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http://dx.doi.org/10.1016/j.neuroscience.2016.03.019
NSC 16983
To appear in:
Neuroscience
Accepted Date:
7 March 2016
Please cite this article as: D-M. Kim, Y-H. Leem, Chronic stress-induced memory deficits is reversed by regular
exercise via AMPK-mediated BDNF induction, Neuroscience (2016), doi: http://dx.doi.org/10.1016/j.neuroscience.
2016.03.019
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Chronic stress-induced memory deficits is reversed by regular exercise via AMPKmediated BDNF induction
Department of Society of Sports & Leisure Studies, Wonkwang University, 460 Iksandea-ro,
Running title: Exercise restores cognitive function in chronic stress through AMPK
Grant sponsor: Wonkwang University in 2014 and National Research Foundation of Korea
funded by the Korean Government, Grant number: NRF-2013R1A1A2062984
ABSTRACT
Chronic stress has a detrimental effect on neurological insults, psychiatric deficits, and
cognitive impairment. In the current study, chronic stress was shown to impair learning and
memory functions, in addition to reducing in hippocampal Adenosine monophosphateactivated protein kinase (AMPK) activity. Similar reductions were also observed for brainderived neurotrophic factor (BDNF), synaptophysin, and post-synaptic density-95 (PSD-95)
levels, all of which was counter-regulated by a regime of regular and prolonged exercise. A
21-day restraint stress regimen (6 h/day) produced learning and memory deficits, including
reduced alternation in the Y-maze and decreased memory retention in the water maze test.
These effects were reversed post-administration by a 3-week regime of treadmill running (19
m/min, 1 h/day, 6 days/week). In hippocampal primary culture, phosphorylated-AMPK
(phospho-AMPK) and BDNF levels were enhanced in a dose-dependent manner by 5amimoimidazole-4-carboxamide riboside (AICAR) treatment, and AICAR-treated increase
was blocked by Compound C. A 7-day period of AICAR intraperitoneal injections enhanced
alternation in the Y-maze test and reduced escape latency in water maze test, along with
enhanced phospho-AMPK and BDNF levels in the hippocampus. The intraperitoneal
injection of Compound C every 4 days during exercise intervention diminished exerciseinduced enhancement of memory improvement during the water maze test in chronically
stressed mice. Also, chronic stress reduced hippocampal neurogenesis (lower Ki-67- and
doublecortin-positive cells) and mRNA levels of BDNF, synaptophysin, and PSD-95. Our
results suggest that regular and prolonged exercise can alleviate chronic stress-induced
hippocampal dependent memory deficits. Hippocampal AMPK-engaged BDNF induction is
at least in part required for exercise-induced protection against chronic stress.
Key words: chronic restraint stress, treadmill running, learning and memory, AMPK, BDNF
1 INTRODUCTION
uptake,
glycolysis,
and
fatty
acid
oxidation.
Correspondingly,
AMPK
phosphorylation also suppresses anabolic process, including the synthesis of fatty acid,
cholesterol, and protein, to restore cellular energy levels (Hadad et al., 2008; Ronnett et al.,
2009). The activation of AMPK through phosphorylation is triggered by ATP depletion
(increased AMP/ATP ratio), metabolic stresses (hypoxia, glucose deprivation, oxidative
stress), and exercise (Culmsee et al., 2001; McCullough et al., 2005; Hardie, 2007).
Furthermore, AMPK is also phosphorylated by Ca2+/calmodulin-dependent protein kinase ,
suggesting that the kinase activity of this molecule is regulated indirectly by intracellular
Ca2+ levels (Woods et al., 2005). As addressed above, AMPK is considered to play a crucial
role in CNS neuronal responses to various physiological and pathological stimuli, particularly
within the hippocampus. Supporting this, modest activation of AMPK in the hippocampus by
diet restriction improves cognitive function and enhances hippocampal neurogenesis (Dagon
et al., 2005). Similarly, Resveratrol treatment elicits activation of hippocampal AMPK and
alleviates prenatal stress-induced memory impairment in pups (Cao et al., 2014). These
articles suggest a potential role for hippocampal AMPK activation in the modulation of
cognitive function. Furthermore, a recent study showed that the induction of unpredictable
chronic mild stress for a 4-week period results in the inactivation of AMPK and the
emergence of abnormal mood-related behaviors (Zhu et al., 2014). The anti-depressive
actions of ketamine appear to require both availability of brain-derived neurotrophic factor
(BDNF) and AMPK activation, with the activation of AMPK causing induction of BDNF
expression (Yoon et al., 2008; Autry et al., 2011; Xu et al., 2013). The neuroprotective role of
BDNF and its link to cognitive function is well established. BDNF contributes to the
promotion of long-term potentiation, enhanced synaptic plasticity, and improved cognitive
function (Conner et al., 1997; Duman and Monteggia, 2006). Judging from these studies,
alterations in hippocampal AMPK activity may be linked to stress-induced memory
impairment.
Physical exercise is renowned for its ability to improve brain function, influencing both
cognitive function and mood (Hillman et al., 2008). In particular, the effect of exercise on
performance in hippocampal dependent memory tasks is believed to be associated with
hippocampal neurogenesis, synaptic plasticity and neurotrophins (Eadie et al., 2005; GomezPinilla et al., 2008; Hillman et al., 2008; van Praag, 2008). In addition, AMPK is highly
expressed in brain regions such as the hippocampus and plays a crucial role in exercise
physiology (Hadie, 2004; Spasic et al., 2009). However, the involvement of hippocampal
AMPK in chronic stress-induced memory impairment and its subsequent reversal with
exercise is still poorly understood. To unravel this issue, we explored whether exercise could
alleviate chronic stress-induced memory impairment using pharmacological activation and/or
inhibition of AMPK.
2 EXPERIMENTAL PROCEDURES
with various restraint durations (2-6 h/day). Water maze test was performed 27 days after the
exposure to stress. In experiment 2 (Fig. 1B), mice were subjected to the 21 consecutive days
of restraint stress. Water maze test was performed 3 or 27 days after the exposure to stress in
independent experiment. In experiment 3 (Fig. 3A), mice were divided into three groups
(control: CON, restraint stress: RST, exercise combined with restraint stress group: RST+Ex)
with each group containing 10 mice. To induce chronic stress by restraint, 8-week-old mice
were individually placed into well-ventilated 50-mL conical tubes, which prevented forward
or backward movement. Control mice remained undisturbed in their home cages during
restraint exposure. Restraint stress was delivered at set times from 1000 to 1600 for 6 h for 21
days. After the stress exposure period, all mice were pre-exercised to acclimate them to
treadmill-running (Myung Jin Instruments Co., Seoul, Korea) from 1000 (Pre-Ex; 12 m/min,
20min/day) for 3 days. Subsequently treadmill exercise was performed at 19 m/min for 60
min/day, 6 days/week for 21 days. Non-exercised mice were placed on the treadmill that was
turned off for 60 min once a day. Two days after the last treadmill session, Y-maze and water
maze tests were performed. In experiment 4 (Fig. 5A), mice were subjected to the 21
consecutive days of restraint stress. Serum CORT and ACTH levels were measured 1 day
after the last exposure to restraint. In experiment 6 (Fig. 5B), mice were subjected to a 21-day
(19 m/min, 1 h/day, 6 days/week) of treadmill running. Serum CORT and ACTH levels were
measured 1 day after the last administration to treadmill running. In experiment 5 (Fig. 7), the
Y-maze test and water maze test was performed 21 days after 7 consecutive days of
intraperitoneal injection with the AMPK agonist 5-amimoimidazole-4-carboxamide riboside
(AICAR, 500 mg/kg, once a day; Tocris Bioscience, Bristol, UK; eight mice per group). In
experimental 6 (Fig. 8), mice were intraperitoneally injected with AICAR (0-500 mg/kg) for
7. Water maze test was performed 14 days after the last treatment with AICAR. In experiment
7 (Fig.9A), mice were divided into four groups (control, restraint stress, exercise combined
with restraint stress group, exercise combined with restraint stress and Compound C; eight
mice per group). The experimental procedure of experiment 9 was equal to that of experiment
3, except on Compound C (10 mg/kg; EMD Chemicals, Gibbstown, NJ) treatment.
Compound C was intraperitoneally injected during exercise intervention every 4 days. Mice
without drugs (AICAR and Compound C) treatment were injected with saline (1% DMSO).
with the
mouse to reach the platform within 60s. On day 5, the mice were subjected to three probe
trials, in which they were required to swim for 60s without a platform. The time required to
reach the previous platform location (escape latency) was recorded for each animal. Each trial
was stored on videotape for subsequent analysis.
On day in vitro (DIV) 1, the cells were incubated in Neurobasal Medium with 1 B27, 1
penicillin/streptomycin, and 1 L-glutaMax. The medium was changed every 2 days.
Cultures taken from DIV 7-9 were used in experiments. In experiment 1, cells were cultured
with AICAR (1, 0.5, 1 mM) for 1 hour, and then BDNF, phospho-AMPK, and AMPK levels
were measured using western blot analysis. In experiment 2, cells were cultured with
Compound C (1, 3, 10 M) for 2 hours before undergoing treatment with AICAR (0, 0.5 mM)
for 1 hour.
2.7 Immunohistochemistry
Anaesthetized mice were perfused with 100 mM phosphate buffer (PBS; pH 7.4), followed
by cold 4% paraformaldehyde in PBS. After perfusion, the brains were removed, fixed for
another 18 h,, and transferred to 1030% sucrose solution. Finally, 40-m-thick sections were
prepared using a vibratome (Leica, Wetzlar, Germany). Every eleventh section was taken
from the region between bregma 1.46 mm and 2.80 mm. Free-floating sections were
incubated with 0.3% hydrogen peroxide (H2O2), permeabilized with 0.3% Triton X-100, and
nonspecific protein binding was blocked by incubation with 3% normal goat serum. Sections
were incubated overnight at 4C with anti-Ki-67 and anti-doublecortin (DCX) primary
antibodies, respectively (Abcam, Cambridge, MA, USA; rabbit polyclonal, 1: 2,000) and
subsequently with biotinylated secondary antibodies (Vector Laboratories; Burlingame, CA,
USA 1: 200, respectively), and then visualized using the ABC method (ABC Elite kit, Vector
Laboratories; Burlingame, CA, USA). The sections were mounted and assessed in digital
images (captured at 100 magnification) using Image J (NIH Image Engineering, Bethesda,
MD).
10
11
3 RESULTS
3.1 Regular and prolonged treadmill running alleviated memory impairments produced
by repeated restraint.
First, we explored chronic stress-induced memory defect according to various durations of
restraint exposure (2-6 hours/day). The exposure to restraint for 6 hours per day resulted in
the decreased escape latency on the final day of water maze testing 4 weeks after the last
exposure to restraint, but not 2-3 hours per day (Fig.1A; for 2h/21d t14 = -0.87, p > 0.05; for
3h/21d t14 = -1.90, p > 0.05; for 6h/21d t14 = -5.71.90, p > 0.01). Next, the escape latency of
stressed mice was enhanced on the final day of water maze testing compared with that of
control mice 1 week after the last exposure to restraint and this change lasted up to 4 weeks,
suggesting that the 21 consecutive days of restraint stress-induced memory deficit at least
lasted until 4 weeks in this experimental paradigm (Fig. 1B; for on day 28 t14 = -11.20, p <
0.01; 53 (t14 = -9.98, p < 0.01 ). The rate of alternation in Y-maze tasks was significantly
enhanced 2 and 9 days after the last exposure to treadmill running relative to that of nontreadmill-run mice, but not 7-day treadmill running (Fig. 2A; for 7-day Ex, t12 = 0.13, p >
0.05; for 21-day Ex, post-day 2, t12 = -3.74, p < 0.01; for 21-day Ex, post-day 9, t12 = -2.82, p
< 0.05). Furthermore, pre-exercise for the acclimation to treadmill running did not enhance
memory performance (Fig. 2A; t10 = 0.01, p > 0.05). Memory function was evaluated using
water maze and Y-maze tests to elucidate whether repeated and regular exercise might
alleviate memory impairments induced by 21 consecutive days of restraint (Fig.3A). In the Ymaze test, the alternation of restrained mice was markedly reduced compared with that of
control mice. This was reversed by treadmill exercise (Fig.3Ba; F2, 27 = 5.637, p < 0.01). In
the water maze test, the escape latency of restrained mice was significantly enhanced
compared with that of control mice, which was reversed by exercise treadmill running (the
12
interaction effect of group x day F8, 108 = 18.13, p < 0.01; the main effect of group F1, 27 =
18218.74, p < 0.01; the main effect of day F4, 108 = 235.49, p < 0.01; Fig. 3Bb-c).
3.4 AICAR treatment enhanced BDNF expression, and Compound C treatment reduced
BDNF expression in the primary hippocampal culture.
13
Since the expression pattern of phospho-AMPK corresponded well to that of mature BDNF,
we assessed mature BDNF expression level in the primary hippocampal culture using the
AMPK agonist, AICAR and AMPK antagonist, Compound C. BDNF expression was
enhanced in a dose-dependent manner and phospho-AMPK expression was simultaneously
increased by AICAR treatment (Fig. 6A; for BDNF F2, 9 = 126.7, p < 0.01; for phosphoAMPK F2,
Compound C (Fig. 6B; for BDNF F4, 15 = 88.11, p < 0.01; for phospho-AMPK F4, 15 = 106.84,
p < 0.01).
14
4 DISCUSSION
15
The current study demonstrates that chronic restraint stress induces hippocampal-dependent
memory deficits, which can be counteracted with regular and prolonged treadmill exercise.
Furthermore, the protective effect of exercise against chronic stress-induced memory
impairment is at least in part dependent on hippocampal AMPK-mediated BDNF induction.
To the best of our knowledge, this is the first study investigating the role of hippocampal
AMPK in this capacity, at least with regard to its up regulation by prolonged exercise, and
subsequent induction of BDNF expression.
To investigate whether chronic stress caused memory defect, mice were subjected to restraint
stress for 21 days with various durations (2-6 hours/day) and subsequently memory function
was measured by water maze test (Fig. 1A). In the first instance, the exposure to restraint for
6 hours per day resulted in the decreased escape latency on the final day of water maze
testing 4 weeks after the last exposure to restraint, but not 2-3 hours per day. The escape
latency of stressed mice was enhanced on the final day of water maze testing compared with
that of control mice 1 week after the last exposure to restraint and this change lasted up to 4
weeks, suggesting that the 21 consecutive days of restraint stress-induced memory deficit at
least lasted until 4 weeks in this experimental paradigm (Fig. 1B). Furthermore, although
species and testing method were different from those of this study, the 21 consecutive days of
restraint stress (6h/day) reduced latency of entrance to the dark chamber in passive avoidance
test until 21 day after the last stress exposure (Radahmadi et al., 2015; Radahmadi et al.,
2013). For this reason, the chronic stress regime with a 6h/21d of restraint was adopted in the
current study. Next, we investigated whether a 21-day regime of treadmill running would
affect memory function. Analysis of memory function suggested that the rate of alternation in
Y-maze tasks was significantly enhanced 2 and 9 days after the last exposure to treadmill
running relative to that of non-treadmill-run mice, but not 7-day treadmill running (Fig. 2A).
16
Furthermore, pre-exercise for the acclimation to treadmill running did not enhance memory
performance (Fig. 2B). Based on this data, the exercise paradigms that mice were subjected
to during treadmill running after pre-exercise were adopted in the current study. We found
that treadmill running for three weeks alleviated memory deficits induced by the 21
consecutive days of restraint stress. Chronic restraint stress elicited a decreased rate of
alternation in the Y-maze test, declined retention of memory of water maze testing. These
effects were reversed by exercise intervention over three weeks (Fig. 3), suggesting that this
experimental paradigm was appropriate for exploring the potential role of exercise in
alleviating chronic stress-induced memory deficits. This result was consistent with our
previous findings that prolonged exercise elicited memory improvement in chronically
restrained mice, although the exercise regimen used was different between both of studies
(Kwon et al., 2013).
Chronic physical or psychological stress can lead to abnormal morphology and function of
hippocampal neurons, which may be attributed to altered synthesis of proteins involved in
synaptic plasticity (Kasai et al., 2010; Surget et al., 2011; Zhu et al., 2014). In the current
study, synaptophysin and PSD-95 levels were profoundly reduced by chronic stress and this
decrease was reversed by regular and prolonged exercise (Fig. 4B). Synaptophysin and PSD95 are mainly localized in pre- and post-synaptic regions, playing a crucial role in regulating
synaptic transmission and organizing post-synaptic densities. Reduced object novelty
recognition and impaired spatial learning performance are evident in synaptophysin knockout
mice (Schmitt et al., 2009). PSD-95 contributes to synaptic formation and stabilization, and
participates in the synaptic trafficking of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) receptors that modulate excitatory synaptic transmission (El-Husseini et al.,
2000; Henley JM and Wilkinson KA, 2013). The aforementioned results support our theory
that chronic stress causes reduced hippocampal synaptophysin and PSD-95 levels. Since
17
these proteins regulate synaptic transmission, strength, and plasticity, a reduction in levels of
synaptophysin and PSD-95 could likely impair hippocampal dependent learning and memory
function. The results of this study suggest that stress-induced memory impairments can be
reversed by exercise, which implies that regular, prolonged physical activity can restore the
reduced expression of both synaptic proteins, resulting in the improved efficacy of neuronal
information transfer and affecting cognitive behavior.
Neurons consume a large amount of energy to perform their physiological functions.
Abnormalities
of
are
strongly associated
with
neurocognitive deficits that result from -amyloid accumulation and chronic stress (Park et
al., 2013; Cao et al., 2014). AMPK modulates cellular energy homeostasis as a transcriptional
regulator in response to ATP depletion, metabolic stresses, intracellular Ca2+ levels, and
exercise (Culmsee et al., 2001; McCullough et al., 2005; Woods et al., 2005; Hardie, 2007;
Kobilo et al., 2015a, 2015b). Our data showed that hippocampal phospho-AMPK levels were
markedly reduced by chronic stress and this decrease was subsequently reversed by exercise
intervention (Fig. 4A). The role of AMPK in cognitive functions has been previously
described by a number of researchers (Dagon et al., 2005; Cao et al., 2014; Zhu et al., 2014).
The above-addressed findings supported our result that chronic stress inactivates
hippocampal AMPK and that regular and prolonged exercise counter-regulates chronic stressinduced deficits.
This chronic stress-provoked hippocampal reduction of AMPK activity may be associated
with HPA axis abnormality. Serum corticosterone (CORT) and adrenocorticotrophic hormone
(ACTH) levels was in chronic stressed mice significantly higher than in control mice at the
rest, when CORT and ACTH were measured 2 days after the 21 consecutive day of restraint
stress (Fig. 5A). This result suggests that chronic stress induces the sustained hypothalamicpituitary-adrenal (HPX) axis activation, which the persisted increase in CORT may affect
18
hippocampal AMPK activity. On the other hand, regular exercise didnt alter basal levels of
both CORT and ACTH, suggesting that regular exercise didnt affect basal HPX axis activity
(Fig. 5B). In our study, AMPK phosphorylation behavior corresponds well to hippocampal
mature BDNF expression. BDNF is a neurotrophic molecule contributing to neuronal growth,
development, plasticity, survival, neuroprotection, and repair, which when stimulated, may
act to improve cognitive ability (Conner et al., 1997; Duman and Monteggia, 2006). To
confirm AMPK-mediated BDNF induction, we analyzed mature BDNF protein levels in
primary hippocampal cell cultures treated with AMPK agonist AICAR and antagonist
Compound C. The mature form of BDNF and phospho-AMPK protein levels were enhanced
in dose-dependent manner by AICAR treatment (Fig. 6A), which was reversed by Compound
C treatment (Fig. 6B). Moreover, 7 days of treatment with AICAR enhanced hippocampal
mature BDNF and phospho-AMPK expression, concomitant with the increase in alternation
in the Y-maze test and the decrease in escape latency in water maze test (Fig. 7). The escape
latency was reduced on day 4-5 in mice treated with ALCAR at 500 mg/kg, but not 100-300
mg/kg (Fig. 8). Although AICAR has low permeability across the blood-brain barrier (BBB),
our study demonstrated an increase in hippocampal AMPK activation. There are two possible
reasons for this change. First, in spite of low permeability across the BBB, a small quantity of
permeable AICAR might linger in the extracellular space due to a limited capacity for
reuptake and degradation. Second, enhanced memory function following intraperitoneal
injection of AICAR both in this study and another (Kobilo et al., 2015b) may be attributed to
release of myokines in the muscles. These secretory molecules may pass across the BBB and
indirectly affect cognitive function (Sakuma and Yamaguchi, 2011; Pedersen and Febbraio,
2012). Judging from our results and other findings, systematically circulating AICAR likely
influences hippocampal AMPK activity, whether directly or indirectly. Several studies have
demonstrated that AMPK activation induces BDNF expression (Yoon et al., 2008; Autry et al.,
19
20
AMPK activity suggests that AMPK is heavily involved in the memory side effects of
exercise such as enhancement of memory function. Additionally, hippocampal AMPK
activation is at least partly required for the induction of exercise-induced neurotrophic effects
such as BDNF expression.
CONFLICT OF INTEREST
ACKNOWLEDGMENTS
This study was supported by Wonkwang University in 2014 and grants from the National
Research
Foundation
of
Korea
funded
by
the
Korean
Government
(NRF-
2013R1A1A2062984)..
REFERENCES
Autry AE, Adachi M, Nosyreva E, Na ES, Los MF, Cheng PF, Kavalali ET, Monteggia LM
(2011) NMDA receptor blockade at rest trigger rapid behavioral antidepressant responses.
Nature 475:91-5.
Calabrese F, Guidotti G, Molteni R, Racagni G, Mancini M, Riva MA (2012) Stress-induced
changes of hippocampal NMDA receptors: modulation by duloxetine treatment. PLoS One
7:e37916-37924.
Cao K, Zheng A, Xu J, Li H, Liu J, Peng Y, Long J, Zou X, Li Y, Chen C, Liu J, Feng Z
21
22
Han TK, Lee JK, Leem YH (2015) Chronic exercise prevents repeated restraint stressprovoked enhancement of immobility in forced swimming test in ovariectomized mice.
Metab Brain Dis 30(3):711-8.
Hardie DG (2004) The AMP-activated protein kinase pathway-new players upstream and
downstream. J Cell Sci 117:5479-5487.
Hardie DG (2007) AMP-activated/SNF1 protein kinases: conserved guardiance of cellular
energy. Nat Rev Mol Cell Biol 8:774-785.
Henley JM, Wilkinson KA (2013) AMPA receptor trafficking and the mechanism underlying
synaptic plasticity and cognitive aging. Dialogues Clin Neurosci 15:11-27.
Hillman CH, Erickson KI, Kramer AF (2008) Be smart, exercise your heart: Exercise effects
on brain and cognition. Nat Rev Neurosci 9:58-65.
Kasai H, Fukuda M, Watanabe S, Hayashi-Takagi A, Noguchi J (2010) Structural dynamics
of dendritic spines in memory and cognition. Trends Neurosci 33:121-129.
Kobilo T, Guerrieri D, Zhang Y, Collica SC, Becker KG, van Praag H (2015a) AMPK agoinst
AICAR improves cognition and motor coordination in young and aged mice. Learning and
Memory 21:119-126.
Kobilo T, Yuan C, van Praag H (2015b) Endurance factors improve hippocampal
neurogenesis and spatial memory in mice. Learning and Memory 18:103-107.
Krishnan V, Nestler EJ (2008) The molecular neurobiology of depression. Nature 455:894902.
Kwon D, Kim B, Chang H, Kim Y, Ahn Jo S, Leem YH (2013) Exercise overcame impaired
cognition by restraint stress-induced oxidative insult and BDNF abnormality. BBRC
23
434:245-251.
Magarinos AM, McEwen BS (1995) Stress-induced atrophy of apical dendrites of
hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino
acid receptors. Neuroscience 69:89-98.
McGaugh JL, Roozendaal B (2002) Role of adrenal stress hormones in forming lasting
memories in the brain. Curr Opin Neurobiol 12:205-210.
McLaughlin KJ, Gomez JL, Baran SE, Conrad CD (2007) The effects of chronic stress on
hippocampal morphology and function: an evaluation of chronic restraint paradigms. Brain
Res 1161:56-64.
Park S, Kim S, Kang S, Moon NR (2013) -Amyloid-induced cognitive dysregulation
impairs glucose homeostasis by increasing insulin resistance and decreasing -cell mass in
non-diabetic and diabetic rats. Metabolism 62:1749-1760.
Pedersen BK, Febbraio MA (2012) Muscle, exercise and obesity: skeletal muscle as a
secretory organ. Nat Rev Endocrinol 8:4574-4565.
Pham K, Nacher J, Hof PR, McEwen BS (2003) Repeated restraint stress suppresses
neurogenesis and induces biphasic PSA-NCAM expression in the adult rat dentate gyrus. Eur
J Neurosci. 17:879-86.
Radahmadi M, Alaei H, Sharifi MR, Hosseini N (2015) Preventive and therapeutic effect of
treadmill running on chronic stress-induced memory deficit in rats. J Bodyw Mov Ther.
19:238-45.
Radahmadi M, Alaei H, Sharifi MR, Hosseini N (2013) The effect of synchronized forced
running with chronic stress on shoet, mid and long-term memory in rats. Asian J Sports Med.
4:54-62.
24
Ronnettt GV, Ramamurthy S, Kleman AM, Landree LE, Aja S (2009) AMPK in the brain: its
roles in energy balance and neuroprotection. J Neurochem 109 Suppl. 1:17-23.
Roy A, Jana M, Kundu M, Corbett GT, Rangaswamy SB, Mishra RK, Luan C, Gonzalez FJ,
Pahan K (2015) HMG-CoA reductase inhibitors bind to PPAR to upregulate neurotrophin
expression in the brain and improve memory in mice. Cell Metabolism 22:253-265.
Sakuma K, Yamguchi A (2011) The recent understanding of the neurotrophin;s role in
skeletal muscle adaptation. J Biomed Biotechnol 2011:201696, doi: 1155/2011/201396.
Sandi C, Pinelo-Nava MT (2007) Stress and memory: behavioral effects and neurobiological
mechanisms. Neural Plast. 2007:1-20.
Schmitt U, Tanimoto N, Seeliger M, Schaeffel F, Leube RE (2009) Detection of behavioral
alterations and learning deficits in mice lacking synaptophysin. Neuroscience 162:234-243.
Sousa N, Lukoyanov NV, Madeira MD, Almeida OF, Paula-Barbosa MM (2000)
Reorganization of the morphology of hippocampal neurites and synapse after stress-induced
damage correlates with behavioral improvement. Neuroscience 97:253-66.
Spasic MR, Callaerts P, Norga KK (2009) AMP-activated protein kinase (AMPK) molecular
crossroad for metabolic control and survival of neurons. Neuroscientist 15:309-316.
Steinberg GR, Kemp BE. 2009. AMPK in health and disease. Physiol Rev 89:1025-78.
van Praag H (2008) Neurogenesis and exercise: Past and future directions. Neuromolecular
Med 10:128-140.
Watanabe Y, Gould E, McEwen BS (1992) Stress induces atrophy of apical densrites of
hippocampal CA3 pyramidal neurons. Brain Res 588:341-345.
Woods A,. Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR, Carlson M,
25
Carling D (2005) Ca2+/calmodulin-dependent protein kinase-beta acts upstream of AMPactivated protein kinase in mammalian cells. Cell Metab 2:21-33.
Xu S, Zhou Z, Li X, Ji M, Zhang G, Yang J (2013) The activation of adenosine
monophosphate-activated protein kinase in rat hippocampus contributes to the rapid
antidepressant effect of ketamine. Behav Brain Res 253:305-309.
Yamada K, Nabeshima T (2003) Brain-derived neurotrophic factor/TrkB signaling in memory
processes. J Pharmacol Sci 91:267-270.
Yoon H, Oh YT, Lee JY, Baik HH, Kim SS, Choe W, Yoon KS, Ha J, Kang I (2008)
Activation of AMP-activated protein kinase by kainic acid mediates brain-derived
neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma
cells. BBRC 371:495-500.
Zhu S, Wang J, Zhang Y, Li V, Kong J, He J, Li X (2014) Unpredictable chronic mild stress
induces anxiety and depression-like behaviors and inactivates AMP-activated protein kinase
in mice. Brain Res 1576:81-90.
Figure Legends
Figure 1. A 21-day of restraint stress (6h/day) caused memory decline and this effect
continued up to 4 weeks, but not 2-3 h/day.
A. Quantitative analysis of the escape latency on the final day of water maze testing. The
escape latency of stressed mice was enhanced compared with that of control mice on day 28
(for 2h/21d t14 = -0.87, p > 0.05; for 3h/21d t14 = -1.90, p > 0.05; for 6h/21d t14 = -5.71.90, p
> 0.01).
B. Quantitative analysis of the escape latency on the final day of water maze testing. The
26
escape latency of stressed mice was enhanced compared with that of control mice on day 28
(t14 = -11.20, p < 0.01) and 53 (t14 = -9.98, p < 0.01).
Data are presented as the mean SEM. ** denote differences at p < 0.01.
Figure 2. Pre-exercise for the acclimation to treadmill running did not affect memory
ability measured by Y-maze test.
A. 21-days of exercise intervention improved memory function, but not 7-days of exercise.
a. Experimental design. Mice were subjected to 7-day (19 m/min, 1 h/day) or 21-day (19
m/min, 1 h/day, 6 day/week) treadmill running 2 days after pre-exercise. Y-maze test was
assessed 2 or 9 days after the last exercise intervention, independently.
b. Quantitative analysis of Y-maze test. A 7-day exercise intervention did not change rates of
alternation in the Y-maze test. However, the 21-day exercise regimen enhanced alternation of
Y-maze test 2 and 9 days after the last exercise intervention (for 7-day Ex, t12 = 0.13, p > 0.05;
for 21-day Ex, post-day 2, t12 = -3.74, p < 0.01; for 21-day Ex, post-day 9, t12 = -2.82, p <
0.05)
B. Experimental design and alternation of Y-maze test between sedentary and pre-exercised
mice.
a. Experimental design. Mice were subjected to treadmill running for 3 days (12 m/min, 20
min/day), and subsequently Y-maze test was assessed 21 days after the last exercise
administration.
b. Quantitative analysis of Y-maze test. Alternative rate of Y-maze test in 3-day of treadmillrun mice was comparable to that of sedentary mice (t10 = 0.01, p > 0.05).
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Figure 5. Chronic stress caused the sustained serum CORT and ACTH levels, but not
the regular and prolonged exercise.
A. Quantitative analysis of serum CORT and ACTH levels following chronic stress.
a. Experimental design. Mice were subjected to restraint for 21 days (6h/day), followed by
the assess of serum CORT and ACTH levels.
b. Quantitative analysis of serum CORT and ACTH levels. Serum CORT and ACTH levels
were significantly enhanced by chronic restraint stress.
B. Quantitative analysis of serum CORT and ACTH levels following chronic stress.
a. Experimental design. Mice were subjected to treadmill running for 21 days (19 m/min, 1
h/day, 6 day/week), followed by the assess of serum CORT and ACTH levels.
b. Quantitative analysis of serum CORT and ACTH levels. Serum CORT and ACTH levels
were no significantly different between groups.
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Data are presented as the mean SEM. * and ** denote differences at p < 0.05 and p < 0.01,
respectively.
Figure 8. AICAR treatment with 500 mg/kg improved memory function, but not 0-200
mg/kg.
A. Experimental design. Mice were treated with AICAR (0-500 mg/kg) for 7 days, and
subsequently memory function were measured by water maze test 14 days after the last
treatment with AICAR.
B. Quantitative analysis of the escape latency of water maze testing. The escape latency of
mice treated with 500 mg/kg AICAR decreased on day 4-5, but not 100-200 mg/kg.
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Data are presented as the mean SEM. ** denote differences from 0 mg/ kg at p < 0.01.
Data are presented as the mean SEM. ** denote differences at p < 0.01. and denote
difference from CON and RST+Ex, respectively at p < 0.01 from
Figure 10. Compound C treatment during exercise intervention blocked exerciseinduced enhancement of neurogenesis in mice subjected to restraint.
A. Quantitative analysis of Ki-67-positive cells.
a. Photomicrograph showing immunohistochemical data for Ki-67.
b. Quantitative analysis of Ki-67-positive cells.
A. Quantitative analysis of doublecortin-positive cells.
a. Photomicrograph showing immunohistochemical data for doublecortin.
b. Quantitative analysis of doublecortin-positive cells.
Data are presented as the mean SEM. ** denote differences at p < 0.01.
Figure 11. Compound C treatment during exercise intervention blocked exerciseinduced enhancement of BDNF, synaptophysin, and PSD-95 mRNA in mice subjected to
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restraint.
a. Photomicrograph showing RT-PCR data for BDNF, synaptophysin, and PSD-95 mRNA.
b. Quantitative analysis of BDNF, synaptophysin, and PSD-95 mRNA.
Data are presented as the mean SEM. ** denote differences at p < 0.01.
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RESEARCH HIGHLIGHTS