Brain Research Bulletin 164 (2020) 198–207

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Brain Research Bulletin

journal homepage: www.elsevier.com/locate/brainresbull

Treadmill exercise ameliorates chronic REM sleep deprivation-induced

anxiety-like behavior and cognitive impairment in C57BL/6J mice
Feng Tai a, 1, Che Wang b, 1, Xin Deng c, 1, Ruojin Li b, Zimeng Guo a, Haiying Quan a, *, Song Li d, e, *
a
School of Physical Education, Liaoning Normal University, Dalian, 116029, China
b
Department of Medicinal Chemistry, School of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian, 116029, China
c
Department of Physical Education, Harbin Engineering University, Haerbin, 150001, China
d
Liaoning Provincial Key Laboratory for Research on the Pathogenic Mechanisms of Neurological Diseases, the First Affiliated Hospital, Dalian Medical University,
Dalian, 116011, China
e
Liaoning Provincial Center for Clinical Research on Neurological Diseases, the First Affiliated Hospital, Dalian Medical University, Dalian, 116011, China

A R T I C L E I N F O A B S T R A C T

Keywords: Various sleep disorders have deleterious effects on mental and cognitive performance. Exercise, as an alternative
Exercise therapeutic strategy, exerts beneficial impacts on human health. In the present study, we aimed to evaluate the
REM sleep deprivation effects of 4 weeks treadmill exercise (4W-TE) on anxiety-like behavior and cognitive performance in mice
Anxiety
exposed to 2 months REM sleep deprivation (2M-SD) (20 h per day). Behavioral performance of mice in elevated
Cognition
plus maze test (EPM), open field test (OFT), Y maze test (YM) and Morris water maze test (MWM) was recorded
Neurotransmitter
Neurotrophic factor and analyzed 28 h after the last day of sleep deprivation. After behavioral tests, various neurotransmitters
including norepinephrine (NE), dopamine (DA), serotonin (5-HT) and γ-aminobutyric acid (GABA) in mouse
hippocampus were quantified using high performance liquid chromatography. The hippocampal levels of insulin-
like growth factor-1 (IGF-1) and brain derived neurotrophic factor (BDNF) were further detected using ELISA.
Behavioral data indicated that 2M-SD exposure induced anxiety-like behaviors and cognitive impairment, as
evidenced by the decreased open-arm entries in EPM, reduced central area travels in OFT, declined spontaneous
alteration in YM and prolonged escaping latency in MWM. In addition, 2M-SD exposure increased NE and DA,
decreased 5-HT and GABA, and reduced IGF-1 and BDNF levels in mouse hippocampus. Interestingly, all these
behavioral, neurochemical and neurobiological changes can be ameliorated by 4W-TE training. In summary,
these findings confirm the beneficial impacts of exercise on health and provide further experimental evidence for
future application of exercise as an alternative therapy against the mental and cognitive problems in patients
with sleep disorders.

1. Introduction
Sleep is an essential biological process for maintaining human health
and the stability of emotion and cognition (Gruber and Cassoff, 2014;
Cruz et al., 2019). Millions of people worldwide experiences sleep dis­
orders or deficiency on a daily basis (Hublin et al., 2001). A
population-based study has indicated that, over the past fifty years, sleep
duration in adult and adolescent Americans has decreased by 1.5 – 2 h
per night, with over 30% reporting sleep of less 6 h per night (Ford et al.,
2015). In older populations, sleep disturbances are even more common.
Poor sleep quality and short sleep duration are gaining attention as
potential risk factors for the pathogenesis and progression of various
mental and cognitive disorders, including anxiety, depression and de­
mentia (Palma et al., 2013; Pires et al., 2016; Pineda, 2017; Zhang et al.,
2017; Zhang et al., 2019; Riemann et al., 2020).
Sleep plays important roles in regulating the complex interactions
between brain functions and various neurotransmitters, such as
noradrenaline (NE), dopamine (DA), serotonin (5-HT) and γ-amino­
butyric acid (GABA) (Stenberg, 2007). Consequently, sleep disturbances
can induce alterations of neurotransmission in the central nervous sys­
tem (CNS), leading to deteriorating neurological or psychological
manifestations (Vogel, 1999; Longordo et al., 2009; Zager et al., 2009;
Fell et al., 2011; Howell and Schenck, 2015; Narwade et al., 2017). For
example, rapid eye movement sleep disturbance alters

* Corresponding authors.
E-mail addresses: quan-haiying@lnnu.edu.cn (H. Quan), lisong@dmu.edu.cn (S. Li).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.brainresbull.2020.08.025
Received 19 February 2020; Received in revised form 12 July 2020; Accepted 22 August 2020
Available online 30 August 2020
0361-9230/© 2020 Elsevier Inc. All rights reserved.
F. Tai et al.
neurotransmission in four distinct brain regions and results in
mania-like behavior in Swiss albino mice (Siddique et al., 2018).
Moreover, sleep deficiency also disrupts the expressions of neurotrophic
factors in CNS, such as brain derived neurotrophic factor (BDNF) and
insulin-like growth factor-1 (IGF-1), both have been reported to closely
correlate with cognitive function and mental health (Rusch and Gill,
2015; Chennaoui et al., 2016; Schmitt et al., 2016; Dal-Pont et al., 2019;
Mohammadipoor-Ghasemabad et al., 2019). Therefore, restoring the
impaired neurotransmission and neurotrophic status in CNS, via either
pharmacological therapies or non-pharmacological strategies, may
provide promising tools for the management of sleep-related disorders.
Physical exercise, as a well-accepted and low-cost alternative to
disease treatment and prevention, has been reported to positively
modify mood, mobility and memory symptoms of various CNS diseases
(Silva et al., 2015; Roh et al., 2016; Chen et al., 2019). Moreover, NE,
DA, 5-HT and GABA are also major neurotransmitters to be modulated
by exercise (Lin and Kuo, 2013; Schoenfeld et al., 2013; Vivar and van
Praag, 2017). Interestingly, recent studies have indicated that exercise
can also enhance BDNF and IGF-1 level (Kohman et al., 2012; Wrann
et al., 2013; Żebrowska et al., 2018). Given that sleep disturbances may
lead to anxiety and cognition decline and exercise improves CNS
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diseases related symptoms, it is important to investigate the neuro­
chemical and neurobiological basis of how exercise affects those
abnormal mood and cognitive performance induced by sleep disorders.
In the present study, we aim to evaluate the effects of 4 weeks treadmill
exercise (4W-TE) on behavior, neurochemical and neurobiological al­
terations in mice subjected to 2 months rapid eye movement (REM)
sleep deprivation (2M-SD) (20 h per day). The Behavioral performance
of mice was recorded and analyzed 28 h after the last day of sleep
deprivation. After behavioral tests, various neurotransmitters in mouse
hippocampus were quantified. The hippocampal levels of insulin-like
growth factor-1 (IGF-1) and brain derived neurotrophic factor (BDNF)
were further detected.
2. Materials and methods
2.1. Animals
One hundred male C57BL/6J mice (2 months of age, 22–25 g body
weight) were obtained from Laboratory Animal Center of Dalian Medi­
cal University. Animals were housed (5 mice per cage) in standard
conditions (12 h/12 h light-dark cycle with lights on at 8:00 AM,
Fig. 1. Schematic illustration of experimental
design. All experimental procedures were sum­
marized as Fig. 1a. Non-sleep deprived (NSD)
control animals were kept in their home cages
(8:00 AM to 12:00 PM) or large platform cages
(12:00 PM to 8:00 AM of next day, Fig. 1b, right
panel). Animals in 2M-SD and SD + TE groups
were exposed to REM sleep deprivation using
small platform protocol (from day 1 to 56,
12:00 PM to 8:00 AM of next day, Fig. 1b, left
panel). After 4 weeks sleep deprivation, mice in
SD + TE group were subjected to treadmill
training for 4 weeks (from day 29 to 56, 2:00
PM to 4:00 PM, Fig. 1c). Training control ani­
mals were subjected to treadmill training for 4
weeks (4W-TE) without sleep disturbance.
From day 50 to 56, a battery of behavioral tests,
including Y maze (YM), open field test (OFT),
elevated plus maze (EPM), and Morris water
maze (MWM) were performed (12:00 PM to
2:00 PM, Fig. 1d). Animals were sacrificed on
day 57 and hippocampus was dissected for
HPLC analysis or ELISA.
F. Tai et al.
temperature 23 ± 1 ◦ C, humidity 50 ± 10 %) with food and water ad
libitum. Animal care and procedures were carried out in accordance with
the Laboratory Animal Care Guidelines approved by the Institutional
Animal Care Committee at Dalian Medical University. The protocol was
approved by the Institutional Animal Care Committee at Dalian Medical
University (LCKY-2014-29).
2.2. Experimental procedures
All experimental procedures were schematically illustrated in
Fig. 1a. Animals were randomized into 4 groups (25 mice per group).
Group 1 was set as non-sleep deprived control group (NSD) with animals
kept in their home cages (8:00 AM to 12:00 PM) or large platform cages
(12:00 PM to 8:00 AM of next day, Fig. 1b, right panel) without sleep
disturbances. Animals in group 2 and group 3 were exposed to REM
sleep deprivation for 2 months (2M-SD) using small platform protocol
(from day 1 to day 56, 12:00 PM to 8:00 AM of next day, Fig. 1b, left
panel). After 4 weeks sleep deprivation, mice in group 3 were subjected
to treadmill exercise for 4 weeks (SD + TE) (from day 29 to day 56, from
2:00 PM to 4:00 PM, Fig. 1c). Animals in group 4 were subjected to
treadmill training for 4 weeks (4W-TE) without sleep disturbance. From
day 50 to 56, a battery of behavioral tests, including open field test
(OFT), elevated plus maze test (EPM), Y maze test (YM) and Morris
water maze test (MWM), were performed (from 12:00 PM to 2:00 PM,
Fig. 1d). Animals were sacrificed 24 hours after behavioral tests (on day
57) and the hippocampus was dissected and frozen for high performance
liquid chromatography (HPLC) or enzyme-linked immunosorbent assay
(ELISA). Behavioral tests, HPLC analysis and ELISA were performed by
blinded examiners.
2.3. Sleep deprivation exposure
REM sleep deprivation was performed according to our previous
protocol (Qiu et al., 2016). Briefly, twenty-four small round platforms (3
cm in diameter, 5 cm in height, and 3 cm from each other) were placed
in a water tank (Fig. 1b, left panel). During 2M-SD process, mice were
placed on these small platforms, and the water tank was filled with
water at 26 to 28 ◦ C, 1 cm beneath the platform. Mice can move from
one platform to another, but would fall into the water if they fall asleep.
All conditions of the NSD control group were similar with those of
2M-SD group, except the 11.5 cm diameter of platform (Fig. 1b, right
panel). There were 5 mice placed in one water tank with food and water
ad libitum. Sleep deprivation treatment was given from 12:00 PM to
8:00 AM of the next day, and all mice were placed back to their home
cages from 08:00 AM to 12:00 PM and gave them an ad libitum sleep
opportunity.
2.4. Treadmill exercise protocol
All groups were habituated on an eight-channel motor-drive tread­
mill (YLS-10B, Yuyan Instruments, Shanghai, China). Treadmill exercise
was conducted during daytime from 2:00 PM to 4:00 PM. After three
days of treadmill familiarization (day 29 to day 31, 10 min/day, 5 rpm),
treadmill exercise was performed with a progressive increase from 5 rpm
to 10 rpm from day 32 to day 35 and continued at 10 rpm for 3 weeks.
The animals in NSD and 2M-SD groups were left in the treadmill,
without running, for the same amount of time as the exercise groups
(4W-TE and SD + TE). To minimize stress associated with treadmill
exercise, only gentle tail touching was used to induce mice to run, and
no electric or voice stimulant was used. None of the animals were visibly
hurt or died during exercise session.
2.5. Behavioral tests
2.5.1. Open field test
The open field consisted of a base (100 × 100 cm) divided into 25 (5
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× 5) identical sectors (20 × 20 cm) by white stripes. The squares were
subdivided into peripheral and central sector, where the central sector
included the 9 central squares (3 × 3) and the peripheral sector con­
tained the squares close to the surrounded wall (20 cm high). The arena
was lit by red-light lamps (60 W). The animals were placed in the central
sector and their activity was video-recorded for 10 min for further
analysis. Motility was scored when an animal crossed a sector border
with both its hind-limbs. The following behavioral parameters were
scored: the total frequency of squares crossed, ratio of central squares
crossed frequency [(central / total) × 100], and ratio of central area
duration [(central / total) × 100]. The open field arena was thoroughly
cleaned with alcohol between each test (Wang et al., 2014).
2.5.2. Elevated plus maze test
EPM consists of two open arms (30 × 5 cm) and two closed arms (30
× 5 cm, surrounded by 15 cm-high walls), with the two pairs of identical
arms emerging from a central platform (5 × 5 cm) positioned opposite
each other. The apparatus was elevated 50 cm above the floor. The test
was initiated by placing the mouse on the central platform of the maze,
facing one of the open arms, and letting it explore the maze freely for 5
min. Mouse behavior was continuously recorded by a video camera
placed above the apparatus and analyzed offline by an observer who was
unaware of the animals grouping. The maze was carefully cleaned with
alcohol and rinsed with water after each test. Behavioral parameters
including the frequencies of total arms entries (arm entry = all four paws
into an arm), ratio of open arms entries [(open /total) × 100], time spent
in open or close arms and ratio of time spent in open arms [(time in open
arms / session duration) × 100] (Wang et al., 2014).
2.5.3. Y maze test
YM task was used to assess basic mnemonic processing by sponta­
neous percent alternation and exploratory activity by total number of
arm choices of mice placed into a Y-shape maze apparatus. The arms of
this maze were 21 × 4 cm with 40 cm high walls. Each mouse was placed
in one of the arms and allowed one 5-min trial of free exploration of the
three arms in the maze. The number of total arm choices and sequence of
arm choices were recorded. Alternation percentage is defined by the
proportion of arm choices that differ from the last two choices. Before
each trial, the maze was cleaned with alcohol to erase any scent cues
(Sawmiller et al., 2017).
2.5.4. Morris water maze
Spatial learning and memory ability of mice were evaluated by
MWM test. Briefly, the maze consisted of a circular water tank (120 cm
diameter, 60 cm height) filled with water (25 ± 1 ◦ C) to a depth of 40
cm. Four equally spaced locations around the edge of the pool were used
as start points, which divided the pool into four equal quadrants. An
escape platform (10 cm in diameter) was placed in the middle of one
randomly selected quadrant, 2 cm below the surface of water, and was
kept in same position throughout the entire training session. The
training session consisted of 16 trials (60 s each, 4 trials per day, with a
trial interval time of 60 s) for 4 days (day 52 to day 55). Once climbing
onto the hidden platform, mice were remained there for additional 15 s.
If the mice failed to locate the hidden platform during 60 s training, it
was gently guided to the platform and allowed to remain there for 15 s.
The latency for each mouse to locate the platform was recorded. During
the probe test (on day 56), animals were allowed to swim freely in the
tank for 60 s, and the time spent in target quadrant, where the hidden
platform previously located, was recorded. A visual cue test was con­
ducted 30 min after the probe test to assess sensorimotor ability and
motivation. For this test, the escape platform was set 1 cm above the
water surface and marked with black tape so that the mice could locate
the platform using a local visual stimulus rather than relying on spatial
orientation to extra-maze cues. The escape latency was recorded.
F. Tai et al.
2.6. Brain sampling and HPLC analysis
On day 57, all mice were anesthetized and hippocampus was
dissected rapidly on ice, homogenized and centrifuged at 12,000 × g for
5 min. The supernatant was filtered through 0.22 μm nylon filters before
injecting into the HPLC sample injector. Monoamines (DA, NE, 5-HT)
and GABA were determined by HPLC with electrochemical detector
according to our previous protocol (Wang et al., 2019). Concentrations
were calculated from the standard curve generated by standard 10–100
ng/mL. The values were expressed as percentage of control group.
2.7. Determination of BDNF and IGF-1 by ELISA
Mouse hippocampus was homogenized in lysis buffer and centri­
fuged at 15,000 × g for 15 min at 4 ◦ C. The supernatants were collected,
and their protein concentrations were measured with a Bio-Rad protein
assay kit (Bio-Rad, Hercules, CA, USA). BDNF and IGF-1 levels were
measured using BDNF Emax Immunoassay kit (Promega, Madison, WI)
and IGF-1 ELISA kit (Abcam, UK) according to the manufacturer’s in­
structions. Absorbance at 450 nm was measured with a Victor3 plate
reader (PerkinElmer, USA). The quantity of BDNF and IGF-1 was
expressed as percentage of NSD control values.
Fig. 2. Treadmill exercise ameliorates anxiety-like behavior induced by 2M-SD as assessed by EPM. (a) total arms entries; (b) ratio of open arms entries; (c) duration
in close arms; (d) ratio of close arms duration. ** p < 0.01, compared with NSD control group; ## p < 0.01, compared with 2M-SD group.
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2.8. Statistical analyses
Descriptive statistics are presented as mean ± standard error. One
way analysis of variance (ANOVA) followed by post hoc Tukey’s test or
repeated measurement was employed to compare results between
multiple groups. Statistical analysis was carried out using GraphPad
Prism 7.04. Statistical significance was set at p < 0.05.
3. Results
3.1. Treadmill exercise ameliorates anxiety-like behavior induced by 2M-
SD
In EPM test, no significant changes of total arms entries were
observed after 2M-SD exposure (p > 0.999, 95% CI -1.676 to 1.756, F (3,
96) = 2.484) or 4W-TE training (p = 0.132, 95% CI -3.156 to 0.2758)
(Fig. 2a), suggesting an intact locomotor and exploring behavior.
However, those sleep-deprived mice showed decreased entries (p <
0.01, 95% CI 13.46 to 23.68, F (3, 96) = 46.75, Fig. 2b) and traveling
time in open arms in EPM (p < 0.01, 95% CI 5.589 to 9.585, F (3, 96) =
56.02, Fig. 2d). In addition, sleep deprivation also caused decreased
squares crossing (p < 0.01, 95% CI 13.26 to 20.74, F (3, 96) = 69.73,
F. Tai et al. Brain Research Bulletin 164 (2020) 198–207

Fig. 3b) and traveling duration in the central section of open field (p <
0.01, 95% CI 1.823 to 3.297, F (3, 96) = 36.84, Fig. 3c) without sig­
nificant change of locomotor activity (total squares crossed, Fig. 3a. p =
0.324, 95% CI -1.48 to 7.08, F (3, 96) = 15.8). All these data suggested
that 2M-SD could induce anxiety-like behavior in mice. As expected, this
elevated anxiety level can be ameliorated by 4 weeks treadmill exercise,
evidenced by the increased open arm entries and open arm traveling
time in EPM (p < 0.01, Fig. 2b, c) and the increased central area trav­
eling in OFT (p < 0.01, Fig. 3b, c).

3.2. Treadmill exercise ameliorates cognitive impairment induced by 2M-

SD

In addition to anxiety assessment, behavioral tests were further

performed to evaluate the learning/memory performance of mice. The
data from MWM task indicated that, compared to those NSD control
mice, 2M-SD-treated mice showed an increased latency to find the
hidden platform during training sessions (Fig. 4a) and a decreased target
quadrant percentage in probe test (Fig. 4b), indicating an impaired
learning and memory ability. This impaired cognitive performance
caused by 2M-SD was further confirmed by Y maze test, as shown by the
decreased percentage of spontaneous alternation (Fig. 4e). Interestingly,
while 4W-TE showed no significant impacts on learning and memory
compared to NSD control (p = 0.222, 95% CI 0.1497 to 2.71, F = 82.95,
Fig.4a; p = 0.255, 95% CI -4.339 to 0.7392, F = 89.33, Fig.4b; p = 0.92,
95% CI -3.354 to 5.434, F = 55.93, Fig.4e), 4W-TE ameliorated the 2M-
SD-induced cognition impairment (p < 0.01, Fig. 4a, b and e).
Escape latency in the visual cue test was decreased compared with
day 4 training and showed no significant difference among 4 groups (p =
0.3269, F (3, 96) = 1.166, Fig. 4d), suggesting a normal sensorimotor
ability and motivation. Moreover, consistent with the intact locomotor
performance in EPM (Fig. 2a) and OFT (Fig. 3a), there were no signifi­
cant differences noted in swim speed (p = 0.8613, F (3, 96) = 0.2498,
Fig. 4c) in MWM and total entries in YM (data not shown) among all
groups, suggesting that the nootropic activity of treadmill exercise is not
due to an altered motor ability.

3.3. Treadmill training reverses 2M-SD-induced alterations in

hippocampal neurotransmission

Exercise and sleep both have close interactions with various neuro­
transmitters, which have been reported to be involved in the regulation
of both emotion and cognition. Therefore, we detected neurotransmit­
ters levels in mouse hippocampus, including NE, DA, 5-HT and GABA.
Our data indicated that 4W-TE training significantly elevated DA (p =
0.0374, F (3, 96) = 88.21), 5-HT (p = 0.0282, F (3, 96) = 103.2) and
GABA (p = 0.0127, F (3, 96) = 170.4) levels in hippocampus without
significant alteration of NE (p = 0.404, F (3, 96) = 143.1) (Fig. 5). 2M-
SD exposure caused significant increases of hippocampal NE (p < 0.01,
95% CI -45.74 to -34.5, Fig. 5a) and DA levels (p < 0.01, 95% CI -38.86
to -26.98, Fig. 5b) together with decreased GABA (p < 0.01, 95% CI
31.23 to 41.97, Fig. 5c) and 5-HT levels (p < 0.01, 95% CI 23.47 to
34.61, Fig. 5d), suggesting an altered hippocampal neurotransmission.
All these 2M-SD-induced neurochemical alterations can be ameliorated
by 4 weeks treadmill exercise (p < 0.01, Fig. 5).

3.4. Treadmill exercise reverses 2M-SD-induced hippocampal BDNF and

IGF-1 decline

The hippocampal levels of neurotrophins BDNF and IGF-1 were Fig. 3. Treadmill exercise ameliorates anxiety-like behavior induced by 2M-SD
measured using ELISA. We found that, compared with NSD control mice, as assessed by OFT. (a) total squares crossed; (b) ratio of central squares
the hippocampal levels of BDNF and IGF-1 were both elevated after 4W- crossed; (c) ratio of central area duration. ** p < 0.01, * p < 0.05, compared
TE training (p < 0.01, 95% CI -9.332 to -1.388, F (3, 96) = 2.655, Fig. 6a; with NSD control group; ## p < 0.01, compared with 2M-SD group.
p = 0.0158, 95% CI -11.31 to -0.8546, F (3, 96) = 2.907, Fig.6b). In
contrast, 2M-SD mice showed significantly decreased levels of BDNF and
IGF-1 in hippocampus (p < 0.01, Fig. 6a, b). Four weeks treadmill

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Fig. 4. Treadmill exercise ameliorates memory impairment induced by 2M-SD as assessed by MWM and Y maze. (a) latency to reach safe platform during training
sessions in MWM; (b) Percentage of time spent in target quadrant during probe test session in MWM; (c) swimming speed in MWM; (d) Escape latency in visual cue
test of MWM; (e) Percentage of spontaneous alternation in YM. ** p < 0.01, compared with NSD control group; # # p < 0.01, compared with 2M-SD group.

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Fig. 5. Treadmill training reverses 2M-SD-induced alterations in hippocampal levels of neurotransmitters. Hippocampal levels of NE (a), DA (b), GABA (c) and 5-HT
(d) were determined by HPLC. * p < 0.05, ** p < 0.01, compared with NSD control group; ## p < 0.01, compared with 2M-SD group.
training ameliorated the declined hippocampal levels of BDNF and IGF-1
in 2M-SD mice (increase 30% and 27% respectively compared with 2M-
SD group, p < 0.01, Fig.6).
4. Discussion
Exercise has been indicated to be beneficial to human health. In the
present study, we found that 4 weeks treadmill exercise ameliorated
anxiety-like behavior and cognitive impairment induced by 2-month
chronic REM sleep deprivation in mice. In addition, our data further
indicated that all these emotional and cognitive benefits of exercise
might be due to the neurochemical and neurotrophic modulations in
mouse hippocampus.
Previous studies have demonstrated a beneficial effect of exercise on
basal levels of anxiety in rodents. For examples, Fulk et al. found that
treadmill training reduces anxiety-like behaviors in rats on EPM and
OFT (Fulk et al., 2004). Schoenfeld et al. also revealed that five weeks of
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wheel running reduced the anxiety level of both GFAP-TK mice and wild
type control mice on EPM and novelty-suppressed feeding test
(Schoenfeld et al., 2016). Moreover, previous clinical trials also revealed
positive impacts of exercise on anxiety in human (Stonerock et al.,
2015). A meta-analysis has suggested that exercise is effective in
improving anxiety symptoms in people with a current diagnosis of
anxiety (Stubbs et al., 2017). Interestingly, in our present study, we
found four weeks mild treadmill training exerted anxiolytic effect in
mice exposed to chronic REM sleep deprivation. These data further
confirmed the beneficial impact of exercise on anxiety under
stress-related conditions. In contrast to these findings, Zielinski et al.,
found that moderate sleep deprivation protocol did not deteriorate
anxiety-related behavior and even exerted an anxielytic tendency as
assessed by EPM (open arm percentage 8.70 ± 1.17 in normal sleep
control group v.s. 14.07 ± 2.74 in Sleep restriction + sedentary group)
(Zielinski et al., 2013). Furthermore, the exercise protocol used in Zie­
linski et al.’s study failed to ameliorate the impacts of REM sleep
F. Tai et al.
Fig. 6. Treadmill exercise reverses 2M-SD-induced BDNF and IGF-1 decline in hippocampus. BDNF (a) IGF-1 (b) were determined by ELISA. ** p < 0.05, * p < 0.05,
compared with NSD control group; # # p < 0.01, compared with 2M-SD group.
deprivation on anxiety-like behaviors. These differences might be due to
the protocols discrepancies in both sleep deprivation and exercise
training. In Zielinski et al.’s study, using a 2 × 2 experimental design,
they aimed to evaluate the beneficial effects of exercise (1 h per day, 6
days per week for 11 weeks) on the cognition and anxiety-like behavior
and further explore the detrimental impacts of chronic but relative mild
sleep deprivation (11 weeks, 4 h per day). Comparing with this
well-designed study by Zielinski et al., our present study aimed to test
the potential ameliorating impacts of treadmill exercise (4 weeks, 2 h
per day) on behavioral alterations induced by REM sleep deprivation (8
weeks, 20 h per day). While Zielinski et al. performed sleep deprivation
and exercise training in a parallel way, we conducted the treadmill
training and sleep deprivation protocol in a therapeutic manner, i.e. 4
weeks after the initiation of sleep deprivation exposure (Fig. 1).
Despite the positive impacts on emotion regulation, previous studies
have also indicated a beneficial effect of exercise on cognition. Meta-
analyses of randomized controlled trials have revealed that exercise
programs might play an important role in cognition and activities of
daily living in patients with dementia (Li et al., 2019). Ichinose et al.
have also demonstrated that both intermittent and continuous exercise
may improve cognitive function (Ichinose et al., 2020). Additionally,
previous studies have further indicated that exercise protects against
cognitive decline in rodents under chronically stressed condition (Kwon
et al., 2013; Lee et al., 2018). In consistent with these findings, in our
present study, we found that 4 weeks treadmill exercise ameliorated the
impaired learning and memory ability in sleep-deprived mice.
Considering the important roles of hippocampus in cognition and
emotion (Femenía et al., 2012; Bannerman et al., 2014; Anacker and
Hen, 2017), we further determined the levels of various neurotrans­
mitters and neurotrophic factors levels in mouse hippocampus, to
further explore the neurochemical and neurobiological mechanisms
underlying the anxiety and amnesia-like behaviors induced by chronic
sleep deprivation and the ameliorating effects of treadmill exercise. We
found that 4W-TE training elevated DA, GABA and 5-HT concentrations.
In addition, 2M-SD exposure in mice increased NE and DA but decreased
5-HT and GABA levels in hippocampus, whereas treadmill training can
ameliorate all these neurochemical alterations (Fig. 5). NE, as a pivotal
stress hormone and monoamine neurotransmitter, has been associated
with general arousal and stress-responses, such as stress-related mood
disorders (Kalk et al., 2011; Liu et al., 2018) and stress-associated
amnesia (Tully and Bolshakov, 2010; Shansky and Lipps, 2013).
Importantly and consistent with our present findings, previous studies
have also documented that sleep deprivation can increase NE level in the
brain (Gulyani and Mallick, 1993; Das and Mallick, 2008; Daniele et al.,
2017), which can be reversed by exercise (Daniele et al., 2017). The
effects of sleep deprivation on dopaminergic systems remain elusive.
While one night sleepless increase DA level in human brain to maintain
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Brain Research Bulletin 164 (2020) 198–207
arousal (Volkow et al., 2008), the DA content remains unaffected in
striatum (Lara-Lemus, 1998) or decreased in hippocampus (Wen et al.,
2013) in chronic sleep deprived rats. Our data revealed that after 8
weeks sleep deprivation, mice showed an increased DA content in hip­
pocampus, which is consistent with the elevated NE concentration
(Fig. 5a,b). The reasons for these conflict findings compare with those by
Wen et al. are possibly due to the different experimental design of sleep
deprivation (18 h daily for 3 weeks v.s. 20 h daily for 8 weeks) or
different animal species (rats v.s. mice). Much more interestingly, in our
present study, we found that sleep-deprivation-caused DA elevation can
be blocked by 4W-TE, whereas 4W-TE alone also increased DA (Fig.5b).
The exact neurochemical mechanisms underlying the modulation of
exercise on sleep-deprivation-induced DAergic alterations need further
exploration. In contrast to the elevated NE and DA, 2M-SD significantly
decreased the hippocampal levels of 5-HT and GABA, both inhibitory
neurotransmitter that has a close correlation with sleep control (Got­
tesmann, 2002; Monti, 2011; Cespuglio, 2018; Hernández-Vázquez
et al., 2019). Interestingly, serotonin and GABA have been reported to
serve as key modulator of anxiety (Lydiard, 2003; Kalueff and Nutt,
2007; Zangrossi and Graeff, 2014; Liu et al., 2019) and cognition
(Strasser et al., 2016; Ma et al., 2018; Schmidt-Wilcke et al., 2018;
Chakraborty et al., 2019). Therefore, the altered neurotransmission
including NE, DA, 5 H T and GABA in hippocampus induced by 2M-SD
may contribute the anxiety- and amnesia-like behavior. Moreover,
moderate exercise can ameliorate these neurochemical alterations in
mouse hippocampus induced by sleep deprivation, providing neuro­
chemical interpretations for the beneficial impacts of exercise on
cognitive and mood disorders in individuals suffering sleep
disturbances.
Despite the neurochemical changes, sleep disorders have been re­
ported to disrupt neurotrophic factors expressions in CNS, such as BDNF
and IGF-1, both have been closely correlated with cognitive function and
mood disorders. Dal-Pont et al. found that 36 h sleep deprivation
decreased the BDNF, NGF, and GDNF levels in the frontal cortex and
hippocampus of mice and induced manic-like behavior (Dal-Pont et al.,
2019). Mohammadipoor-Ghasemabad et al. suggested that sleep depri­
vation can decrease hippocampal BDNF expression together with an
impaired spatial cognition in ovariectomized rats (Mohammadipoor-­
Ghasemabad et al., 2019). Sleep deprivation protocol in Zielinski et al.’s
study showed a tendency of declined BDNF expression in CA1 region of
mouse hippocampus (Zielinski et al., 2013). Consistent with these pre­
vious findings, our data confirmed the impacts of sleep deprivation on
the expressions of neurotrophic factors. This declined neurotrophic level
in hippocampus after chronic stressful stimuli may result in an impaired
neurogenesis and synaptic plasticity, which consequently leading to
mood disorders (Jiang and Salton, 2020) and cognitive damage (Toda
and Gage, 2018). Interestingly, recent studies have indicated that
F. Tai et al.
exercise can enhance BDNF and IGF-1 level (Kohman et al., 2012; Wrann
et al., 2013; Żebrowska et al., 2018). All these prior works further
supported our present data that exercise can reverse sleep
deprivation-induced anxiety-like behavior and amnesia possibly via
restoring impaired neurotrophic level.
5. Conclusion
In conclusion, our present study showed that exercise can reduce
anxiety-like behavior and ameliorate cognitive decline induced by
chronic REM sleep deprivation. These present findings add to the un­
derstanding of behavior and brain alterations in connection with sleep
disorders and provide experimental evidence for the therapeutic po­
tential of exercise against sleep-related disorders.
Fundings
This work was supported by Science and Technology Project of
Liaoning Department of Education, China, Natural Science Foundation
of Liaoning Department of Science and Technology, China
(20180550306 and 2019-ZD-0467), Liaoning Provincial Project for
Innovative Talents, China, the Talent Program of the First Affiliated
Hospital Dalian Medical University, China (2017YC005).
CRediT authorship contribution statement
Feng Tai: Conceptualization, Investigation, Methodology, Writing -
original draft. Che Wang: Data curation, Funding acquisition, Investi­
gation, Methodology, Writing - original draft. Xin Deng: Data curation,
Investigation, Methodology. Ruojin Li: Data curation, Investigation,
Methodology. Zimeng Guo: Data curation, Investigation, Methodology.
Haiying Quan: Conceptualization, Project administration, Supervision,
Validation, Writing - review & editing. Song Li: Conceptualization,
Formal analysis, Funding acquisition, Project administration, Resources,
Software, Supervision, Validation, Visualization, Writing - original draft,
Writing - review & editing.
Declaration of Competing Interest
The authors report no declarations of interest.
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