Glucocorticoids are not responsible for paradoxical sleep deprivation-induced memory impairments.

Abstract

To evaluate whether paradoxical sleep deprivation-induced memory impairments are due to release of glucocorticoids, by means of corticosterone inhibition with metyrapone.
The design was a 2 (Groups [control, paradoxical sleep-deprived]) x 2 (Treatments [vehicle, metyrapone]) study, performed in 2 experiments: Acute treatment (single injection given immediately after 96 hours of sleep deprivation) and chronic treatment (8 injections, twice per day, throughout the sleep-deprivation period). Animals were either paradoxical sleep-deprived or remained in their home cages for 96 hours before training in contextual fear conditioning and received intraperitoneal injections of a corticosterone synthesis inhibitor, metyrapone. Memory performance was tested 24 hours after training.
Three-month old Wistar male rats. Measurements: Freezing behavior was considered as the conditioning index, and adrenocorticotropic hormone and corticosterone plasma levels were determined from trunk blood of animals sacrificed in different time points. Animals were weighed before and after the paradoxical sleep-deprivation period.
Acute metyrapone treatment impaired memory in control animals and did not prevent paradoxical sleep deprivation-induced memory impairment. Likewise, in the chronic treatment, paradoxical sleep-deprived animals did not differ from control rats in their corticosterone or adrenocorticotropic hormone response to training, but still did not learn as well, and did not show any stress responses to the testing. Chronic metyrapone was, however, effective in preventing the weight loss typically observed in paradoxical sleep-deprived animals.
Our results suggest that glucocorticoids do not mediate memory impairments but might be responsible for the weight loss induced by paradoxical sleep deprivation.

Full text

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A LARGE NUMBER OF ANIMAL STUDIES SUGGEST
A RELATIONSHIP BETWEEN SLEEP AND MEMORY.
MOST OF THEM USE STRATEGIES SUCH AS EXPLOR-
ING common events occurring during learning and subse-
quent sleep, comparing the performance of individuals before
and after a period of sleep, and examining the outcomes of
partial or total sleep deprivation on the performance in mem-
ory tasks.1-3
Employment of the sleep deprivation procedure frequently
demonstrates that this manipulation has deleterious effects on
memory performance in both animals and humans.4,5 Studies
from our laboratory have shown that 96 hours of paradoxical
sleep deprivation (PSD) before training impairs the performance
of rats in memory tasks, such as inhibitory avoidance and con-
textual fear conditioning.6-10 PSD also disrupts the performance
in other tasks, including the spatial version of the Morris Water
Maze (MWM) ,11 8-arm/box maze,12,13 and appetitive discrimi-
nation task.14
The studies supporting a relationship between paradoxi-
cal sleep and memory that have used PSD have been strongly
criticized because of the confounding nonspecific effects of
the method, e.g., increased locomotor activity and activity of
the hypothalamic-pituitary-adrenal (HPA) axis, which could
be responsible for producing the alterations in performance
observed.15-17 The key components of the HPA axis include the
corticotropin-releasing hormone (CRH) neurons of the paraven-
tricular nucleus, which stimulate the secretion of adrenocortico-
tropic hormone (ACTH) from the anterior pituitary. Circulating
ACTH acts on the adrenal cortex, where it stimulates the re-
lease of glucocorticoids (cortisol in humans and corticosterone
in rodents), which then feed back to the brain and pituitary to
shut off the stress response. In addition to the neuroendocrine
limb, the stress also involves activation of the sympathetic
adrenomedullary system. The procedures commonly used to
induce sleep deprivation result in hypertrophy of the adrenal
glands and increased ACTH and corticosterone levels, indicat-
ing its stressful characteristics.18,19 Therefore, it is difficult to
distinguish between the outcomes of stress and sleep loss on
memory performance.
It is well known that the effects of glucocorticoids on cog-
nition are dependent on their circulating levels. Very high or
very low levels of this hormone are prejudicial to memory con-
solidation, a relationship known as an inverted U-shape.20,21
Conrad and colleagues22 tested the effects of corticosterone or
its agonists (aldosterone and RU362) and antagonists (RU318
and RU555) on a Y-maze task and reported that both blockade
of glucocorticoid receptors and high levels of corticosterone
or its agonists impair memory performance, suggesting that
Glucocorticoids and Paradoxical sleeP dePrivation
Glucocorticoids Are Not Responsible for Paradoxical Sleep Deprivation-Induced
Memory Impairments
Paula Ayako Tiba; Maria Gabriela de Menezes Oliveira, PhD; Vanessa Contatto Rossi; Sergio Tufik, MD, PhD; Deborah Suchecki, PhD
Department of Psychobiology, Universidade Federal de São Paulo, São Paulo, Brazil
study objectives: To evaluate whether paradoxical sleep deprivation-
induced memory impairments are due to release of glucocorticoids, by
means of corticosterone inhibition with metyrapone.
design: The design was a 2 (Groups [control, paradoxical sleep-de-
prived]) × 2 (Treatments [vehicle, metyrapone]) study, performed in 2
experiments: Acute treatment (single injection given immediately after
96 hours of sleep deprivation) and chronic treatment (8 injections, twice
per day, throughout the sleep-deprivation period). Animals were either
paradoxical sleep-deprived or remained in their home cages for 96
hours before training in contextual fear conditioning and received intra-
peritoneal injections of a corticosterone synthesis inhibitor, metyrapone.
Memory performance was tested 24 hours after training.
subjects: Three-month old Wistar male rats.
Measurements: Freezing behavior was considered as the condition-
ing index, and adrenocorticotropic hormone and corticosterone plasma
levels were determined from trunk blood of animals sacrificed in differ-
ent time points. Animals were weighed before and after the paradoxical
sleep-deprivation period.
results: Acute metyrapone treatment impaired memory in control
animals and did not prevent paradoxical sleep deprivation-induced
memory impairment. Likewise, in the chronic treatment, paradoxical
sleep-deprived animals did not differ from control rats in their corticos-
terone or adrenocorticotropic hormone response to training, but still did
not learn as well, and did not show any stress responses to the testing.
Chronic metyrapone was, however, effective in preventing the weight
loss typically observed in paradoxical sleep-deprived animals.
conclusions: Our results suggest that glucocorticoids do not medi-
ate memory impairments but might be responsible for the weight loss
induced by paradoxical sleep deprivation.
Keywords: paradoxical sleep deprivation; learning; memory; me-
tyrapone; contextual fear conditioning; corticosterone; weight loss, rat.
citation: Tiba PA; de Menezes Oliveira MG; Rossi VC; Tufik S;
Suchecki D. Glucocorticoids are not responsible for paradoxical sleep
deprivation-induced memory impairments. SLEEP 2008;31(4):505-
515.
disclosure statement
This was not an industry supported study. The authors have indicated no
conflicts of interest.
submitted for publication october, 2007
accepted for publication december, 2007
Address correspondence to: Deborah Suchecki, Department of Psychobi-
ology, Universidade Federal de São Paulo, Rua Napoleão de Barros, 925,
Vila Clementino, São Paulo, SP 04024-002, Brazil; Tel: 55 11 2149 0159;
Fax: 55 11 5572 5092; E-mail: suchecki@psicobio.epm.br
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altered occupancy by corticosterone or its agonists of differ-
ent receptors (glucocorticoid/ mineralocorticoid) could under-
lie the bimodal action of corticosterone on memory. A recent
study shows that chronic stress-induced memory impairment
is due to hypersecretion of corticosterone at the time of train-
ing, since treatment with metyrapone (an inhibitor of the corti-
costerone synthesizing enzyme 11-β-hydroxylase) immediately
before training in a Y-maze prevents the deleterious effect of
chronic stress, indicating that this effect is mediated by HPA
axis dysregulation, such as reduced hippocampal glucocorti-
coid receptor expression, rather than by structural changes in
the hippocampus.23 Removal of the adrenal glands (adrenalec-
tomy or systemic administration of metyrapone, impairs the
performance of rats on the MWM task.24,25 Glucocorticoid re-
ceptor antagonists induce similar deficits in this task,26 whereas
posttraining administration of dexamethasone, a glucocorticoid
receptor agonist, enhances the performance of animals in tasks
such as aversive and appetitive contextual fear conditioning.27
This impairment observed after adrenal removal is reversed by
corticosterone administration, suggesting that glucocorticoids,
rather than other adrenal hormones, are directly responsible for
adrenalectomy-induced memory impairments.28
Given that sleep deprivation impairs learning in the rat, and
that it also results in augmented secretion of glucocorticoids,
which can interfere with memory consolidation processes, a
recent paper addressed this issue by eliminating the adrenal
stress response by means of adrenalectomy. Stable physiologic
levels of corticosterone were maintained in adrenalectomized
rats with an implanted pellet. Adrenalectomy plus corticos-
terone replacement does not modify the effect of 72 hours of
sleep deprivation on acquisition in the MWM, demonstrating
that deficits in spatial task acquisition are not affected by the
adrenal stress response.29 It is important to bear in mind that
adrenalectomy also eliminates other adrenal hormones, such as
epinephrine, which interacts with corticosterone in the modu-
lation of memory consolidation30-32; moreover, adrenalectomy
performed before training also inhibits corticosterone release
during all phases of memory: acquisition, consolidation, and
retrieval. Using a more refined approach, the present study ad-
dressed the question of whether the effects of PSD-induced
memory impairments would be mediated by corticosterone, by
administrating metyrapone acutely or chronically during the
sleep-deprivation period and, therefore, eliminating the inter-
ference produced by increased glucocorticoids levels.
Methods
subjects
Male Wistar rats, aged 3 months, were obtained from the De-
partment of Psychobiology breeding colony. The animals were
kept in groups of 4 in plastic cages filled with sawdust bedding
in a room under controlled temperature (23°C ± 2°C) and light/
dark cycle (lights on from 07:00 to 19:00), with food and water
ad libitum. Animal studies were approved by the Animal Care
and Use Committee of UNIFESP and were in accordance with
National Institutes of Health guidelines on animal care (CEP #
0071/05).
Psd Procedure
Rats were sleep deprived by the modified multiple platform
method33 for 96 hours. Sleep deprivation was conducted by
placing 8 rats in a large water tank (145 × 30 × 41 cm) con-
taining 12 narrow platforms (6 cm in diameter). This proce-
dure completely abolishes paradoxical sleep but also decreases
slow-wave sleep by approximately 35%.34 The presence of mul-
tiple platforms avoids the lack of movement and isolation as-
sociated with earlier techniques of sleep deprivation. Animals
in the control group remained in their home cages in the same
room where the sleep deprivation procedure took place and
were placed daily in the water tank for 1 hour, between 12:00
and 13:00.
apparatus
The contextual fear conditioning (CFC) test apparatus con-
sisted of an acrylic box, measuring 30 × 21 × 30 cm. The ap-
paratus had black walls with white visual patterns (2 squares
measuring 5.5 × 5.5 cm and 3 measuring 4.0 × 4.0 cm made of
white cardboard). The top was covered with transparent acryl-
ic. The floor consisted of a metal grid (0.4-cm diameter rods
placed 1.2 cm apart) connected to a shock generator and control
module (AVS—Projetos Especiais, Sao Paulo, Brazil), through
which foot shocks could be delivered.
contextual Fear conditioning
The task was carried out during 2 consecutive days. On the
first day (training), the animals were individually placed in the
black box, where they remained for 2 minutes. The behavior of
each animal was recorded continuously by measuring the sec-
onds it remained in freezing (defined as complete immobility
and absence of vibrissae movements) minute by minute. After
this period, rats received 5 foot shocks (0.8 mA, 1-s duration)
at 30-second intervals and were removed from the apparatus
1 minute after the last foot shock. Behavioral reaction to foot
shock, measured by paw flinch and vocalization, was recorded
as a sensitivity parameter. CFC tests were performed on the
second day, 24 hours after training. All rats were placed in the
same training context, and no foot shock was delivered. The
time in freezing was recorded minute by minute for 5 minutes.
The freezing/minute ratio was taken as a measure of contextual
conditioning.
drug preparation and administration
The dose of 100 mg/kg (in a volume of 2.0 mL/kg) of 11-
β-hydroxylase inhibitor metyrapone [2-methyl-l,2-di-3-pyri-
dyl-1-propanone (Sigma)] was chosen for being effective to
inhibit corticosterone secretion in a pilot study. The drug was
dissolved in polyethylene glycol and diluted with a 0.9% sa-
line solution to reach the appropriate concentration. The final
concentration of polyethylene glycol was 40%. The vehicle
solution contained the same polyethylene glycol concentra-
tion. The drug and vehicle solution were administered intra-
peritoneally.
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were then sacrificed 15 minutes after testing (Figure 1). Plasma
was collected for determination of ACTH and corticosterone lev-
els (n = 8/group per time point). All animals were weighed at the
beginning of the experiment (initial) and before (final weight)
the sacrifice.
Blood sampling and hormone Plasma levels
Trunk blood was collected in precooled plastic tubes con-
taining 0.1 mL of ethylenediaminetetraacetic acid (60 mg/mL).
Blood was centrifuged at 2300 revolutions per minute for 20
minutes at 4°C. Plasma was collected in polycarbonate tubes
and frozen at -20°C.
Corticosterone levels were determined, in duplicate, by a
double antibody radioimmunoassay method, specific for rats
200
180
160
140
120
100
80
60
40
20
0
250
200
150
100
50
0
CTL VEH CTL MET PSD VEH PSD MET
Group
CTL VEH CTL MET PSD VEH PSD MET
Group
#
#
*#
CORT
CORT(ng/ml)
ACTH(pg/ml)
ACTH
a
B
#
*
Figure 2— Adrenocorticotropic hormone (ACTH) (A) and cor-
ticosterone (CORT) (B) plasma levels, assessed 90 minutes after
metyrapone (MET) or vehicle (VEH) acute injection in paradoxi-
cal sleep deprivation (PSD) or control (CTL) groups. *Different
from CTL group (analysis of variance group effect). #Different
from VEH group (ANOVA treatment effect). n = 8/group.
experimental Procedure
Rats were randomly distributed into 1 of 4 groups: paradoxical
sleep-deprived injected with metyrapone (PSD MET) or vehicle
(PSD VEH) and control treated with metyrapone (CTL MET)
or vehicle (CTL VEH). PSD began in the morning (07:00). In
Experiment 1, a single metyrapone (or vehicle) injection was ad-
ministered at 07:00 of the last day of sleep deprivation, e.g., at
the end of 96 hours of sleep deprivation, and, 90 minutes later,
animals were either sacrificed or submitted to CFC training and
sacrificed 15 minutes after the test. In Experiment 2, animals
received chronic administration of metyrapone or vehicle, and
the first injection was given at the onset of the sleep-deprivation
period (07:00). Rats received 2 injections per day (07:00 and
19:00) throughout the 4 days of sleep deprivation, for a total of
8 injections. The half-life of metyrapone is very short (about 1.9
± 0.7 hours), but, apparently, metyrapol, an active metabolite,
shows very similar effects to metyrapone and, therefore, may be
involved in the pharmacologic activity of metyrapone in vivo.35
In this way, this experimental design allowed us to cease admin-
istration on the night before training, thus preventing the memory
impairment induced by acute metyrapone administration. PSD
and control animals were sacrificed 90 minutes after the last in-
jection (08:30) or after the sleep-deprivation period (07:00). The
remaining animals were submitted to training in CFC. Half of the
animals were sacrificed 15 minutes after training (posttraining)
and the other half were tested (posttest) 24 hours later. Animals
Exp. 1. Acute metyrapone
Exp. 2. Chronic metyrapone
training
training
sacrifice
PSD
CTL
CTL VEH/MET
PSD VEH/MET
MET
VEH
sacrifice
test
sacrifice
sacrifice
test - sacrifice
Figure 1—Experimental procedure of experiment 1 (acute me-
tyrapone [MET]) and experiment 2 (chronic MET). White rect-
angles represent light phase and black rectangles represent dark
phase of the light-dark cycle. In both experiments, animals were
distributed in 4 groups: control (CTL) receiving vehicle (VEH) or
MET and paradoxical sleep deprivation (PSD) receiving VEH or
MET. In the acute treatment, animals received 1 single injection
after 96 hours of sleep deprivation and were sacrificed 90 min-
utes later or were submitted to contextual fear conditioning (CFC)
training and test. The trained animals were sacrificed 15 minutes
after test, 24 hours after the training. In the chronic experiment,
animals received 2 injections per day (07:00 and 19:00) during 4
days of sleep deprivation (or at the equivalent time point for the
CTL group). Animals were sacrificed either 90 minutes after the
last injection (21:00); immediately after sleep deprivation (07:00);
15 minutes after training or 15 minutes after the test in CFC.
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30
25
20
15
10
5
0
30
25
20
15
10
5
0
0-11-22-33-44-5
120
100
80
60
40
20
0
350
300
250
200
150
100
50
0
CTL VEH CTL MET PSD VEH PSD MET CTL VEH CTL MET PSD VEH PSD MET
Training
Training
Test
Minute Minute
afterbefore
CORT
ACTH
CTL VEH
CTL MET
PSD VEH
PSD MET
*
¥
¥
¥
¥
¥
¥
*
*
Freezing (5) Freezing (5)
ACTH(pg/ml)
CORT(ng/ml)
a
c
B
d
Figure 3—Freezing response (mean/min) before and after foot shock during contextual fear conditioning (CFC) training (A) and during 5
minutes (mean/min) of CFC test (B). Adrenocorticotropic hormone (ACTH) (C) and corticosterone (CORT) (D) plasma levels were obtained
15 minutes after CFC test session of paradoxical sleep deprivation (PSD) (96 hours of PSD - circles, full line) and CTL (control - squares,
dotted line) animals, which received an acute injection of MET (metyrapone - dark squares/circles) or VEH (vehicle - open squares/circles) 90
minutes before training. *Different from CTL group (analysis of variance [ANOVA] group effect). ¥Different from the other groups (analysis
of variance group × treatment interaction effect). n = 8/group.
ACTH and corticosterone in Experiment 1 (acute treatment)
were analyzed by a 2-way ANOVA, with Group and Treatment
as main factors. Hormone data from Experiment 2 were ana-
lyzed separately for each time-point (post-last injection; post-
PSD; posttraining and posttest) by a factorial two-way ANOVA
with Group (PSD x control) and Treatment (metyrapone x ve-
hicle) as main factors.
For body weight analysis, we used an index (final weight
× 100/ initial weight), and statistical analysis was conducted
using a 2-way ANOVA, with Group (PSD × control) and Treat-
ment (metyrapone × vehicle) as factors.
A Pearson correlation test was carried out between hormone
parameters (ACTH and corticosterone levels) and total freezing
time during the test session.
When necessary, a posthoc analysis was performed by the
Newman-Keuls test, with a P value ≤ 0.05 being considered
statistically significant.
and mice, using a commercial kit (ICN Biomedicals, Costa
Mesa, CA). The sensitivity of the assay is 3.125 ng/mL, and
intraassay and interassay variations are, respectively, 10.3%
and 7.1%. ACTH was assayed by a sequential chemilumines-
cence immunometric method using a monoclonal murine an-
tibody specific for ACTH (DPC Immulite, Los Angeles, CA).
The sensitivity of the assay is 9 pg/mL and intraassay and in-
terassay variations are 9.6% and 9.4%, respectively.
statistical analysis
Data concerning the behavior of rats in the CFC were ana-
lyzed by a 3-way analysis of variance (ANOVA) for repeated
measures, with Group (PSD × control), Treatment (metyrapone
× vehicle), and Minute (repeated measure = before and after
shock for training and each of the 5 minutes for test) as main
factors.
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5
3
1
-1
-3
-5
-7
-9
-11
-13
-15
CTL VEH CTL MET PSD VEH PSD MET
Group
Weight Var (% initial)
Weight Variation
¥
Figure 4—Weight variation (final × 100/initial) of sleep deprived
(PSD) and control (CTL) animals injected chronically with me-
tyrapone (MET) or vehicle (VEH). ¥Different from other the
groups (analysis of variance group × treatment interaction effect).
n = 20-25/group.
× Treatment interaction (F1, 28 = 7.9; P = 0.009). Posthoc analy-
sis indicated that all groups displayed less corticosterone levels
than control-vehicle animals (P = 0.0005). No difference was
found for paw flinch or vocalization indices among groups.
correlations
A significant correlation was found for total freezing during
test session and ACTH (r = 0.49, n = 8) and corticosterone levels
(r = 0.56, n = 8). When considering only the control group, a cor-
relation was still found for corticosterone levels (r = 0.53, n = 8).
experiment 2. chronic Metyrapone treatment
Weight variation
Main effects of Group (F1, 88 = 4.07; P = 0.05), Treat-
ment (F1, 88 = 10.12; P = 0.002), and a Group × Treatment in-
250
200
150
100
50
0
300
250
200
150
100
50
0
CTL VEH CTL MET PSD VEH PSD MET
Group
CTL VEH CTL MET PSD VEH PSD MET
Group
#
#
CORT
CORT(ng/ml)
ACTH(pg/ml)
ACTH
a
B
Figure 5—Adrenocorticotropic hormone (ACTH) (A) and cor-
ticosterone (CORT) (B) plasma levels of animals sacrificed 90
minutes (20:30) after receiving the last (8th) metyrapone (MET)
or vehicle (VEH) injection. PSD refers to paradoxical sleep de-
privation; CTL, control. #Different from VEH group (analysis of
variance treatment effect). N = 7-8/group.
results
experiment 1. acute Metyrapone treatment
hormone Parameters
Figure 2 presents ACTH (A) and corticosterone (B) levels of
control and PSD animals, which received metyrapone or vehi-
cle 90 minutes before the sacrifice. Two-way ANOVA revealed
group (F1, 27 = 5.39; P = 0.028) and treatment effects (F1, 27 =
22.07; P < 0.001), in which PSD animals presented greater
ACTH release than control animals and metyrapone-treated
animals released more ACTH than vehicle-treated ones. For
corticosterone levels, ANOVA revealed a treatment effect (F1, 27
= 7.01; P = 0.01), in which metyrapone-treated animals released
less corticosterone than did vehicle-treated ones.
contextual Fear conditioning
Figure 3A shows the behavioral results of acute metyrapone
treatment on CFC training. ANOVA showed a Group (F1, 28 =
6.09; P = 0.02), Minute (F1, 28 = 62.9; P = 0.00001), and Group
× Minute interaction (F1, 28 = 4.61; P = 0.04). Newman-Keuls
posthoc analysis indicated that foot shock induced freezing
response, but, in PSD animals, this response was lower than
in control animals. No treatment effect was found. During the
test session (Figure 3B), ANOVA revealed a Group (F1, 28 = 8.3;
P = 0.008) and Treatment (F1, 28 = 5.27; P = 0.04) effect, in which
PSD animals displayed less freezing time than control animals
(P = 0.008), and metyrapone-treated rats froze less than vehi-
cle-treated animals (P = 0.03). Figure 3C presents ACTH levels
15 minutes after the test session. ANOVA detected a Group ef-
fect (F1, 28 = 19.9; P = 0.0002), in which PSD animals showed
lower ACTH levels than control animals. Corticosterone levels
are shown in Figure 3D, and ANOVA revealed a Group (F1, 28 =
13.08; P = 0.001), Treatment (F1, 28 = 6.78; P = 0.01), and Group
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160
140
120
100
80
60
40
20
0
450
400
350
300
250
200
150
100
50
0
CTL VEH CTL MET PSD VEH PSD MET
Group
CTL VEH CTL MET PSD VEH PSD MET
Group
*
*
*
*
CORT
CORT(ng/ml)
ACTH(pg/ml)
ACTH
a
B
Figure 6—Adrenocorticotropic hormone (ACTH) (A) and cor-
ticosterone (CORT) (B) plasma levels of animals submitted to
paradoxical sleep deprivation (PSD) or control (CTL) condition,
chronically treated with metyrapone (MET) or vehicle (VEH).
Animals were sacrificed in the morning (07:00), 12 hours after the
last injection. *Different from CTL group (analysis of variance
group effect). n = 7-8/group.
for ACTH (F1,28 = 10.33; P = 0.003) and corticosterone (F1,28 =
6.03; P = 0.02) was detected, in which PSD animals secreted
more ACTH and corticosterone levels than did control animals
(Figure 6 A and 6B).
contextual Fear conditioning
A Group (F1,57 = 10.85; P=0.002), Minute (F1,57 = 253,5;
P < 0.00001), and a Group × Minute interaction was found in
the training session (F1,57 = 5,89; P = 0.018), indicating that all
animals exhibited more freezing time after foot shock than be-
fore and that control group froze more during the last minute
than PSD group (P < 0.0002). No statistical differences were
found for ACTH and corticosterone levels for animals sacri-
ficed after training (Figures 7A-C).
Again, during the test session, a Group (F1,27 = 15.08;
P = 0.0006) and a Group × Minute interaction was found (F4,108
= 5,22; P = 0.0007). Regardless of the treatment, PSD animals
showed less freezing time throughout the entire test session
(P ≤ 0.01). A Group effect was found in 2-way ANOVA for
ACTH levels (F1,26 = 21.88; P < 0.0001), in which PSD ani-
mals displayed less ACTH secretion than control animals af-
ter the test session. For corticosterone, a Group (F1,26 = 29.15;
P < 0.0001) and Treatment effect (F1,26 = 4.74; P = 0.04) was
detected, in which PSD animals exhibited less corticosterone
secretion than control ones and metyrapone-treated animals
displayed more corticosterone secretion than vehicle-treated
ones (Figures 8A-C). No difference was found for paw flinch or
vocalization indices among groups.
correlations
A significant correlation was found for total freezing during
test session and ACTH (r = 0.50, n = 8) and corticosterone lev-
els (r = 0.75, n = 8). When considering only the control group,
a correlation was still found for corticosterone levels (r = 0.66,
n = 8).
discussion
The main finding of the present study was that acute or
chronic inhibition of corticosterone release immediately after
or during PSD, respectively, did not prevent the impairment
of memory induced by this adverse situation. Chronic treat-
ment, however, was effective in preventing the weight loss
frequently observed in paradoxical sleep-deprived animals.33,36
To the best of our knowledge, this is the first study showing
that metyrapone abolishes the loss in body weight so typically
found in sleep-deprived animals. Repeated stress,37 as well as
PSD,33,36,38,39 has been shown to decrease body weight, although
the issue regarding increased food intake is under scrutiny. Re-
cently, the alleged PSD-induced hyperphagia was questioned
by Martins and coworkers,39 who observed intense food waste
that had been interpreted as food intake. Nonetheless, with the
appropriate corrections of assessment, it is still possible to ob-
serve increased food intake by PSD rats, especially on the last
days of sleep deprivation.36,39 It was expected that sleep depri-
vation would act as a stressor, either by itself or because of
the methodology applied. Since chronic metyrapone treatment
teraction effect (F1, 88 = 8.69; P = 0.004) were shown. Posthoc
analysis pointed out that PSD-vehicle animals lost weight dur-
ing the 4-day period, being different from the other groups
(P < 0.002).
hormone Parameters
Figure 5 shows the results of ACTH (A) and corticosterone
(B) from animals sacrificed 90 minutes after the last injection
of metyrapone or vehicle (e.g., during the dark phase, at 21:00).
Two-way ANOVA revealed a treatment effect for ACTH
(F1,27 = 31.45; P < 0.0001), in which metyrapone-treated ani-
mals presented higher ACTH plasma levels than vehicle-treated
ones. For corticosterone, no effect was found.
When the sacrifice was performed immediately after the
PSD period (during the light phase, at 07:00 h), a group effect
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prevented the weight loss, our findings strongly suggest that
the effect of PSD on weight loss is mediated by elevated corti-
costerone levels, whereas memory impairment may result from
other factors.
Our results with acute metyrapone treatment are in agree-
ment with others showing that administration of this drug be-
fore training impairs acquisition in the water maze task.23,40 Re-
cently, a study showed that acute administration of metyrapone
prevented chronic stress-induced impairments on spatial mem-
ory. In the present study, acute treatment did not inhibit PSD-
induced memory impairment; on the contrary, it also impaired
the performance of control rats. This effect might have been
due to the dose used (100 mg/kg), which was higher than those
used in Wright’s study (35 and 75 mg/kg).23
In the present study, corticosterone secretion was attenuated
during sleep deprivation only when metyrapone was adminis-
tered chronically, e.g., the drug was not present at training or
test moments. A similar treatment for control rats did not inter-
fere with their performance during both training and test ses-
sions. In addition, all rats exhibited a similar hormone response
to training, which is expected, since corticosterone is important
for memory consolidation.28 These results are in line with those
reported by Ruskin and coworkers,29 who showed that adrena-
lectomy does not modify the effect of sleep deprivation on the
MWM acquisition. Collectively, these results demonstrate that
the adrenal stress response is not the source of deficits in spa-
tial task acquisition induced by pretraining sleep deprivation.
The choice of using chronic metyrapone treatment instead of
adrenalectomy was done because the former induces a tem-
porary, reversible, and dose-dependent inhibition of glucocor-
ticoid synthesis. When using surgical removal of the adrenal
gland, corticosterone release is inhibited during all phases of
memory, and the purpose of the present study was to prevent
elevated corticosterone levels only during sleep deprivation but
not during memory acquisition, consolidation, or retrieval. An-
other advantage of the metyrapone treatment over adrenalec-
tomy is that metyrapone does not disrupt synthesis and release
of the adrenomedullary hormone epinephrine, which is also im-
portant for memory consolidation.30
The fact that even chronic metyrapone treatment failed to
prevent the memory deficit indicates that PSD-induced memory
impairment is not due to elevated corticosterone levels. Because
acute treatment with metyrapone induced memory impairment,
we used 8 administrations, the last 1 taking place on the night
before training. Perhaps, for this reason, we did not observe
the inhibiting effect of metyrapone on corticosterone secretion
when the animals were sacrificed in the following morning,
e.g., before training. We refrained from using the ninth admin-
istration because in Experiment 1, we showed the acute effect
of the substance on memory. In a pilot study, we observed that
9 injections effectively eliminated the corticosterone response
in paradoxical sleep-deprived animals (data not shown). From
this result, added to the fact that ACTH levels were elevated
and that chronic treatment prevented weight loss, we presumed
that metyrapone was acting during the PSD period. One might
speculate that the elevated levels of corticosterone at 19:00 (last
injection) and immediately after PSD (07:00, before training)
might be responsible for the memory impairment observed
in sleep-deprived animals; however results from our lab have
Metyrapone and Memory Impairment in Sleep Deprived Rats—Tiba et al
50
45
40
35
30
25
20
15
10
5
0
140
120
100
80
60
40
20
0
CTL VEH CTL MET PSD VEH PSD MET
CTL VEH CTL MET PSD VEH PSD MET
Training
Minute
After Before
ACTH
Group
Group
CORT
Freezing (5)
ACTH(pg/ml)
CORT(ng/ml)
a
B
c
CTL VEH
CTL MET
PSD VEH
PSD MET
450
400
350
300
250
200
150
100
50
0
Figure 7—(A) Freezing response (mean/min) before and after foot
shock during training in the contextual fear conditioning (CFC).
Adrenocorticotropic hormone (ACTH) (B) and corticosterone (C)
plasma levels in chronically treated animals sacrificed 15 minutes
after training. PSD refers to 96 hours of paradoxical sleep depriva-
tion; CTL, control; MET, metyrapone; VEH, vehicle. * Indicates
that both PSD groups, regardless of the treatment, are different
from their respective CTL groups. n = 14-16 for behavioral results
and 7-8 for hormone results.

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SLEEP, Vol. 31, No. 4, 2008
512
shown that 24 hours of pretraining sleep deprivation does not
affect inhibitory avoidance task.6
Interestingly, we found no differences in corticosterone lev-
els between PSD and control animals sacrificed at night (af-
ter the eighth injection). But it should be kept in mind that, in
most of the studies carried out in our laboratory, animals were
sacrificed during the day (in which PSD animals always pres-
ent elevated corticosterone levels).19,33,36 Since baseline corti-
costerone peak occurs during the activity period (dark phase
for rodents), generally, responses to stress applied at night are
smaller in magnitude than those seen following stress applied
at the beginning of the light cycle.41 Therefore, during the night,
corticosterone levels are so high that they could mask the sleep
deprivation affect. Nevertheless, it was possible to indirectly
observe the treatment effect because ACTH levels were elevat-
ed in metyrapone-treated animals.
In Experiment 2, all animals displayed similar ACTH and
corticosterone levels in the posttraining period, as well as similar
behavioral reaction to foot shock (e.g., paw flinch and vocaliza-
tion), supporting the idea of a comparable reaction to the shock
delivered during training and that the effect of the metyrapone
had worn off during training. The similar hormone release af-
ter training also supports the idea that memory acquisition or
consolidation is not impaired by a distinct corticosterone avail-
ability, precluding the idea that differences in training-induced
corticosterone secretion could be responsible for the memory
impairment resulting from PSD. Sleep-deprived animals pre-
sented lower ACTH and corticosterone levels during the test,
which indicates that the environment was less aversive to these
rats, which ultimately reflects the decreased learning during the
training, despite the fact that the hormone response of PSD rats
at the time of training was similar to that of control animals.
After the test session, a clear correlation between performance
and hormone response was observed when analyzing all groups
together. In both acute and chronic experiments, the animals
that displayed high freezing behavior were those that secreted
more ACTH and corticosterone in response to the exposure to
the aversive context. The correlation was also valid when con-
sidering only the control group. Because deprived animals did
not remember the aversive environment (Experiment 2), a floor
effect might have flattened the hormone release, and no correla-
tion between corticosterone or ACTH levels and freezing time
was found. These results are in agreement with others show-
ing the close relationship between release of stress hormones
and memory performance.42 Animals chronically treated with
metyrapone showed an increase in corticosterone release after
the test session. This effect could be due to a supersensitiv-
ity of the adrenal gland to its tropic hormone, ACTH, insofar
as chronic inhibition of corticosterone release diminished the
negative feedback on the hypothalamus and pituitary, increas-
ing ACTH secretion, which could result in upregulation of its
own receptor in the adrenal gland. Thus, when the treatment
ceased, even smaller doses of ACTH (e.g., reduced release in
deprived animals) would induce greater corticosterone release.
Another explanation for this effect involves a likely rebound ef-
fect of the 11-β-hydroxylase enzyme after cessation of the treat-
ment, with increase activity of the enzyme leading to enhanced
corticosterone production and, possibly, release in chronically
treated animals.
Metyrapone and Memory Impairment in Sleep Deprived Rats—Tiba et al
50
45
40
35
30
25
20
15
10
5
0
300
250
200
150
100
50
0
CTL VEH CTL MET PSD VEH PSD MET
CTL VEH CTL MET PSD VEH PSD MET
Test
Minute
0-1 1-2 2-33-44-5
ACTH
Group
Group
CORT
*
*
#
*
*#
Freezing (5)
ACTH(pg/ml)
CORT(ng/ml)
a
B
c
CTL VEH
CTL MET
PSD VEH
PSD MET
400
350
300
250
200
150
100
50
0
Figure 8—(A) Freezing response (mean/min) during 5 minutes
(mean/min) of contextual fear conditioning (CFC) test. ACTH (B)
and corticosterone (C) plasma levels in animals after 96 hours of
paradoxical sleep deprivation (PSD). CTL refers to control; MET,
animals that received chronic administration of metyrapone;
VEH, vehicle chronically treated animals. Rats were sacrificed 15
minutes after the CFC test. *Indicates that both PSD groups, re-
gardless of the treatment, are different from their respective CTL
groups. #Different from VEH group (analysis of variance treat-
ment effect). n = 7-8/group.

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SLEEP, Vol. 31, No. 4, 2008
513
Our results suggest that glucocorticoids per se are not in-
volved in PSD-induced memory deficit, although there is the
likelihood that increased CRH resulting from blockade of cor-
ticosterone synthesis affects memory performance. Which al-
terations provoked by sleep deprivation other than stress could
lead directly to memory deficits still remain uncertain, but one
possible candidate mechanism is that sleep deprivation induces
changes in cholinergic neurotransmission, with reductions in
acetylcholine release, acetylcholinesterase activity, and recep-
tor sensitivity.55-58 The cholinergic system is closely related to
cognitive function,59 and previous results from our laboratory
have shown that sleep deprivation-induced memory impair-
ment can be blocked by treatment with the cholinergic agonist
pilocarpine during the sleep deprivation period.7 It is possible,
therefore, that sleep deprivation alters cholinergic neurotrans-
mission, leading, in turn, to memory impairment.
acKnoWledGMents
The authors are in debt to Ricardo Borges Machado and Mar-
cos Vinicius Buncheidt for assistance in the behavioral testing,
injections and for the animal care and to Adriana Fernandes Far-
ia for helping with ACTH hormone determinations. This work
was supported by grants from Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP - CEPID# 98/14303-3) and
Associação Fundo de Incentivo à Psicofarmacologia (AFIP).
Paula Ayako Tiba is the recipient of a Ph.D. fellowship from
Coordenação de Aperfeiçoamento de Pessoal de Nível Supe-
rior (CAPES). Vanessa Contatto Rossi is the recipient of an
Undergraduate Research Fellowship from FAPESP. Deborah
Suchecki, Maria Gabriela Menezes de Oliveira and Sergio Tu-
fik are the recipients of research fellowships from Conselho Na-
cional de Desenvolvimento Científico e Tecnológico (CNPq).
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