induced an increase in sleep (Kalinchuk et al.
2003). Recently, it has been shown that NO can inhibit
neuronal energy production (Brorson and Zhang 1999;
Maletic et al. 2004; Rosenberg et al. 2000) and stimulate
adenosine release from forebrain neurones (Rosenberg 2000
et al.), leading us to consider the possible role of NO in the
regulation of behavioural state, and specifically in the
induction of recovery sleep.
NO is an intercellular signalling molecule that regulates
both physiological and pathophysiological processes in the
CNS (Garthwaite and Boulton 1995; Gross and Wolin 1995;
Keynes and Garthwaite 2004). NO concentrations undergo
state-dependent modulation during the sleep–wake cycle
both in the thalamus and the cortex (Burlet and Cespuglio
1997; Williams et al. 1997), but there are no measurements
of NO concentrations during prolonged wakefulness in any
brain area. Several previous studies have shown that
intraperitoneal, subcutaneous or intracerebroventricular
administration of inhibitors of the NO-synthesizing enzyme
NO synthase (NOS) decreases spontaneous sleep (Kapas
et al. 1994; Dzoljic et al. 1996; Monti et al. 1999, 2001;
Ribeiro et al. 2000; Monti and Jantos 2005; Ribeiro and
Kapas 2005; Cavas and Navarro 2006) whereas NO donors
increase it (Kapas and Krueger 1996; Monti and Jantos
2004a), suggesting that NO may have a role as a sleep-
facilitating agent. Local injections of NOS inhibitors into the
pons, including the cholinergic laterodorsal/pedunculopon-
tine tegmental nuclei (LDT/PPT) as well as the dorsal raphe
nucleus, have generally also decreased either REM sleep or
both NREM and REM sleep (Datta et al. 1997; Leonard and
Lydic 1997; Hars 1999; Monti et al. 1999, 2001). Studies
employing local manipulations of NO level in the BF have
provided controversial results: injections of NOS inhibitors
into the BF have been reported to decrease NREM sleep and
increase wakefulness (Monti and Jantos 2004b) or have no
effect on sleep (Vazquez et al. 2002), and injection of the NO
precursor
L
-arginine or a NO donor have been reported to be
ineffective (Monti and Jantos 2004b). Some studies have also
suggested that NO has a pro-arousal (Pape and Mager 1992;
Marino and Cudeiro 2003) effect. However, the role of NO in
the induction of recovery sleep after SD has been addressed
in only one study, in which the NOS inhibitor N
x
-nitro-
L
-
arginine methyl ester (L-NAME) administered intraperiton-
eally decreased NREM sleep recovery (Ribeiro et al. 2000).
We hypothesized that release of NO locally in the BF
during prolonged wakefulness, either from intrinsic cells or
from terminals of projecting neurones such as those from the
LDT/PPT, may be critical for the subsequent increase in
sleep, and that the NO release is associated with changes in
energy metabolism. To test this hypothesis we either
decreased the amount of NO produced during prolonged
wakefulness or pharmacologically increased it, and measured
the effect of these manipulations on metabolites of energy
metabolism and sleep. We also measured the concentrations
of NO
2
and NO
3
(collectively termed NO
x
) in the BF
during SD, thereby assessing directly the question of whether
NO levels change during prolonged wakefulness.
Materials and methods
Animals and surgery
Male Wistar rats (300–400 g) were kept under constant temperature
(23.5–24°C) conditions on a 12-h light–dark cycle with lights on at
08.30 hours. Water and food were provided ad libitum. Under
general anaesthesia (0.1 mg/kg s.c. medetomidine + 30 mg/kg i.p.
pentobarbital), rats were implanted with electrodes for recording
electroencephalogram (EEG) and electromyogram (EMG) and with
a unilateral guide cannula for microdialysis probes (CMA/11 Guide;
CMA/Microdialysis, Stockholm, Sweden) targeting the BF cho-
linergic area, including the horizontal limb of the diagonal band of
Broca (HDB), substantia innominata and magnocellular preoptic
area (MCPO) (anterior ¼ ) 0.3; lateral ¼ 2.0; vertical ¼ 5; Paxi-
nos and Watson 1998), and as a control, neighbouring areas that do
not contain cholinergic cells (Figs 1a and b). After a recovery and
adaptation period of 2 weeks, before microdialysis probe insertion, a
30-h baseline EEG recording was obtained for each rat. The
experimental protocol was accepted by the Ethical Committee for
Animal Experiments at the University of Helsinki and the provincial
government of Uusimaa, Finland, and was in accordance with the
laws of Finland and the European Union.
Microdialysis experiments
Microdialysis probes (CMA/11, membrane length and diameter 2
and 0.24 mm respectively; CMA/Microdialysis) were inserted into
the target areas at least 20 h before the start of experiments
(Porkka-Heiskanen et al. 1997) and stayed in place permanently
during the 2-week experimental period. If a probe became
clogged or dried out, it was replaced by a new one. For
experiments, animals were connected to microdialysis leads
(EEG/EMG leads combined with microdialysis tubing) for 6 h
starting after lights on at 09.00–09.30 hours. Artificial cerebro-
spinal fluid (aCSF; 147 m
M
NaCl, 3 m
M
KCl, 1.2 m
M
CaCl
2
,
1.0 m
M
MgCl
2
) or solutions of the studied drugs (dissolved in
aCSF) were pumped through the microdialysis probe at 1 lL/min.
The microdialysis leads were disconnected after 1 h of recovery
sleep following SD at 15.30–16.00 hours and replaced by
ordinary EEG/EMG recording leads. When metabolite concentra-
tions were measured during 2 h of recovery sleep after SD,
microdialysis tubing was disconnected at 16.30–17.00 hours. The
30-min periods during which microdialysis tubing was being
replaced were excluded from analysis. Four types of microdialysis
experiments were performed.
Experiment type 1
SD was performed between 11.30 and 14.30 hours; changes in
metabolite concentrations during SD as well as changes in
subsequent sleep after SD (‘recovery sleep’) were measured
(Kalinchuk et al. 2003).
484 A. V. Kalinchuk et al.
Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
Ó 2006 The Authors
Experiment type 2
A drug was infused for 3 h during the spontaneous sleep–wake
cycle (11.30–14.30 hours); changes in metabolite concentrations
during treatment and in subsequent sleep after treatment were
measured.
Experiment type 3
A drug was infused during SD; its effects on changes in metabolite
concentrations during SD and on recovery sleep after SD were
measured and compared with baseline and the SD effect. Drug
infusions to be combined with SD were started 1 h before the SD at
10.30 hours and continued through the deprivation until 14.30
hours.
Experiment type 4
A drug (drug B) was infused simultaneously with another drug,
whose effects had already been tested (drug A). Infusion of drug B
started 1 h before the infusion of drug A at 10.30 hours and
continued for 4 h until 14.30 hours.
Analysis
On each experimental day microdialysis samples for the analysis of
metabolites were collected at 30-min intervals from 09.30 hours to
15.30 or 16.30 hours (see above). The first recording was always a
baseline aCSF infusion for 6 h. All subsequent experiments were
preceded by a daily pretreatment baseline period of aCSF infusion
for 2 h, during which samples were collected for metabolite
analysis. For each analysed metabolite (NO
x
, adenosine, lactate
and pyruvate), averages of concentrations from two (experiment 3)
or three (experiments 1 and 2) samples collected during the
pretreatment period and from three samples collected during
treatment were compared.
The EEG was recorded for 30 h (continuing for 24 h after the
treatment). One animal was used in two to four experiments
(including the aCSF baseline run) during a 2-week period with a
minimum of 48 h between experiments. The EEG was also recorded
during the non-experimental days, and the records were evaluated to
ensure that there was no carry-over effect of the drugs on sleep.
Baseline was recorded from all animals (n ¼ 25) and SD was
performed for 18 animals. For the final analysis, SD data from
animals with probes in the BF and outside BF were combined,
giving n ¼ 18 for the SD group. In the other groups n ¼ 4–8 (see
figure legends).
SD protocol
The animals were trained to human presence for at least 1 week
before the experiments. During the daily training sessions the
animals were actually handled, by taking them out of the cage and
letting them play with the researcher, and then returned to the cage.
The daily sessions lasted up to 10 min, and the animals were
regarded as trained when they did not show a fear reaction when the
researcher entered the room and approached the cage.
Rats were sleep-deprived for 3 h between 11.30 and 14.30 hours
using a gentle handling procedure (Franken et al. 1991), which
included introduction of new objects into the cages in order to keep
the animals occupied and replacing them by new ones when the
animals appeared to become sleepy. Any physical contact with
animals was excluded. Monitoring of food consumption did not
show significant differences between spontaneous wakefulness and
SD periods. Continuous monitoring of EEG/EMG during the
deprivation period was used to assess the behavioural state of the
animals.
EEG recording and analysis
The EEG/EMG signals were amplified and sampled at 104 Hz as
described previously (Kalinchuk et al. 2003). EEG recordings were
scored using the Spike 2 program (version 5.11; Cambridge
Electronic Devices, Cambridge, UK) in 30-s bins semi-automatic-
ally for NREM sleep, and manually for REM sleep and wakefulness.
Semi-automatic scoring of NREM sleep was performed based on
quantification of EEG power in the delta band (0.5–4Hz), sigma
band (11–15Hz) and gamma band (30–45Hz) using custom scripts
Fig. 1 Location of microdialysis probe tips. (a) Camera lucida drawing of the microdialysis probe tips in the BF (n ¼ 16, filled circles) or control
areas outside the BF (n ¼ 9, open circles). ac, anterior commissure; ox, optic chiasm. (b) Photograph of the track of a representative probe tip
located in the BF (HDB area).
Nitric oxide and recovery sleep 485
Ó 2006 The Authors
Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
for power spectral analysis as described previously (Stenberg et al.
2003). Scoring of NREM sleep was validated by correlating the
results of the semi-automatic scoring with results of manual scoring
for 14 records (30 h each); the mean ± SEM correlation was
91.4 ± 0.6%. Manual analysis of wakefulness, NREM sleep (for
validation of results of semi-automatic scoring) and REM sleep was
performed in accordance with classical criteria: wakefulness was
identified by the presence of low-amplitude desynchronized activity
in the EEG and high-amplitude activity in the EMG; NREM sleep
was identified by the presence of high-amplitude slow waves in the
EEG and decreased activity in the EMG compared with wakeful-
ness; REM sleep was distinguished as a state with regular theta
activity (5–8Hz) in the EEG and decreased muscle tone compared
with wakefulness. The 30-h recordings were divided into 3- and 6-h
bins; the amounts of NREM sleep, REM sleep and EEG power in
the delta range during NREM episodes (delta power) in each bin
during the experimental day were compared with the corresponding
time bin on the baseline day and percentage differences were
calculated. A period of 18 h after the treatments (14.30–08.30 hours,
the shaded area in figures) was used for the final quantitative
analysis as this was the period of maximal change from baseline in
sleep and delta power.
To compare EEG power density spectra during recovery sleep
(n ¼ 13) and NO donor infusion (n ¼ 8), vigilance states were
manually scored for 4-s epochs during 6 h after treatment. EEG
power spectra (Fast Fourier transform routine, Hanning window)
were calculated within the frequency range of 0.4–20 Hz with
resolution 0.4 Hz.
Definitions of baselines
Baseline EEG 1 (30 h) was recorded before probe insertion to
ensure that the animal had recovered completely from the operation.
Baseline EEG 2 (30 h) was recorded before the experiments. The
probe was inserted at least 20 h before the start of recording.
Microdialysis leads were attached and aCSF was infused for 6 h
between 9.30 and 15.30 hours. As there was no difference in sleep
between the two baselines, baseline EEG 2 was used when
normalizing the sleep data.
Daily pretreatment baseline was determined for metabolite
(adenosine, lactate and pyruvate) measurements. The daily baseline
values for each animal were monitored throughout the 2-week
experimental period to ascertain the stability of the preparation.
HPLC analysis
Adenosine was measured using HPLC coupled to a UV detector or
a McPherson fluorimeter [when the infused molecules, i.e. L-
NAME and (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]
diazen-1-ium-1,2-diolate (DETA/NO) prevented UV detection].
Details of the adenosine assays have been published previously
(Porkka-Heiskanen et al. 1997; Rosenberg et al. 2000). The
amounts of lactate and pyruvate were measured as described by
Hallstro¨m et al. (1989). The detection limits of the assays were
0.8 n
M
for adenosine (signal to noise ratio 2 : 1), 0.6 l
M
for
pyruvate (3 : 1) and 10 l
M
for lactate (3 : 1) (Grob 1985).
Concentrations of the samples collected during SD or drug
infusions were normalized to the mean concentration of samples
collected during the baseline pretreatment period when aCSF was
infused (¼ 100%). If a drug interfered with the adenosine assay
(2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,
cPTIO), adenosine was not measured.
NO
x
measurements
As no endogenous source other than NO is known for NO
2
and
NO
3
(collectively NO
x
) this metabolite has generally been taken as
indicative of NO production (Mackenzie et al. 1996). The dietary
intake of nitrate can considerably affect the plasma nitrate
concentrations, but as these anions do not penetrate the blood–
brain barrier, their effect on brain concentrations is not significant
(Clark et al. 1996). NO
x
concentrations were measured using a
Nitrate/Nitrite Fluorometric Assay kit (Cayman Chemical Com-
pany, Ann Arbor, MI, USA) according to the manufacturers
instructions. The detection limit of the assay was 0.06 l
M
in the
final reaction mixture. The results were calculated as NO
x
.
Concentrations of the samples collected during SD or drug
infusions were normalized to the mean concentration of samples
collected during the baseline pretreatment period when aCSF was
infused (¼ 100%). Measurement of NO
x
during DETA/NO
infusion was not performed because the values would not reflect
correct tissue concentrations.
Materials
To decrease NO concentration in the BF we used either a NO
scavenger, cPTIO (potassium salt; Tocris, Ballwin, MO, USA)
(Akaike et al. 1993) at concentrations 1, 5 and 10 m
M
, or a non-
selective NOS inhibitor, L-NAME (Sigma, St Louis, MO, USA).
After a dose-finding study (data not shown) a concentration of
0.6 m
M
L-NAME was chosen for the experiments. N
x
-nitro-
D
-
arginine methyl ester (D-NAME) (Sigma), an inactive isomer of L-
NAME, was infused at 0.6 m
M
as a control. Infusions were
performed both during the normal sleep–wake cycle and during SD.
To increase the local NO concentration in the BF during the
spontaneous sleep–wake cycle, we infused the NO donor DETA/NO
(Sigma) at 1 m
M
into the BF (Beltran et al. 2000).
To block effects mediated by adenosine we infused caffeine, a
non-specific antagonist of adenosine receptors (Fluka, Basel,
Switzerland) at 1 m
M
, which was estimated to give an effective
tissue concentration of 0.1 m
M
. The effect of adenosine at this dose is
predominantly adenosine receptor antagonism, whereas higher doses
also act as phosphodiesterase inhibitors (Daly and Fredholm 1998).
According to the instructions of the manufacturer of CMA probes
and our previous measurements (Porkka-Heiskanen et al. 1997), the
probe recoveries for most substances are 10–15%. Thus, the
effective BF concentrations of drugs were estimated to have been
about one tenth of those in the infusion solutions. The concentra-
tions indicated above are concentrations in the infusion solution.
Histological verification of the probe locations
After the experiments, the probe tip locations were verified
histologically, as described previously (Kalinchuk et al. 2003)
(Fig. 1). Figure 1(a) shows the locations of the probe tips in the BF
(n ¼ 15) and in the area outside the BF (n ¼ 10). Fig. 1(b) shows a
representative probe scar in the brain.
Data presentation
To emphasize the homeostatic component of sleep regulation
(Borbely 1982), we normalized the sleep data from experimental
486 A. V. Kalinchuk et al.
Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
Ó 2006 The Authors
days to corresponding time bins from the baseline day a procedure
that eliminated the circadian variation in the data presented. The
effects of this procedure on the original data are exemplified in
Fig. 4. Because data presented either in 3- or 6-h bins described the
results equally, 6-h bins were chosen for presentation. For statistical
analysis, three 6-h mean values obtained during the first 18 h after
the treatment were averaged and normalized to the respective three
6-h mean values obtained during the baseline day. As the levels of
metabolites did not change during the 6-h baseline collection (data
not shown), we used a daily baseline protocol to evaluate the
changes in metabolite levels during the SD and drug infusions.
Concentrations of samples collected during the baseline pretreat-
ment period (average, ¼ 100%) and the experimental period
(average) were compared, and the difference was expressed as a
percentage increase/decrease compared with baseline. Data are
expressed as mean ± SEM.
Statistical analysis
Statistical analysis was performed using SigmaStat 3.0 statistical
software (SPSS Inc., Chicago, IL, USA). To evaluate the statistical
significance of the effects of SD and drug infusions on NREM/
REM sleep and delta power for comparisons of two groups we
used the Mann–Whitney rank sum test and for comparisons
between more than two groups we used one-way
ANOVA
followed
by Student–Newman–Keuls post hoc test (for normally distributed
values) or Kruskal–Wallis one-way
ANOVA
on ranks, followed by
Dunn’s post hoc test (for non-normally distributed values).
Comparison of EEG power spectra was performed using
ANOVA
on ranks for repeated measures followed by Holm–Sidak post hoc
test.
To evaluate the effects of different treatments on concentrations
of adenosine, lactate, pyruvate and NO
x
before and after the
treatments we used a paired t-test or Wilcoxon signed rank test (for
non-normally distributed data).
Results
Effects of SD
NO
x
concentration in the BF
We hypothesized that production of NO in the BF during SD
is causally related to the generation of recovery sleep. As an
initial test of this hypothesis, we measured the concentration
of NO
2
+NO
3
(NO
x
), oxidative degradation products of
NO, in microdialysis fluid taken from a probe implanted into
the BF. NO
x
concentrations in samples collected before the
SD (during the daily baseline collection, average of three
samples) were compared with those measured during the 3 h
of SD (average of three samples). The basal concentration of
NO
x
measured in samples collected before SD was
0.6 ± 0.1 l
M
. During the 3 h of SD, NO
x
concentrations
increased by 100 ± 51% compared with baseline predepri-
vation values (n ¼ 7, paired t-test, t ¼ 2.589, p < 0.05)
(Fig. 2a; BF). Separate analysis of samples collected at 1, 2
and 3 h of SD revealed increasing concentrations of NO
x
during the course of SD, with the highest value reached by
the end of SD (Fig. 3a). Concentrations gradually returned to
baseline during 2 h of recovery sleep.
Adenosine, lactate and pyruvate concentrations in the BF
The basal predeprivation concentration of adenosine in
microdialysis samples was 3.1 ± 0.5 nmol/L. During the
3 h of SD the adenosine concentration increased by
254 ± 70% compared with baseline predeprivation values
(n ¼ 7, paired t-test, t ¼ 5.179, p < 0.01) (Fig. 2b; BF). In
agreement with previous data (Porkka-Heiskanen et al.
1997), the adenosine level gradually increased during the
course of SD reaching its maximum by the third hour and
returned to the baseline level during 2 h of recovery sleep
(Fig. 3b), closely resembling the pattern of NO
x
in the course
of SD and recovery (Fig. 3a).
In agreement with our previous data (Kalinchuk et al.
2003), the concentration of lactate and pyruvate in samples
collected during SD were increased by 45 ± 8% (paired
Fig. 2 Changes in NO
x
,
adenosine, lactate and pyruvate concentra-
tions in the BF and outside the BF during SD. In the BF area (BF; n ¼
7) concentrations of NO
x
(a), adenosine (b), lactate (c) and pyruvate
(d) during SD were significantly higher than before SD; outside the BF
(nBF; n ¼ 7) they were not changed. Values are mean ± SEM.
*p < 0.05 versus control (predeprivation baseline level).
Nitric oxide and recovery sleep 487
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Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
t-test, t ¼ 5.020, p < 0.01) and 35 ± 5% (paired t-test, t ¼
5.371, p < 0.01) respectively, compared with the predepriva-
tion level (Figs 2c and d; BF).
Adenosine, lactate, pyruvate and NO
x
concentrations
outside the BF
The basal level of NO
x
detected outside BF did not differ
significantly from the level in the BF (0.5 ± 0.1 l
M
; t-test,
t ¼ 0.611, p > 0.5) but, in contrast to the effect observed in
the BF, outside the BF SD did not induce any changes in
NO
x
(n ¼ 7, non-significant decrease by 2 ± 16% compared
with predeprivation level) (Fig. 2a; nBF).
In agreement with our previously published data (Kalin-
chuk et al. 2003), adenosine, lactate and pyruvate concen-
trations were not changed during SD outside the BF
(Figs 2b–d; nBF).
Sleep and delta power (recovery sleep)
In order to characterize the effect of SD on the generation of
NREM and REM sleep we combined data collected from
animals with microdialysis probes located in the BF and
outside the BF (n ¼ 18). The effect of the SD on sleep was
evaluated by comparing the EEG recording obtained on the
SD day to that obtained on the baseline day (30-h recording).
SD significantly increased subsequent NREM sleep, which
was increased by 32 ± 3% compared with baseline (Mann–
Whitney rank sum test, T ¼ 551.000, p < 0.01) (Figs 4a–c)
and led to a 44 ± 8% increase in delta power (0.4–4Hz)
intensity during NREM sleep (T ¼ 352.000, p < 0.001)
(Figs 4d–f). SD also increased REM sleep by 41 ± 7%,
compared with baseline (T ¼ 494.000, p < 0.001) (Figs
4g–i).
In summary, SD increased both NREM and REM sleep as
well as delta power (recovery sleep) after the cessation of the
3-h SD period. During the deprivation, adenosine, lactate,
pyruvate and NO
x
concentrations were raised in microdi-
alysates collected from the BF area, but not in those collected
outside the BF area.
Effect of increase in NO concentration in the BF
If production of NO in the BF during SD plays a causal role
in the generation of recovery sleep, then we would expect
that instillation of NO donor into the BF should mimic the
effect of SD. For this purpose we infused the NO donor,
DETA/NO, through the microdialysis probe (experiment type
2). The effect of drug on metabolite concentrations was
evaluated by comparison of average concentrations from
pretreatment (¼ 100%) and treatment periods. The effect of
the drug on sleep was evaluated by comparing the EEG
record obtained on the drug infusion day to that obtained on
the baseline day (30-h recording) and SD day. The distribu-
tions of spectral power during spontaneous sleep, recovery
sleep and NO-induced sleep were also compared.
Adenosine, lactate and pyruvate
During DETA/NO infusion into the BF, the adenosine
concentration increased by 312 ± 89% compared with the
preinfusion baseline level (n ¼ 7, paired t-test, t ¼ 2.807,
p < 0.05) (Fig. 5a; BF), to a level similar to that encountered
during SD. The levels of lactate and pyruvate were
significantly increased by 54 ± 12% (paired t-test, t ¼
2.795, p < 0.05) and 31 ± 4% (paired t-test, t ¼ 4.723,
p < 0.05), respectively (Fig. 5b; BF).
Sleep and delta power
The treatment increased NREM sleep by 35 ± 4% compared
with the baseline (n ¼ 8, one-way
ANOVA
, F ¼ 24.575,
p < 0.001; Student–Newman–Keuls post hoc test, q ¼
Fig. 3 Changes in NO
x
and adenosine concentrations in the BF dur-
ing SD and recovery sleep. Values are mean ± SEM of seven rats. (a)
The concentrations of both adenosine (a) and NO
x
(b) increased
gradually in the course of SD and then gradually decreased during the
recovery sleep.
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Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
Ó 2006 The Authors
(a) (b) (c)
(f)(e)(d)
(i)(h)(g)
Fig. 4 Effects of SD on NREM sleep, REM sleep and delta power. (a,
d, g) Distribution of NREM/REM sleep (minutes) and EEG power
density (lV
2
) in the delta range (0.5–4 Hz) at different time points
during a baseline day and during a SD day. (b, e, h) For presentation
of the data in 6-h time bins, two 3-h mean values were averaged to
obtain 6-h mean values. (c, f, i) The bars show quantitative changes in
sleep/delta power during the first 18 h of recovery sleep after the SD
period. x-axis open bar, light period; hatched bar, SD during the light
period; black bar, dark period. (a–c) SD increased NREM recovery
sleep with a maximum at 9–12 h after SD. (d–f) Maximal increase in
delta power intensity during NREM sleep was observed at 3–6 h after
SD. (g–i) REM sleep was not changed during the first 6 h after SD but
was increased at 9–12 h after SD. *p < 0.05, **p < 0.001 versus
control (baseline EEG recording). Values are mean ± SEM.
Nitric oxide and recovery sleep 489
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Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
8.531, p < 0.001) (Fig. 5d). This effect was not quantita-
tively different from that of SD (Student–Newman–Keuls
post hoc test, q ¼ 0.748, p > 0.05).
Infusion of DETA/NO into the BF increased delta power
during NREM sleep by 46 ± 21% (Kruskal–Wallis one-way
ANOVA
on ranks, H
2
¼ 19.966, p < 0.001; Dunn’s post hoc
test, Q ¼ 2.577, p < 0.05) (Fig. 5e). Comparison with the
effect induced by SD showed no difference between
treatments (Dunn’s post hoc test, Q ¼ 0.267, p > 0.05).
Analysis of EEG power spectra during the first 6 h after
SD or DETA/NO infusion into the BF revealed that both
treatments induced increases in slow wave activity in the
range of 0.4–1.6Hz, showing that SD and DETA/NO induced
similar changes in the power spectrum, increasing the low
range of delta power, which is a typical change in sleep after
SD (Cirelli et al. 2005) (Fig. 6).
In contrast to the effect on NREM sleep, infusion of
DETA/NO into the BF led to significant decrease in REM
sleep by 30 ± 9% (one-way
ANOVA
, F ¼ 19.869, p < 0.001;
Student–Newman–Keuls post hoc test, q ¼ 3.157, p < 0.05)
(Fig. 5f).
Effect of increase in NO concentration outside the BF
Adenosine, lactate and pyruvate
Infusion of DETA/NO into the non-BF areas induced similar
effects as in the BF area: adenosine concentration was
increased by 265 ± 133% compared with baseline (n ¼ 7,
Wilcoxon signed rank test, T ¼ 27.000, p < 0.05), and
concentrations of lactate and pyruvate were increased by
34 ± 6% (paired t-test, t ¼ 4.894, p < 0.05) and 30 ± 5%
(paired t-test, t ¼ 2.915, p < 0.05) respectively (Figs 5a–c;
nBF).
Sleep and delta power
Infusion of DETA/NO into the non-BF areas did not
induce any significant changes in NREM or REM sleep
(Figs 5d–f).
(a) (b) (c)
(f)(d) (e)
Fig. 5 Changes in metabolite concentrations, sleep and delta power
after DETA/NO infusion into the BF and outside the BF, and after
pretreatment with caffeine. (a–c) Changes in metabolite concentra-
tions during the DETA/NO infusion. Concentrations of adenosine (a),
lactate (b) and pyruvate (c) during the 3 h of DETA/NO infusion into
the BF (n ¼ 7) and outside the BF (n ¼ 7) were significantly increased
compared with pretreatment baseline values. *p < 0.05 versus base-
line. (d–f) Changes in sleep/delta power and REM sleep after the
DETA/NO infusion and DETA/NO + caffeine infusion. Probes were
placed in the BF or outside the BF (nBF). Infusion of DETA/NO into
the BF (n ¼ 8) induced significant increases in NREM sleep
(p<0.001) (d) and delta power (p<0.05) (e), which were similar to
increases observed in recovery sleep after SD. Pretreatment with
caffeine (DETA + caffeine BF, n ¼ 4) blocked the DETA/NO effect.
REM sleep after DETA/NO infusion into the BF was significantly de-
creased (p<0.05) (f). Infusion of DETA/NO outside the BF (n ¼ 7) did
not change time spent in NREM sleep (d), delta power during NREM
sleep (e) or time spent in REM sleep (f). Values are mean ± SEM.
490 A. V. Kalinchuk et al.
Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
Ó 2006 The Authors
Subsequent NREM sleep differed from the response
evoked by DETA/NO infusion into the BF (n ¼ 7, one-
way
ANOVA
, F ¼ 62.728, p < 0.001; Student–Newman–
Keuls post hoc test, q ¼ 14.310, p < 0.001) but did not
differ from control (Student–Newman–Keuls post hoc test,
q ¼ 1.574, p > 0.05) (Fig. 5d).
Delta power during NREM sleep after DETA/NO infusion
outside the BF was not changed compared with the control
(decrease by 10 ± 18%; Kruskal–Wallis one-way
ANOVA
on
ranks, H
2
¼ 17.437, p ¼ 0.004; Dunn’s post hoc test, Q ¼
1.171, p > 0.05) but was significantly different from the
effect observed after DETA/NO infusion on to the BF
(Dunn’s post hoc test, Q ¼ 3.549, p < 0.05) (Fig. 5e).
The amount of REM sleep did not differ from that of the
control (one-way
ANOVA
, F ¼ 8.175, p < 0.05; Student–
Newman–Keuls post hoc test, q ¼ 0.539, p > 0.05) and
significantly differed from the effect of DETA/NO infusion
into the BF (Student–Newman–Keuls post hoc test, q ¼
4.469, p < 0.05) (Fig. 5f).
Effects of adenosine receptor antagonist infusion into the
BF on DETA/NO-induced sleep
In order to verify that the effect of DETA/NO on NREM
sleep can be mediated by adenosine we continuously infused
the non-specific antagonist of adenosine receptors, caffeine
(1 m
M
), into the BF for 4 h, starting 1 h before DETA/NO
infusion (experiment type 4). Pretreatment with caffeine was
able to block the increase in NREM sleep, which was
significantly different from effects induced by DETA/NO
infusion ( n ¼ 4, one-way
ANOVA
, F ¼ 10.961, p < 0.001;
Student–Newman–Keuls post hoc test, q ¼ 6.078,
p < 0.001) and SD (Student–Newman–Keuls post hoc test,
q ¼ 6.271, p < 0.001) (Fig. 5d).
Pretreatment with caffeine also diminished the increase in
delta power compared with the effect of DETA/NO infusion
(one-way
ANOVA
, F ¼ 7.596, p < 0.05; Student–Newman–
Keuls post hoc test, q ¼ 4.664, p < 0.05) (Fig. 5e), and the
effect of SD (Student–Newman–Keuls post hoc test, q ¼
5.357, p < 0.001).
In summary, infusion of the NO donor DETA/NO into the
BF and areas outside the BF during the spontaneous sleep–
wake cycle, a treatment that locally increases NO concen-
tration, induced increases in adenosine, lactate and pyruvate
concentrations. However, subsequent increases in NREM
sleep and delta power were observed only when DETA/NO
was infused into the BF area. In contrast to the effect of SD,
REM sleep was decreased after DETA/NO infusion into the
BF, whereas infusion outside the BF area had no effect on
subsequent REM sleep. The effects of DETA/NO on NREM
sleep and increase in delta power were blocked by caffeine,
a non-specific adenosine antagonist.
Effect of decreasing NO in the BF during SD
We wanted to test whether NO production in the BF during
SD is necessary for the generation of recovery sleep. We
approached this in two ways, one testing the effect of
pharmacological inhibition of NOS and second testing the
effect of a NO scavenger instilled during SD.
Effect of a NOS inhibitor L-NAME
The effects of L-NAME infusion into the BF were compared
with those of D-NAME (the non-active isomer of L-NAME)
infusion into the BF and infusion of L-NAME outside the BF
area. Drug infusions started 1 h before SD and continued for
4 h throughout SD (experiment type 3).
Infusion of L-NAME at 0.6 m
M
into the BF during SD
inhibited the increase in NO
x
concentration characteristic of
SD: the level was actually decreased by 14 ± 22% compared
with the pretreatment baseline level (n ¼ 6, paired t-test,
t ¼ 0.850, p > 0.05) (Fig. 7a). Infusion of D- NAME was
ineffective and did not prevent the increase in NO
x
level in
the course of SD (117 ± 58% above baseline; n ¼ 6,
Wilcoxon signed rank test, T ¼ 0.036, p < 0.05).
L-NAME infusion into the BF prevented the adenosine
increase (no difference from predeprivation value; Wilcoxon
signed rank test, T ¼ 0.361, p > 0.5) whereas during D-
NAME infusion the adenosine level was significantly
increased by 288 ± 65% (paired t-test, t ¼ 3.424, p < 0.05)
(Fig. 7b).
Infusion of L-NAME into the BF prevented accumulation
of lactate and pyruvate during SD whereas D-NAME did not
change the increase in metabolite concentrations; pyruvate
reached 29 ± 10% and was significantly different from the
Fig. 6 Effect of SD and DETA/NO infusion into the BF on EEG power
spectra in NREM sleep. Values were obtained by normalizing the
average power density during NREM sleep in the first 6 h after SD
(n ¼ 13) or DETA/NO infusion (n ¼ 8) to the average power in the
same frequency bin during the baseline EEG recording (¼ 100%).
Both SD and DETA/NO infusion induced an increase in the low-
frequency range of delta activity (0.4–1.6Hz) compared with baseline;
there were no differences between the two experimental groups.
Values are mean ± SEM. *p < 0.05, **p < 0.001 versus baseline.
Nitric oxide and recovery sleep 491
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Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
pretreatment level (paired t-test, t ¼ 4.240, p < 0.05); lactate
could not be measured because the drug interfered with the
assay (Figs 7c and d).
Infusion of L-NAME into the BF during SD significantly
inhibited NREM sleep recovery compared with SD (n ¼ 7,
Kruskal–Wallis one-way
ANOVA
on ranks, H
3
¼ 27.070,
p < 0.001; Dunn’s post hoc test, Q ¼ 3.464, p < 0.05)
whereas D-NAME did not influence recovery sleep: NREM
was significantly increased by 24 ± 4% compared with
baseline (Dunn’s post hoc test, Q ¼ 3.068, p < 0.05) and did
not differ from the effect of SD on NREM sleep (Dunn’s post
hoc test, Q ¼ 0.810, p > 0.05) (Fig. 7e).
Infusion of L-NAME into the BF during SD significantly
inhibited the increase in delta power compared with SD
between 0 and 18 h after treatment (n ¼ 7, one-way
ANOVA
,
F ¼ 5.645, p < 0.01; Student–Newman–Keuls post hoc test,
q ¼ 4.215, p < 0.05) (Fig. 7f). After infusion of D-NAME,
delta power was still increased by 39 ± 14% compared with
baseline. The response was different from that after L-NAME
infusion (Student–Newman–Keuls post hoc test, q ¼ 3.093,
p < 0.05) and was not different from that of SD alone
(Student–Newman–Keuls post hoc test, q ¼ 0.472,
p > 0.05).
Infusion of L-NAME also inhibited REM sleep recovery
as compared with SD (Kruskal–Wallis one-way
ANOVA
on
ranks, H
2
¼ 12.480, p < 0.05; Dunn’s post hoc test, Q ¼
2.700, p < 0.05) decreasing to baseline level (Dunn’s post
hoc test, Q ¼ 0.089, p > 0.05) (Fig. 7g).
Effect of the NO scavenger cPTIO
Similar to the L-NAME protocol, cPTIO infusion started 1 h
before SD and continued for 4 h up to the end of SD
(a) (c) (b) (d)
(g) (f) (e)
Fig. 7 Effect of L-NAME infusion into the BF and outside the BF
during SD on metabolite concentrations, NREM/REM recovery sleep
and delta power. (a–d) Changes in metabolite concentrations during
SD accompanied by L-NAME or D-NAME infusions. L-NAME infusion
into the BF (n ¼ 6) prevented increases in NO
x
(a), adenosine (b),
lactate (c) and pyruvate (d) levels during SD, whereas D-NAME infu-
sion had no effect. L-NAME infusion outside the BF (n ¼ 6) did not
induce significant changes in adenosine concentration. The level of
pyruvate was significantly decreased during L-NAME infusion outside
the BF compared with pretreatment baseline values. *p < 0.05 versus
baseline. (e–g) Changes in sleep/delta power and REM sleep after SD
accompanied by L-NAME or D-NAME infusion. Infusion of L-NAME
into the BF during SD (n ¼ 7) significantly reduced NREM recovery
sleep (e) and delta power (f) compared with SD (both p < 0.05)
whereas D-NAME did not block the SD effect (p > 0.05). (g) REM
sleep recovery was significantly decreased after L-NAME infusion into
the BF during SD (p < 0.05). Infusion of L-NAME outside the BF (n ¼
7) during SD did not effect NREM recovery sleep, delta power or REM
recovery sleep compared with SD (all p > 0.05). Values are
mean ± SEM.
492 A. V. Kalinchuk et al.
Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
Ó 2006 The Authors
(experiment type 3). The effects on sleep of three different
doses were tested.
Infusion of cPTIO at 10 m
M
during SD into the BF totally
prevented the increase in NO
x
concentrations: in fact, the
NO
x
level was significantly decreased by 73 ± 22% com-
pared with pretreatment baseline values (p<0.01) (data not
shown).
Infusion of cPTIO decreased NREM sleep recovery in a
concentration-dependent manner (Fig. 8a). After infusion of
cPTIO at the lowest dose (1 m
M
), sleep was still significantly
increased by 22 ± 2% compared with baseline (n ¼ 7,
Kruskal–Wallis one-way
ANOVA
on ranks, H
4
¼ 33.481,
p < 0.001; Dunn’s post hoc test, Q ¼ 3.102, p < 0.05) and
did not differ from the effect of SD (Dunn’s post hoc test,
Q ¼ 0.824, p > 0.05). Infusion of cPTIO at 5 m
M
substan-
tially blocked recovery sleep (increase by 12 ± 2%), which
was not different from baseline (n ¼ 7, Dunn’s post hoc test,
Q ¼ 1.733, p > 0.05). Infusion of cPTIO at 10 m
M
totally
inhibited recovery sleep compared with SD (n ¼ 7, Dunn’s
post hoc test, Q ¼ 1.173, p < 0.05). Similarly, infusion of
cPTIO dose-dependently decreased the increase in delta
power after SD (Fig. 8b). cPTIO blocked the increase in
delta power compared with SD at 5 m
M
(one-way
ANOVA
,
Student–Newman–Keuls post hoc test, q ¼ 4.059, p < 0.05)
and 10 m
M
(Student–Newman–Keuls post hoc test, q ¼
5.219, p < 0.05). In both cases delta power was no different
from that of the control (Student–Newman–Keuls post hoc
test, all q < 0.754, p > 0.05).
cPTIO also inhibited REM recovery sleep. The effect was
significant at 1 m
M
(Kruskal–Wallis one-way
ANOVA
on
ranks, H
4
¼ 16.356, p < 0.05; Dunn’s post hoc test, Q ¼
3.174, p < 0.05); at higher concentrations, similar reductions
were observed, but they were not significantly different from
SD (Dunn’s post hoc test, all Q < 1.597, p > 0.05) (Fig. 8c).
They were also not significantly different from the control.
Effect of NO decrease in the non-BF area during SD
Effect of the NOS inhibitor L-NAME
In contrast to the BF, the NO
x
level outside the BF, which
was not increased during SD alone (Fig. 2a), was signifi-
cantly decreased after L-NAME infusion by 31 ± 7% (n ¼
6, paired t-test, t ¼ 4.454, p < 0.05) (Fig. 7a).
In contrast to the BF, outside the BF there was no increase
in adenosine with SD. Infusion of L-NAME into the non-BF
areas during SD induced a small and insignificant decrease in
the level of adenosine (by 23 ± 19% compared with
predeprivation value; n ¼ 6, Wilcoxon signed rank test,
T ¼ 0.418, p > 0.05) and lactate (by 27 ± 20%, paired t-test,
t ¼ 1.867, p > 0.05). The concentration of pyruvate was
significantly decreased by 38 ± 10% (paired t-test, t ¼
4.943, p < 0.05) (Figs 7b–d).
The increase in NREM sleep after L-NAME infusion
outside the BF during SD was significant compared with the
control (by 33 ± 2%; n ¼ 7, Kruskal–Wallis one-way
ANOVA
on ranks, H
3
¼ 27.082, p < 0.001; Dunn’s post hoc test,
Q ¼ 3.338, p < 0.05) and did not differ from recovery sleep
after normal SD (Dunn’s post hoc test, Q ¼ 0.415, p > 0.05)
(Fig. 7e).
L-NAME infusion outside the BF was not effective in
decreasing delta power (increase by 43 ± 20% compared
with baseline; one-way
ANOVA
, F ¼ 3.523, p < 0.05; Stu-
dent–Newman–Keuls post hoc test, q ¼ 3.642, p < 0.05);
the effect was not different from that of SD (Student–
Newman–Keuls post hoc test, q ¼ 9.104, p > 0.05)
(Fig. 7f).
(a)
(b) (c)
Fig. 8 Effect of cPTIO infusion into the BF during SD on NREM/REM
recovery sleep and delta power. Infusion of cPTIO at 1, 5 and 10 m
M
(n ¼ 7) induced dose-dependent suppression of NREM recovery
sleep (a) and delta power (b). Infusion of cPTIO at 1 m
M
induced a
significant decrease in REM sleep recovery (c); infusions of cPTIO at 5
and 10 m
M
decreased REM but the changes were not significant.
Values are mean ± SEM.
Nitric oxide and recovery sleep 493
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Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
Infusion outside the BF did not block REM recovery
sleep, that increased by 44 ± 19% (compared with SD;
Kruskal–Wallis one-way
ANOVA
on ranks, H
2
¼ 10.401,
p < 0.05; Dunn’s post hoc test, Q ¼ 0.525, p > 0.05) (Fig. 7g).
In summary, treatment with L-NAME in the BF during SD
blocked the increase in NO
x
concentrations, prevented
adenosine, lactate and pyruvate accumulation in the BF, and
abolished the development of NREM recovery sleep,
increase in delta power and REM sleep recovery. Infusion
of L-NAME outside the BF was not effective in blocking
recovery sleep. Similarly, scavenging NO in the BF during
SD by cPTIO blocked the development of NREM sleep
recovery, decreased delta power and diminished REM sleep
recovery in a dose-dependent manner. These studies indicate
that NO production in the BF is necessary for the generation
of recovery sleep.
Effects of decreased NO in the BF on spontaneous sleep
Finally, we tested the effects of the NOS inhibitor L-NAME
on spontaneous sleep, and measured the respective changes
in metabolite concentrations in the BF and outside. The
results were confirmed by infusion of the NO scavenger
cPTIO into the BF. Drugs were infused for 3 h between
11.30 and 14.30 hours (experiment type 2).
Effect of the NOS inhibitor L-NAME
Infusion of L-NAME at 0.6 m
M
into the BF during the
spontaneous sleep–wake cycle induced a significant decrease
in adenosine level by 38 ± 7% compared with pretreatment
baseline level (n ¼ 6, paired t-test, t ¼ 3.312, p < 0.05)
(Fig. 9a; BF)
After infusion of L-NAME into the BF the lactate level
was significantly decreased by 38 ± 6% (Wilcoxon signed
rank test, T ¼ 21.000, p < 0.05) and the pyruvate level was
significantly decreased by 37 ± 10% (Wilcoxon signed rank
test, T ¼ 21.000, p < 0.05) (Figs 9b and c; BF).
L-NAME significantly decreased NREM sleep by
17 ± 3% (n ¼ 5, Mann–Whitney test, T ¼ 15.00; p <
0.01) (Fig. 9d) and decreased delta power by 58 ± 4%
(Fig. 9e) compared with baseline (Mann–Whitney test, T ¼
(a) (b) (c)
(f)(e)(d)
Fig. 9 Effect of L-NAME infusion into the BF and outside the BF on
spontaneous sleep. (a–c) Changes in metabolite concentrations dur-
ing L-NAME infusion. Infusions of L-NAME into the BF (n ¼ 6) and
outside the BF (nBF; n ¼ 6) decreased adenosine in the BF (a), and
lactate and pyruvate in both BF and outside BF. *p < 0.05 versus
pretreatment baseline. (d–f) Changes in NREM sleep/delta power and
REM sleep after the L-NAME infusion. NREM sleep (d) and delta
power (e) were significantly decreased after infusions of L-NAME into
the BF (n ¼ 5, both p < 0.01) but not outside (n ¼ 5, p > 0.05). (f)
REM sleep was not changed after L-NAME infusions into either area
(all p > 0.01). Values are mean ± SEM.
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Ó 2006 The Authors
15.00; p < 0.01), whereas the decrease of 25 ± 12% in REM
sleep was not significant (Mann–Whitney test, T ¼ 21.00;
p > 0.05) (Fig. 9f).
Effect of the NO scavenger cPTIO
Infusion of 10 m
M
cPTIO into the BF decreased NREM
sleep by 19 ± 7% compared with baseline (n ¼ 4, Mann–
Whitney test, T ¼ 26.000, p < 0.05) (Fig. 10) but the
decrease in delta power of 22 ± 19% was not significant
(Mann–Whitney test, T ¼ 22.00; p > 0.05). cPTIO at 1 m
M
slightly decreased NREM sleep by 8.2 ± 3.2%; the differ-
ence was not statistically significant (data not shown).
Infusion of cPTIO at 10 m
M
decreased REM sleep by
27 ± 12%. The difference was not statistically significant
(Mann–Whitney test, T ¼ 22.00; p > 0.05) (Fig. 10).
Effects of decreased NO outside the BF on spontaneous
sleep
Effect of the NOS inhibitor L-NAME
Infusion of L-NAME into the non-BF area decreased the
adenosine level by 38 ± 7% but the change was not
statistically significant (n ¼ 6, paired t-test, t ¼ 2.086,
p ¼ 0.105). Infusion of L-NAME outside the BF induced a
significant decrease in lactate level by 36 ± 1% (Wilcoxon
signed rank test, T ¼ 21.000, p < 0.05) and the pyruvate
level was significantly decreased by 23 ± 7% (paired t-test,
t ¼ 3.961, p < 0.05) (Fig. 9a–c; nBF).
L-NAME infusion outside the BF did not induce signifi-
cant changes in NREM sleep (decreased by 1 ± 4%
compared with baseline; n ¼ 5, Mann–Whitney test, T ¼
27.00; p > 0.05) and in delta power (increased by 11 ± 5%;
Mann–Whitney test, T ¼ 45.00; p > 0.05) (Figs 9d and e).
The change in REM sleep was also non-significant (decrease
by 11 ± 18%; Mann–Whitney test, T ¼ 33.00; p > 0.05)
(Fig. 9f).
Discussion
The main finding of the present study is that the production
of NO in the basal forebrain during SD is necessary and
sufficient to explain the production of NREM recovery sleep.
NO
x
concentration in the BF rose during SD and fell during
recovery sleep; infusion of a NO donor into the BF produced
an increase in NREM sleep comparable to that produced by
SD, and infusion of a NOS inhibitor or a NO scavenger into
the BF prevented the generation of NREM recovery sleep.
Remarkably, these effects were restricted to the BF; outside
this structure the treatments failed to have effects on sleep.
Moreover, increases in adenosine, lactate and pyruvate
concentrations in the BF preceded the increase in sleep,
whereas inhibition of recovery sleep was not associated with
an increase in metabolite concentrations, suggesting a
connection between energy metabolism and production of
recovery sleep in the basal forebrain area.
We have previously established that SD is accompanied by
an increase in extracellular adenosine concentration in the BF
(Porkka-Heiskanen et al. 1997; Kalinchuk et al. 2003). In
the present study, an NO-associated increase in NREM sleep
was accompanied by increased concentrations of adenosine,
whereas inhibition of NO synthesis or use of an NO-
scavenging compound was associated with unchanged levels
of adenosine and no NREM sleep recovery, suggesting that
the effect of NO on NREM sleep recovery is mediated
through an increase in extracellular adenosine in the BF. This
view was further confirmed by the observation that blocking
of adenosine receptors with caffeine prevented the DETA/
NO-induced increase in sleep. An increase in extracellular
adenosine level is a signal of discrepancy between energy
demand and availability (Dunwiddie and Masino 2001). We
have previously shown that experimentally induced energy
depletion in the BF increases local adenosine, lactate and
pyruvate levels and induces NREM sleep (Kalinchuk et al.
2003). Inhibition of energy production as a consequence of
local NO production may be a specific pathway for
generating adenosine release in the vicinity of structures
capable of producing sleep. In vitro, NO donors stimulate
glycolysis, increasing adenosine, lactate and pyruvate levels,
and inhibit oxidative phosphorylation, resulting in depletion
of total energy production and a decrease in the ATP/ADP
ratio (Rosenberg et al. 2000; Maletic et al. 2004). Neurones
and astrocytes respond differently to the increase in NO:
Fig. 10 Effect of cPTIO infusion into the BF on spontaneous sleep.
Data were obtained by normalizing cPTIO infusion day values for
NREM sleep and REM sleep to the baseline day values (¼ 100%) in 6-
h bins for each rat. Three 6-h mean values from the period 18 h after
the 3-h cPTIO infusion (shown by the grey shaded area) were aver-
aged and taken for final quantitative analysis. cPTIO infusion at 10 m
M
(n ¼ 4) induced a significant decrease in NREM sleep (p<0.05) and a
non-significant decrease in REM sleep (p > 0.05). Values are mean ±
SEM.
Nitric oxide and recovery sleep 495
Ó 2006 The Authors
Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
astrocytes are able to maintain their energy production by
increasing glycolysis by activating 6-phosphofructo-1-kin-
ase, whereas neurones appear to be unable to do this
(Almeida et al. 2004). Active inhibition by NO of energy
production in a specific brain region the BF may be
considered as a specific mechanism for induction of sleep in
response to prolonged wakefulness. We propose that the
concentration of NO increases during prolonged wakeful-
ness, resulting in inhibition of local energy metabolism and
production of adenosine, which is the signal for NREM sleep
induction.
NO production undergoes state-dependent modulations
during the sleep–wake cycle both in the thalamus and the
cortex, probably due to state-dependent changes in the
activity of neuronal NOS-containing projection neurones
(Burlet and Cespuglio 1997; Williams et al. 1997); the NO
values in both structures were lower during sleep than during
waking, suggesting an activity-related NO increase in the
brain. NO concentrations during SD have not been assessed
previously. Our results, showing an increased level of NO in
the BF during SD, support the hypothesis that NO production
is critical for homeostatic sleep regulation. One previous
study has shown a decrease in recovery sleep after L-NAME
administration during SD (Ribeiro et al. 2000). In that study
the recovery sleep was attenuated but not completely
abolished as in the present study. Systemic administration
of the drug in the previous study, as opposed to local
administration in the present study, may explain the partially
different outcomes of the two studies.
Our results are in agreement with those of many previous
studies, in which either intracerebroventricular or systemic
routes of administration of NO donors or NOS inhibitors
were used, most often during the spontaneous sleep–wake
cycle (reviewed in Gautier-Sauvigne et al. 2005). In the
present study, administration of both the NOS inhibitor and
the NO scavenger during spontaneous sleep decreased
NREM sleep, indicating that a certain NO level is required
for the appearance of a normal amount of NREM sleep, and
further that the effects of the drugs during SD may have two
components: effects on the mechanisms of recovery sleep
production (sleep homeostasis) and effects on spontaneous
sleep. Remarkably, L-NAME decreased spontaneous sleep
only when administered into the BF area, indicating that the
effect of NO on spontaneous sleep is also at least partially
localized. Two previous studies, employing local microin-
jections of NOS inhibitors into the BF during spontaneous
sleep, reported conflicting data (Vazquez et al. 2002; Monti
and Jantos 2004b); one reported changes in sleep and the
other did not. The discrepancies can most probably be
explained by the sharp localization of the effect, as shown in
the present study. Previous studies have also shown that local
administration of L-NAME into the pons modulates sleep
(Datta et al. 1997; Leonard and Lydic 1997; Hars 1999).
NOS is co-localized with acetylcholine in most of the BF
nuclei, as well as in the LDT/PPT nuclei that project to the
BF (Vincent and Kimura 1992). Both the BF and the LDT/
PPT area of the pons contain cholinergic neurones, which
regulate the vigilance state by release of acetylcholine (Jones
1991; Leonard and Lydic 1997; Hars 1999). As it has been
shown that NO modulates acetylcholine release (Vazquez
et al. 2002; Leonard and Lydic 2005), it is possible that the
effects of NO on sleep are mediated through cholinergic
neurones (Leonard and Lydic 2005).
Adenosine, through A
1
receptors, is an inhibitory neuro-
modulator (Fredholm 1995). Clinically, adenosine is used for
neuroprotection in connection with seizures (Boison 2005)
and as anti-arrhythmic treatment of supraventricular tachy-
cardia (Hutchinson and Scammells 2004). Adenosine is also
a powerful vasodilator (Biaggioni 2004). It stimulates
neuronal activity through A
2A
receptors (Fredholm 1995).
Adenosine appears to have a specific function in the
regulation of recovery sleep (Porkka-Heiskanen et al.
1997). The site-specificity of the effects in the present study
is in agreement with earlier studies, in which the increase in
adenosine levels during SD was found to be restricted to the
BF (Porkka-Heiskanen et al. 2000; Kalinchuk et al. 2003).
The sites at which adenosine, lactate, pyruvate and NO
increases were found during SD closely correspond to the
area where the cholinergic cells of the BF are situated the
HDB, substantia innominata and MCPO. The present results
suggest that recovery sleep may be regulated through
waking-active cholinergic cells, whereby adenosine acting
on A
1
receptors would decrease their firing rate (Rainnie
et al. 1994). However, sleep homeostasis was not affected in
mice with A
1
receptor knockout (Stenberg et al. 2003), and a
specific lesion of cholinergic cells did not affect sleep (Kapas
et al. 1996; Gerashcenkko et al. 2001). Another possibility is
that adenosine could disinhibit the inhibitory GABAergic
neurones of the nearby ventrolateral preoptic nucleus (Sherin
et al. 1996; Chamberlin et al. 2003) thus promoting sleep.
A
2A
receptor agonist injection into the subarachnoid space
promoted sleep (Scammell et al. 2001), suggesting that
adenosine could also modulate sleep through activation of
A
2A
receptors. In addition, the effects of caffeine were
recently shown to be mediated through A
2A
receptors (Huang
et al. 2005). It is probable that during SD these mechanisms
work in synchrony: the increased adenosine level inhibits the
waking-active neurones and disinhibits sleep-active neurones
through A
1
receptors, while activating sleep-active neurones
through A
2A
receptors. The sleep response to increased
adenosine levels continues for several hours (Porkka-
Heiskanen et al. 1997). We have previously shown that
increased adenosine levels in the BF increase the expression
of A
1
receptors, providing one possible mechanism for the
prolonged sleep-promoting effect (Basheer et al. 2001).
An alternative explanation that we considered is that the
drugs used in this study might have non-specific effects on
sleep that subtract from recovery sleep without actually
496 A. V. Kalinchuk et al.
Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 483–498
Ó 2006 The Authors
affecting the fundamental process. For example, infusion of
glutamate might block recovery sleep but this does not
necessarily mean that glutamate plays a specific role in
homeostatic sleep regulation. We do not think that the effects
of the drugs used in this study are non-specific because: (i)
drugs of varied chemical structure were found to act in a
predictable way based on their effect on NO levels: a NOS
inhibitor and a NO scavenger inhibited recovery sleep,
whereas a NO donor mimicked recovery sleep; (ii) NO
2
/NO
3
levels increased with SD, indicative of an association
between NO production and recovery sleep; and (iii) the
effects of drugs blocking recovery sleep were long-lasting.
The effects were typically at a maximum not during or
immediately after administration, which was during the light/
rest period, when a stimulant would be expected to have
maximal effect, but during the subsequent dark/active period
(Figs 7 and 8). Interestingly, reduction of NO levels in the
BF also had long-lasting effects on spontaneous sleep (Figs 9
and 10), suggesting that NO production is a consequence of
waking per se and is central in the generation of sleep drive
or Process S.
The present results, in addition to confirming NO as a
powerful sleep-facilitating agent, provide strong evidence
that NO is a critical part of the homeostatic sleep control
mechanism regulating effects of prolonged wakefulness, and
is, in fact, necessary and sufficient for the generation of
recovery sleep. The results further support the hypothesis that
local energy depletion in the BF, as reflected in increases in
adenosine, lactate and pyruvate levels specifically in this area
during SD, may be the initiator of the chain of events that
culminate in the induction of recovery sleep. Adenosine, one
of the metabolites indicative of energy depletion, and which
during SD accumulates in the extracellular space where it can
activate adenosine receptors, appears to be the key molecule
in the final induction of recovery sleep.
Acknowledgem ents
We thank Mr Ernst Mecke, Mrs Pirjo Saarelainen and Mrs Sari
Levo-Siitari for excellent technical assistance. The work was funded
by NIH grants P50 HL60292 and P01 HD18655, the Academy of
Finland, European Union grant MCRTN-CT-2004–512362, Finska
La¨karesa¨llskapet, the Sigrid Juselius Foundation and the European
Sleep Research Society Sanofi-Synthelabo Research Award to AK.
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    • "Slow wave, lowfrequency electroencephalography (EEG); likely represent a measure of sleep intensity – extended waking leads to increases in slow-wave energy (SWE) during the subsequent recovery night and the extent of this SWE increase is a function of prior wake duration (Åkerstedt et al., 2009; Brunner et al., 1990). In addition to SWE, other biological markers of sleep homeostasis have been identified including extracellular adenosine, central nitrous oxide levels and salivary amylase; levels of these markers increase with prolonged sleep wakefulness and thus may reflect an increased sleep drive (Kalinchuk et al., 2006; Kalinchuk et al., 2011; Scharf et al., 2008; Seugnet et al., 2006). The homeostatic process of sleep-wake regulation interacts with but is independent from circadian control (Dijk et al., 1989b). "
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