J. Sleep Res.

(2009) 18, 357–364 Mice sleep: build up of process S

doi: 10.1111/j.1365-2869.2008.00728.x

Sleep and sleep homeostasis in constant darkness in the rat

TOM DE BOER
Laboratory for Neurophysiology, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands

Accepted in revised form 17 November 2008; received 9 July 2008

SUMMARY According to the two-process model of sleep regulation, a homeostatic Process S increases
during waking and decreases during sleep. The time course of Process S can be derived on
the basis of changes in vigilance states and changes in electroencephalogram slow-wave
activity (SWA, activity below 4 Hz) during non-rapid eye movement (NREM) sleep. In
most mouse strains, an optimal fit between S and SWA was achieved with one increasing
(active during waking and REM sleep) and one decreasing time constant (active during
NREM sleep) for Process S. However, in the rat, systematic deviations in the light and
dark periods were observed, which were resolved by introducing different decreasing time
constants between the light and dark periods. The present study shows that this difference
between the rest (light) and active (dark) phases remains, and may even be larger, after
animals are adapted to constant dark conditions for at least a week. In addition, the data
show that the build-up rate of SWA at the onset of a NREM sleep episode is slow
compared with the increase rate under light–dark conditions, and that this build-up rate
changes with the circadian phase. The slow build-up rate introduces a systematic error
between the simulation of Process S and SWA in NREM sleep. The circadian modulation
of the build-up rate may, together with circadian changes in NREM sleep episode
duration, be the source of the necessity of introducing a difference in the decreasing time
constant between the rest and active phases.
k e y w o r d s electroencephalogram slow-wave activity, rat, simulation, sleep
deprivation, sleep homeostasis, sleep regulation

function of prior waking duration (Deboer and Tobler, 2003;

INTRODUCTION
In mammals slow waves (1–4 Hz) in the electroencephalogram
(EEG) during non-rapid eye movement (NREM) sleep reflect
synchronized burst-pause firing patterns of hyperpolarized
thalamocortical neurons (Steriade et al., 1993). The activity of
these slow waves (slow-wave activity, SWA) can be quantified by
a spectral analysis of the EEG. SWA positively correlated with
arousal thresholds (Neckelmann and Ursin, 1993) and nega-
tively with NREM sleep fragmentation (Franken et al., 1991a)
and is therefore seen as a measure of NREM sleep intensity.
NREM sleep SWA is dependent on the duration of prior
waking and sleep. Sleep deprivation (SD) results in an increase
in SWA in subsequent NREM sleep and this increase is a
Correspondence: T. de Boer, PhD, Laboratory for Neurophysiology,
Department of Molecular Cell Biology, LUMC S-05-P, PO Box 9600,
2300 RC Leiden, The Netherlands. Tel.: +31 71 526 9771; fax:
+31 71 526 8270; e-mail: tom.de_boer@lumc.nl
Dijk et al., 1987; Huber et al., 2000; Lancel et al., 1991;
Strijkstra and Daan, 1998; Tobler and Borbely, 1986), and a
nap in the afternoon results in attenuated SWA during NREM
sleep in the subsequent evening (Werth et al., 1996). SWA
decreases over the course of a sustained sleep period,
independent of the circadian phase (Dijk and Czeisler, 1995).
These and other observations have been interpreted as
evidence that NREM sleep is homeostatically regulated and
that SWA reflects the need for NREM sleep (Borbely, 1982;
Daan et al., 1984; reviewed by Borbely and Achermann, 2005).
The dynamics of this homeostatic process (Process S) has been
studied extensively and mathematical models have been
applied successfully in humans (Achermann et al., 1993), rat
(Franken et al., 1991b) and mouse (Franken et al., 2001;
Huber et al., 2000).
In contrast to the mathematical model applied in humans,
where Process S is estimated based on the actual SWA

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358 T. de Boer
expressed by the subjects (Achermann et al., 1993), the models
in rodents estimate Process S purely on the basis of prior
sleep–wake history and correlate the resulting level of Process
S with the expressed SWA (Franken et al., 1991b, 2001; Huber
et al., 2000; Vyazovskiy et al., 2007). In these simulations, the
time course of S is determined iteratively on the basis of the
vigilance states. During waking and REM sleep, S increases
according to a saturating exponential function with an upper
asymptote of 1 (Franken et al., 1991b; Huber et al., 2000) or
an upper asymptote derived from the SWA data (Franken
et al., 2001; Vyazovskiy et al., 2007). During NREM sleep, S
decreases according to an exponential function with a lower
asymptote of 0 (Franken et al., 1991b; Huber et al., 2000) or a
lower asymptote derived from the data (Franken et al., 2001;
Vyazovskiy et al., 2007). The time constants of the increase (si)
and decrease (sd), and the initial value at the start of the
experiment (S0) are estimated by optimizing the linear corre-
lation between S and SWA and minimizing the mean square of
the difference between S and SWA on the basis of hourly
values.
For mice, this approach was sufficient to reach an optimal fit
in most strains (Franken et al., 2001; Huber et al., 2000).
However, in the rat, an additional modulation of sd was needed
(Franken et al., 1991b). SWA was consistently higher than S in
the light period and lower than S in the dark period. Similarly,
in a simulation of the effects of a 6-h SD at the start of the dark
period, SWA was lower than S in the dark period in the rat
(Vyazovskiy et al., 2007). Absence of light was shown to
increase SWA in the rat (Alfoldi et al., 1990; Tobler et al., 1994)
and therefore a higher sd was assumed in the light compared
with in the dark period. This adaptation increased the success of
subsequent simulations considerably (Franken et al., 1991b).
Besides the influence of light on sleep and SWA (Alfoldi
et al., 1990; Tobler et al., 1994) it was proposed that the
discrepancies between S and SWA could be caused by a
difference in the duration of NREM sleep episodes between the
light and dark periods. In addition, at the start of an NREM
sleep episode, it takes a couple of minutes before SWA reaches
an asymptotic level (AL) (Trachsel et al., 1989). When the
increase rate differs between the light and dark periods, this
will cause an under- or overestimation of Process S, depending
on the time of day.
To eliminate the effect of light and other exogenous stimuli,
animals were released in constant dark conditions for at least a
week and a baseline day was recorded followed by an SD of
6 h and a recovery period of 18 h. The time course of SWA
within an NREM sleep episode was analyzed and the hourly
values of SWA were simulated with and without a circadian
modulation of sd. The environmental conditions are, from a
chronobiological point of view, adequate to rule out any
environmental influence on daily modulation of vigilance
states or EEG variables, which means that any daily modu-
lation observed must be endogenous, e.g. from within the
animal itself (Mistlberger and Rusak, 2005). The present
conditions enable to determine whether under constant dark
conditions: (1) a circadian modulation is still necessary in the
simulation, (2) a circadian modulation is present in vigilance
state episode frequency and duration, (3) a circadian modu-
lation is present in the increase rate of SWA at the onset of an
NREM sleep episode.
MATERIALS AND METHODS
All experiments were performed under the approval of the
Animal Experiments Ethics Committee of the Leiden University
Medical Center. Male Wistar rats (n = 11), 300 g at the time of
surgery, were implanted under deep anesthesia with EEG and
electromyogram (EMG) electrodes. For the EEG, screw elec-
trodes (Plastics One, Roanoke, VA, USA) were screwed through
the skull on the dura over the right parietal cortex and the
cerebellum. For the EMG, two wires with suture patches
(Plastics One, Roanoke) were inserted between the skin and neck
muscle tissue.
Electroencephalogram and EMG recording techniques were
as described previously (Deboer et al., 2003, 2007). In short,
the EEG and EMG were continuously recorded and amplified
(amplification factor 2000), band-pass filtered (0.5–30 Hz,
)40 dB ⁄ decade) and subjected to analog-to-digital conversion
(sampling rate 100 Hz). All data were recorded simultaneously
in 10-s epochs and stored on a computer hard disk.
The animals were connected to the recording system by a
flexible cable and a counterbalanced swivel system, and then
allowed to remain on the cable for at least 1 week (9–15 days)
before the start of the recording. During that time and during
the experimental recordings, the animals were maintained in
continuous darkness. Drinking rhythms were continuously
recorded and an estimate of the circadian phase was obtained
by visual inspection of drinking onset. Under constant
conditions, a 24-h baseline day was recorded. Subsequently,
the animals were sleep deprived for 6 h, starting at rest onset
(CT 0), followed by 18-h recovery.
During the 6-h SD, the animals were observed with an
infrared camera in addition to the online EEG recording.
Whenever the animals appeared drowsy or the EEG exhibited
slow waves, they were mildly disturbed by moderate noise, by
the experimenter entering the recording room, and, if neces-
sary, by introducing fresh food, fresh drinking water or nesting
material into the cage. The animals were never touched, and
never disturbed during feeding and drinking.
Offline EEG power density spectra were calculated with a
Fast Fourier Transform routine within the frequency range of
0.25–25.0 Hz in 0.1-Hz bins. EMG signals were integrated
over 10-s epochs. Three vigilance states, waking, NREM sleep
and REM sleep were determined on the basis of standardized
EEG and EMG criteria for rats (Deboer et al., 2003, 2007;
Franken et al., 1991a). Epochs containing artifacts in the EEG
signal were excluded from the analysis of the spectrum.
All EEG power density data were standardized relative to
the mean 24-h baseline value in NREM sleep (=100%).
Hourly values of vigilance states and SWA in NREM sleep
were calculated. To analyze changes in SWA at transition into
NREM sleep, NREM sleep episodes lasting at least 7 min were
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 Sleep and sleep homeostasis in constant darkness 359

selected based on episode duration criteria published previ-
ously (Deboer and Tobler, 1996; Huber et al., 2000). The
analysis was performed on consecutive 6-h intervals of baseline
(CT 0–6; 6–12; 12–18; 18–24) and recovery (CT 6–12; 12–18;
18–24). The time course of SWA within the NREM sleep
episode was approximated by fitting a saturating exponential
function (Eqn 1) to the empirical values (Trachsel et al., 1989):
si = 8.6 h, sd = 3.2 h and S0 = 0.55. This resulted in a
significant correlation between SWA and S (r = 0.555).
However, SWA was consistently higher than S in the rest
period and lower than S in the active period (Fig. 1, top
panel). Systematically varying si, sd and S0 did not result in
one single optimal solution (data not shown); however, a local

SWA ¼ Að1  et=T Þ þ SWA0 ð1Þ (a)

where SWA is relative SWA, A is the asymptote, t is the time

after an NREM sleep episode onset counted in 10-s epochs, T
is the time constant of increase and SWA0 is the initial value of
SWA at t = 0 (onset of NREM sleep). The parameters to be
determined are A and T. The AL of SWA is the sum of A and
SWA0.
In the simulation of Process S, the time course of S was
determined iteratively on the basis of vigilance states (Franken (b)
et al., 1991b; Huber et al., 2000). In 10-s epochs scored as
waking or REM sleep, S increased according to a saturating
exponential function with an upper asymptote 1 (Eqn 2). In
epochs scored as NREM sleep, S decreased according to an
exponential function with a lower asymptote of 0 (Eqn 3). S
was computed according to Franken et al. (1991b) and Huber
et al. (2000):
Stþ1 ¼ 1  ð1  St ÞeDt=si ð2Þ
Dt=sd (c)
Stþ1 ¼ St e ð3Þ

where St and St+1 are values of S for consecutive epochs, si

and sd the time constants of the increase and decrease,
respectively, and Dt the 10-s time interval of the iteration. For
all animals, the same parameters were used. Initially, the time
constants (si = 8.6, sd = 3.2) and the initial value
(S0 = 0.55) were taken from the publication by Franken
et al. (1991b). Subsequently, the time constants si and sd and
the initial value S0 were estimated by optimizing the linear
correlation between the hourly values of SWA in NREM sleep
and S in baseline and recovery for all animals. Finally, si and
S0 were set to 8.6 h and 0.55, and sd was increased in the rest
phase (CT 0–12) and reduced in the active phase (CT 12–24)
according to the values provided by Franken et al. (1991b). To
compare SWA and S, SWA was transformed according to a
linear regression. For plotting purposes, data from Process S
were standardized relative to the mean 24-h baseline values of
S. Values of r presented are mean r-values over all animals
after FisherÕs z transformation of the individual r-values.
Overall effects were analyzed by two-way anova with factors
ÔtimeÕ (1- and 6-h intervals) and ÔconditionÕ (baseline or
recovery day). Contrasts were tested by post hoc two-tailed t-
test only if the main factor or interaction of the anova reached
significance.
RESULTS
The first simulation of Process S was based on the initial values
and time constants obtained by Franken et al. (1991b). In this,
Figure 1. A 48-h record of EEG slow-wave activity (SWA, EEG
power density between 1.1 and 4.0 Hz) and simulation of Process S
with the time constants obtained by Franken et al. (1991b). Curves
connect 1-h mean values (±SEM) for 24-h baseline, 6-h SD and 18-h
recovery. SWA in NREM sleep and the level of Process S are expressed
as a percentage of the 24-h baseline value (=100%, see Materials
and methods). (a) Comparison between SWA and Process S without
circadian modulation of the decreasing time constant sd. (b) Com-
parison between SWA and Process S with circadian modulation of sd,
where sd = 3.9 during the rest phase and 2.5 during the active phase
(Franken et al., 1991b). (c) Comparison between SWA and Process S
after optimizing the circadian modulation of sd, where sd = 4.8 h
during the rest phase and 1.57 h during the active phase. Triangles in
panel A indicate where SWA was increased above baseline values
during recovery (open triangles P < 0.05; solid triangles P < 0.01,
two-tailed paired t-test after significant anova). Asterisks indicate sig-
nificant differences between data and simulation (P < 0.05, two-tailed
paired t-test after significant anova). Note that SWA is consistently
higher during the rest phase and consistently lower during the active
phase compared with the simulation of Process S in the top panel. This
difference decreases when a circadian modulation of sd is introduced
(middle panel) and virtually disappears when this difference is
optimized (bottom panel).

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360 T. de Boer
maximum was obtained close to the values obtained by
Franken et al. (1991b). It therefore was decided to continue
with those values. Making sd variable by increasing sd in the
rest phase (sdr) and decreasing sd in the active phase
(sdr = 3.9, sda = 2.5, Franken et al., 1991b) reduced the
amount of 1-h intervals with significant differences between S
and SWA and increased the value of r to 0.682 (Fig. 1, middle
panel). Subsequently, the difference in sd between the rest and
active phases was iteratively increased, keeping the other
variables (si and S ) constant. This resulted in an optimal
solution with sdr = 4.8, sda = 1.57. Only a few 1-h intervals
still show a significant difference between S and SWA and the
value of r increased to 0.749 (Fig. 1, bottom panel).
The average time course of SWA within the first 7 min after
the start of a NREM sleep episode is shown in Fig. 2. SWA
exhibited an initial rapid increase after NREM sleep onset and
reached an AL within 2–5 min. During baseline, the AL
(Table 1) decreased from the first 6 h (143%) to the second 6 h
(100%), remained constant from the second to third 6-h
period (93%) and increased again from the third to fourth
6-h period (127%). These changes in the AL over the baseline
day were significant (P < 0.05, anova factor Ôtime of dayÕ).
After the 6-h SD, the AL was significantly increased to 167%
in the second 6-h period of the rest phase (CT 6–12) and
then decreased progressively reaching levels significantly
below that at baseline in the last 6 h of the active period
(101%). The amount of waking interspersed in the NREM
sleep episodes did not differ within the first 5 min after
the start of the NREM sleep episodes across the circadian
phase.
The time constant T of the increase in SWA at the onset of
NREM sleep changed significantly over the day (Table 1) with
the slowest T in the first 6 h of the rest phase (2 min and 13 s).
T was shorter in the second 6-h interval (1 min and 25 s)
reaching the fastest T in the first 6 h of the active phase (40 s)
and slowing after that (1 min and 33 s). The changes over the
day in T were significant (P < 0.05, anova factor Ôtime of
dayÕ); however, T was not influenced significantly by SD,
reaching similar values in recovery after SD compared with
that at baseline.
Slow-wave activity in NREM sleep was significantly
increased for several hours immediately after SD (Fig. 1).
Moreover, NREM sleep and REM sleep were increased above
baseline levels for several 1-h intervals throughout the 18-h
recovery period (Fig. 3). The first and second 6-h intervals
after SD showed significant increments in NREM and REM
sleep, compared with that at baseline (Table 1).
Waking episode duration was significantly shorter in the rest
phase compared with that in the active phase (P < 0.05, anova
factor Ôtime of dayÕ), but the frequency of waking episodes did
not change over the day (Table 2). As a mirror image, NREM
sleep episode duration was significantly longer in the rest phase
compared with that in the active phase (P < 0.05, anova
factor Ôtime of dayÕ). NREM sleep episode frequency decreased
significantly from the rest to active phase (P < 0.05 anova
factor Ôtime of dayÕ). Similarly, REM sleep episode frequency
and duration decreased significantly from the rest to the active
phase (P < 0.05 anova factor Ôtime of dayÕ). SD mainly
influenced vigilance state episode frequency with less waking
episodes and more NREM and REM sleep episodes in the
recovery period (Table 2). Waking episode duration was
significantly reduced in the first 6 h of the active phase of the
recovery period.
DISCUSSION
The present analysis shows that SWA in the NREM sleep
EEG needs time to come to full expression after the start of a
Figure 2. Time course of slow-wave activity
(SWA; EEG power density between 1.1 and
4.0 Hz) within NREM sleep episodes during
baseline (circles) and recovery after 6-h SD
(dots). Plotted are the first 7 min of NREM
sleep episodes with a minimal duration of
7 min and the preceding minute of waking or
REM sleep. SWA is expressed as a percentage
of the 24-h baseline value of SWA in NREM
sleep (=100%). From left to right, consecu-
tive 6-h intervals are plotted. Data points
represent mean values of SWA of successive
10-s epochs (±SEM). Arrows indicate the
value of the rise rates (T) for baseline (arrows
point up) and recovery (arrows point down).
Horizontal thin solid lines indicate ALs of
SWA for baseline (BL) and recovery (RE-
C) ± SEM (dotted lines). Values of T and the
ALs are given in Table 1.
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 Sleep and sleep homeostasis in constant darkness 361

Table 1 Amount of vigilance states and the rise rate (T) and Table 2 Episode frequency and duration of the different vigilance
asymptote of SWA in the course of an NREM sleep episode in 6-h states in 6-h intervals
intervals
Episode duration (min) Episode frequency (h)1)
Waking NREM REM Asymptote
(%) (%) (%) T (min) (%) Waking NREM REM Waking NREM REM

Baseline Baseline
1 31.2 (2.3) 57.3 (1.8) 11.5 (1.0) 2.22 (0.23) 143.7 (14.1) 1 2.3 (0.1) 7.5 (0.4) 2.1 (0.1) 7.8 (0.3) 5.1 (0.2) 3.4 (0.3)
2 25.2 (1.1) 60.4 (1.2) 14.3 (0.5) 1.42 (0.14) 99.6 (8.7) 2 1.4 (0.1) 7.0 (0.4) 1.9 (0.1) 9.8 (0.5) 5.8 (0.3) 4.5 (0.3)
3 67.6 (4.0) 28.6 (3.1) 3.8 (1.0) 0.67 (0.08) 92.6 (4.9) 3 6.5 (0.9) 5.0 (0.5) 1.6 (0.1) 7.0 (0.6) 4.0 (0.3) 1.4 (0.3)
4 61.7 (3.4) 33.6 (2.7) 4.6 (0.7) 1.56 (0.24) 126.8 (14.6) 4 5.4 (0.5) 5.5 (0.3) 1.6 (0.2) 6.8 (0.4) 4.1 (0.3) 1.8 (0.3)
SD recovery SD recovery
1 92.2 (1.0) 7.8 (1.0) 0 (0.0) – – 1 – – – – – –
2 15.7 (1.8)* 67.0 (1.5)* 17.3 (1.0)* 1.24 (0.13) 166.6 (7.6)* 2 1.5 (0.3) 8.4 (0.4) 2.2 (0.1) 6.1 (0.4)** 5.0 (0.2)* 4.8 (0.3)
3 50.8 (3.2)* 41.3 (2.6)* 7.9 (0.8)* 0.77 (0.14) 93.5 (8.1) 3 3.7 (0.3)** 5.4 (0.4) 1.7 (0.1) 8.0 (0.4) 5.2 (0.2)** 2.9 (0.3)**
4 58.4 (2.5) 36.4 (2.1) 5.2 (0.5) 1.20 (0.29) 101.1 (17.4)* 4 4.3 (0.3) 5.1 (0.4) 1.7 (0.2) 8.2 (0.6)* 4.7 (0.2)* 2.0 (0.2)

Significant differences from baseline (*P < 0.01, two-tailed paired *P < 0.05 or **P < 0.01 indicate significant differences from
t-test after significant anova). baseline (two-tailed paired t-test after significant anova).
NREM, non-rapid eye movement; REM, rapid eye movement; SD, NREM, non-rapid eye movement; REM, rapid eye movement; SD,
sleep deprivation. sleep deprivation.

NREM sleep episode. In our experimental conditions, it took

Figure 3. Time course over 48 h of waking, non-rapid eye movement
(NREM) sleep and rapid eye movement (REM) sleep across the
baseline day, during 6-h sleep deprivation and 18-h recovery in 1-h
mean values (±SEM). The vigilance states are expressed as a per-
centage of total recording time (=100%). Asterisks indicate where
recovery significantly differed from baseline (P < 0.05, two-tailed
paired t-test after significant anova).
2–5 min before SWA reached plateau levels and this duration
was a function of the time of day, but not of sleep pressure or
previous time awake. A similar analysis at the end of a
NREM sleep episode did not reveal an effect of SD or the time
of day on the drop of SWA at the end of an NREM sleep
episode (data not shown). In the simulation of Process S, sd is
ÔonÕ from the first scored 10-s NREM sleep epoch. The level of
SWA is thought to represent the decrease rate of Process S.
However, the present rodent models do not represent what is
happening with SWA (and therefore the decrease rate of
Process S) at the onset of a NREM sleep episode. This may be
one of the reasons why there is a systematic circadian
modulation of the difference between S and SWA in the initial
simulation.
This is schematically drawn for the baseline situation in
Fig. 4. sd of Process S (in gray) is here set to the AL of each
NREM sleep episode in the 4.6-h baseline episodes obtained
in Fig. 2. The actual mean values of SWA are plotted in
front of it, relative to this AL. Because SWA needs time to
build up to its full expression for several minutes, the
simulation is systematically overestimating SWA during the
first minutes of a NREM sleep episode. After optimizing of
sd and si, this will result in a systematic underestimation of
sd. This by itself will not cause a circadian modulation in the
difference between S and SWA. However, the rise rate of
SWA at the onset of a NREM sleep episode is shown to
change over the circadian day and therefore the amount of
underestimation of sd also changes with the time of day.
Fig. 4 clearly shows that the differences in the area under the
curve between SWA and the ALs are larger in the first and
last 6-h episodes compared with 6-h episodes between CT6
and CT18 and this difference is significantly changing with
the time of day (P < 0.05, anova). This circadian change in
T introduces circadian fluctuations in the accuracy of the
simulation when a constant sd is applied.

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362 T. de Boer

Figure 4. Time course of slow-wave activity (SWA; EEG power density between 1.1 and 4.0 Hz) within NREM sleep episodes during the baseline
day (white area). Plotted are the first 7 min of NREM sleep episodes with a minimal duration of 7 min and the preceding minute of waking or
REM sleep. From left to right consecutive 6-h intervals are plotted. Data points represent mean values of SWA of successive 10-s epochs. SWA is
expressed as a percentage of the AL of SWA plotted in Fig. 2 (=100%). The gray area behind SWA represents the hypothetical level of Process S.
Discrepancies between hourly values of the model and SWA are probably caused by differences in the build-up rate of SWA at entrance into
NREM sleep, which changes as a function of the time of day. The difference between model and data is significantly smaller between CT12 and
CT18 (smaller gray area) compared with that between CT0 and CT6 (larger gray area, P < 0.05, Duncan after significant anova).

The rise rates of SWA at the initiation of NREM sleep in the
present experiment are considerably slower than those obtained
previously by Trachsel et al. (1989). The main difference
between the two experiments is the availability of light in the
rest phase in the previous experiments (Trachsel et al., 1989). In
the absence of light, rats are known to express more SWA during
NREM sleep in the rest phase (Tobler et al., 1994). Possibly, the
absence of light during the rest phase enables the increase in T. In
that case, plateau levels will be reached later, resulting in an
increase in SWA in the mean hourly values compared with SWA
in the light condition. This finding is in accordance with the
notion that the daily changes in the rise rate influence the success
of the simulation. Longer rise rates in the present study,
particularly at the beginning of the rest phase, are paralleled by a
larger difference between sdr and sda compared to Franken et al.
(1991b) where the animals were in a light–dark cycle.
A second modulating source could be NREM sleep episode
duration, which also shows a circadian modulation with
shorter episodes in the active phase compared with that in the
rest phase. With shorter episodes, the relative contribution of
the rising phase of SWA becomes larger.
The present method of simulation has been applied earlier in
Sprague–Dawley rats (Franken et al., 1991b) and in three
different mouse strains (Huber et al., 2000) kept under
12-h : 12-h LD conditions. Although the strain and LD
conditions differed from Franken et al. (1991b), the present
simulations were able to find a local maximum close to the
values obtained by Franken et al. (1991b). That the simula-
tions did not result in one single optimum solution may be
caused by a difference in the increase rate of SWA at the
entrance into NREM sleep, which was markedly slower
compared with the values found by Trachsel et al. (1989)
obtained under LD conditions. As mentioned previously,
slower increase rate will increase the discrepancy between data
and simulation and will reduce the fit between data and
simulation.
Applying a different method, Franken et al. (2001) were able
to successfully simulate Process S in mice. The latter may
indicate a species difference between rats and mice. However,
Huber et al. (2000) were able to apply the present method to
C57 ⁄ BL6 and the 129 ⁄ SvJ mouse strain, but were not able to
simulate the time course of SWA in the 129 ⁄ Ola mouse strain,
indicating that the applied simulation method may be an
important factor as well. In addition, NREM sleep episode
duration is more than 2 min shorter in 129 ⁄ Ola mice
compared with that in C57 ⁄ BL6 and 129 ⁄ SvJ mice (4.7, 7.1
and 7.1 min, respectively; Huber et al., 2000) supporting the
notion that this variable may influence the success of the
simulation. Whether 129 ⁄ Ola mice display a slower increase
rate of SWA at the entrance into NREM sleep remains to be
determined.
It has been suggested that quality of waking also may cause
differences in the sleep homeostatic response, with more
exploratory behavior, or social stress inducing higher SWA
in subsequent NREM sleep (Huber et al., 2006; Meerlo et al.,
1997). As rats probably explore more during the active phase

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than during the rest phase, this also could induce a circadian
modulation in the relation between simulation and data.
Nevertheless, it is clear that the models applied in rodents do
not fit the data at the initiation of an NREM sleep episode due
to the time SWA needs to come to full expression.
In humans, it had been observed that the build-up rate of
SWA within an NREM sleep episode is dependent on the prior
duration of sleep and waking (Dijk et al., 1990). With longer
previous waking, T is faster in humans. As this effect is similar
to the effect of prior waking duration on SWA, this increase in
T seems to be caused by increased sleep pressure. Therefore, a
change in T has been implemented in the simulations of
Process S and is coupled to the level of Process S at the time of
initiation of an NREM sleep episode (Achermann et al., 1993).
This refinement enables a detailed and quantitative prediction
of the changes in SWA in the course of an NREM sleep
episode in different experimental protocols.
With a similar approach in rats possibly the circadian
adjustment of sd is no longer necessary. However, the present
data seem to indicate a difference between rats and humans. In
the rat, T is not influenced by changes in sleep pressure. After a
24-h SD, a clear increase in the AL was found, but T did not
differ significantly from that at baseline (Trachsel et al., 1989).
In the present experiment with a 6-h SD, a clear increase in the
AL is visible (more than 66% above baseline). However, no
significant change in T was found and SWA reached plateau
levels after approximately 3 min in both conditions (Fig 2,
CT6–CT12 condition).
The present analysis shows that under constant dark
conditions SWA in NREM sleep is a function of prior waking
duration, which is in accordance with the two process model of
sleep regulation (Borbely, 1982; Daan et al., 1984). Remark-
ably, the constant conditions did not eliminate the necessity of
a systematic circadian modulation in sd, which is therefore
probably an endogenous property of rat sleep. The data
suggest that this modulation may be caused by a circadian
modulation in the rise rate of SWA at the onset of an NREM
sleep episode; however, other factors influencing the circadian
modulation, like NREM sleep episode duration or a difference
in the quality of waking between the rest and active phases
may also contribute to this phenomenon. Data obtained after
SD indicate that in the rat, the rise rate of SWA at the onset of
a NREM sleep episode is independent of the prior amount of
sleep and wakefulness, indicating that incorporating this into
the modeling of sleep homeostasis in a similar way as is done in
humans (Achermann et al., 1993) is not possible in the rat.
ACKNOWLEDGEMENTS
The study was supported by the European Union (Grant
LSHM-CT-2005-518189).
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