chronic stress-induced memory deficits is reversed by regular exercise via ampk-mediated bdnf...

Upload: iluquiitasi

Post on 03-Mar-2018

218 views

Category:

Documents


0 download

TRANSCRIPT

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    1/44

    Accepted Manuscript

    Chronic stress-induced memory deficits is reversed by regular exercise via

    AMPK-mediated BDNF induction

    Dong-Moon Kim, Yea-Hyun Leem

    PII: S0306-4522(16)00242-6

    DOI: http://dx.doi.org/10.1016/j.neuroscience.2016.03.019

    Reference: 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

    This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

    we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

    review of the resulting proof before it is published in its final form. Please note that during the production process

    errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

    http://dx.doi.org/10.1016/j.neuroscience.2016.03.019http://dx.doi.org/http://dx.doi.org/10.1016/j.neuroscience.2016.03.019http://dx.doi.org/http://dx.doi.org/10.1016/j.neuroscience.2016.03.019http://dx.doi.org/http://dx.doi.org/10.1016/j.neuroscience.2016.03.019http://dx.doi.org/http://dx.doi.org/10.1016/j.neuroscience.2016.03.019http://dx.doi.org/10.1016/j.neuroscience.2016.03.019
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    2/44

    1

    Chronic stress-induced memory deficits is reversed by regular exercise via AMPK-

    mediated BDNF induction

    Dong-Moon Kim1, Yea-Hyun Leem

    2,*

    1Department of Society of Sports & Leisure Studies, Wonkwang University, 460 Iksandea-ro,

    Iksan, Jeonbuk, Republic of Korea

    2Department of Molecular Medicine and TIDRC, School of Medicine, Ewha Womens

    University, Seoul 158-710, Republic of Korea

    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

    *Corresponding author: Yea-Hyun Leem, Ph.D.,

    Department of Neuroscience and TIDRC, Ewha Womans University, Mokdong Hospital,

    911-1 Mok-Dong, Yangcheon-Ku, Seoul 158-710, Republic of Korea.

    Tel: +82-2-2650-5749, Fax: +82-2-2650-5850, Email: [email protected]

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    3/44

    2

    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 monophosphate-

    activated protein kinase (AMPK) activity. Similar reductions were also observed for brain-

    derived 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 5-

    amimoimidazole-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 exercise-

    induced 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.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    4/44

    3

    Key words: chronic restraint stress, treadmill running, learning and memory, AMPK, BDNF

    1 INTRODUCTION

    Stress causes widespread changes to the neurochemical, neurobiological, and behavioral

    responses of the brain. Accumulating evidence also suggests that chronic stress negatively

    affects neural plasticity to produce deficits in memory and learning processes (Sandi and

    Pinelo-Nava, 2007; Krishnan and Nestler, 2008). The detrimental effect of chronic stress on

    cognitive function is suggested to be modulated by corticosterone, neurotrophins, oxidative

    stress, and various neurotransmitters (McGaugh and Roozendaal, 2002; Yamada and

    Nabeshima, 2003; Sandi and Pinelo-Nava, 2007; Calabrese et al., 2012; Kwon et al., 2013).

    Among the brain structures affected, the hippocampus is a region commonly implicated in

    repeated or chronic stress-triggered abnormalities of neural plasticity. Such abnormalities

    include hippocampal atrophy, decreased neurogenesis, and impaired synaptic plasticity

    (Watanabe et al., 1992; Sousa et al., 2000; Pham et al., 2003; Han et al., 2015).

    Since a large amount of energy is required to fulfill the physiological demands of neurons in

    the central nervous system (CNS), dysregulation of energy metabolism will deleteriously

    affect their survival and function. Adenosine monophosphate-activated protein kinase

    (AMPK) is an energy metabolite-sensing protein kinase that contributes to regulating cellular

    energy homeostasis (Spasic et al., 2009; Steinberg and Kemp, 2009). The phosphorylation of

    AMPK on threonine 172, producing phospho-AMPK, stimulates catabolic processes such as

    glucose uptake, glycolysis, and fatty acid oxidation. Correspondingly, AMPK

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    5/44

    4

    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.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    6/44

    5

    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; Gomez-

    Pinilla 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

    2.1 Experimental subjects

    Male 7-week-old C57BL/6 mice were obtained from Daehan Biolink, Inc. (Eumsung,

    Chungbuk, Korea) and housed in clear plastic cages in specified pathogen-free conditions

    under a 12:12-h light-dark cycle (lights on at 0800 and off at 2000). Mice had free access to

    standard irradiated chow (Purina Mills, Seoul, Korea). Ewha Womans University Animal

    Care and Use Committee granted the approval for all experimental procedures involving

    animals.

    2.2 Experimental design

    In experimental 1 (Fig.1A), mice were subjected to the 21 consecutive days of restraint stress

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    7/44

    6

    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

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    8/44

    7

    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).

    2.3 Y-maze test

    The Y-maze consisted of three equal-sized arms made of white PVC. The arms measured

    38.5-cm long, 3-cm wide, and 13-cm high, and were oriented at 60 angles from each other

    (JEUNG DO Bio & Plant Co. LTD, Seoul, Korea). The Y-maze test was performed under

    moderate lighting conditions (200 Lux) with moderately loud background white noise (40

    dB). Mice began a single trial at the end of one arm and were allowed to explore the Y-maze

    freely for 8 minutes. The number and sequence of arm visits were recorded manually by an

    observer. Alternation was defined as a consecutive entry in three different arms. The

    alternation percentage was calculated with the following formula: (number of

    alternations/total number of arm visits) 2.

    2.4 Water maze test

    The test was performed using the SMART-CS (Panlab, Barcelona, Spain) program in an air-

    conditioned room. The water maze experiment was carried out in a 1.5m diameter plastic

    circular pool with 22C water containing powdered milk to obstruct the platform visibility.

    Escape latency was monitored by a computer, using the SMART-LD program, which was

    connected to a ceiling-mounted camera directly above the pool. The training schedule

    consisted of two trials per day over 4 days of testing, and each trial assessed the ability of the

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    9/44

    8

    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.

    2.5 Western blot analysis

    Hippocampal tissue was homogenized with lysis buffer (50 mM HEPES pH 7.5; 150 mM

    NaCl;

    10% glycerol; 1% TritonX-100; 1 mM PMSF; 1 mM EGTA; 1.5 mM MgCl26H2O; 1 mM

    sodium orthovanadate; 100 mM sodium fluoride). Protein samples (20 g) were

    electrophoretically separated on 10% polyacrylamide gels, transferred to nitrocellulose

    membranes (Amersham Bioscience, Buckinghamshire, UK), and incubated with a primary

    antibody in a blocking buffer at room temperature overnight. The next day, the samples were

    rinsed with a washing buffer and incubated with horseradish peroxidase-conjugated

    secondary antibody for 2 hours at room temperature. The optical density of each band was

    measured using the SCION program (NIH Image Engineering, Bethesda, MD, USA).

    Anti-phospho-AMPK and anti-AMPK antibodies were obtained from Cell Signaling Tech.

    Inc. (Danvers, MA, USA), anti-BDNF was from Santa Cruz Biotechnology (Santa Cruz, CA,

    USA), and anti-synaptophysin and anti-PSD-95 were obtained from Abcam (Cambridge, UK).

    2.6 Primary hippocampal culture

    Primary hippocampal cell cultures were prepared from E17 ICR mice. Dissociated single

    cells were plated in a solution containing RF media, DMEM with 10% FBS, 1

    penicillin/streptomycin, 1.4 mM L-glutamine, and 0.6% glucose, in 12-well plates for 1 day.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    10/44

    9

    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).

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    11/44

    10

    2.8 CORT and ACTH measurements

    Blood samples were centrifuged at 1000 gfor 15 min to obtain serum. CORT and ACTH

    levels were measured from serum using CORT enzyme immunoassay kits (Cayman Chemical,

    Ann Arbor, MI, USA), ACTH ELISA kit (Sigma-Aldrich, MO. USA).

    2.9 RT-PCR

    Total RNA was extracted using Trizol reagent kit (Invitrogen, CA, USA). The RNA was

    reverse transcribed with reverse transcriptase and a random hexamer primer (Promega, CA,

    USA). cDNA was amplified with the following sense and antisense primers (53): for

    BDNF sense 5-TGG CTG ACA CTT TTG AGC AC-3and antisense 5-GTT TGC GGC

    ATC CAG GTA AT-3; for synaptophysin sense, 5-TAA CCC GAG TAA GAA TGT C-3

    and antisense 5-CCC TAC ATT CAC CCA CTT CTC C-3; for PSD-95 sense 5-TGC ACT

    CTT GAT GTA TCA GC-3 and antisense 5-ACG GAT GAA GAT GGC GAT AG-3;for

    GAPDH for sense 5-TCC ATG ACA ACT TTG GCA TT-3 and antisense 5-GTT GCT

    GTT GAA GTC GCA GG-3. PCR products were analyzed on 1.5% agarose gel, and the

    intensity of each band was measured using Image J (NIH Image Engineering, Bethesda, MD).

    2.10 Statistical analysis

    Statistical analysis was performed with SPSS (SPSS for Windows, version 18.0, Chicago, IL,

    USA), using one-way ANOVA, two-way repeated measured ANOVA, and independent t-tests

    to assess for significance. Post-hoc comparisons were made using Newman-Keuls tests. All

    values are reported as mean standard error (SE). Statistical significance was set at p < 0.05.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    12/44

    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

    0.05; 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 Y-

    maze 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

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    13/44

    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).

    3.2 The expression of BDNF, phospho-AMPK, and synaptic proteins such as

    synaptophysin and PSD-95 were reduced by chronic restraint, and this change was

    counter-regulated by regular and prolonged treadmill running.

    Chronic stress in the form of repeated restraint was found to down-regulate hippocampal

    mature BDNF and phospho-AMPK expression. This effect was reversed by regular and

    prolonged treadmill running (Fig. 4A; for BDNF F2, 21= 70.90, p < 0.01, for phospho-AMPK

    F2, 21 = 85.30, p < 0.01). Additionally, the expression of two synaptic proteins, synaptophysin

    (a presynaptic marker) and PSD-95 (a postsynaptic marker), was reduced by chronic stress,

    which was similarly counter-regulated by regular and prolonged exercise (Fig. 4B; for

    synaptophysin F2, 21= 184.06, p < 0.01; for PSD-95 F2, 21 = 49.59, p < 0.01).

    3.3 Chronic stress enhanced serum corticosterone (CORT) and adrenocorticotrophic

    hormone (ACTH)

    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; for

    CORT, t8= -4.74, p < 0.01; for ACTH, t8= -3.70, p < 0.01). Regular exercise didnt alter

    basal levels of both CORT and ACTH (Fig. 5B; for CORT, t8= 0.17, p > 0.05; for ACTH, t8=

    -0.24, p > 0.05).

    3.4 AICAR treatment enhanced BDNF expression, and Compound C treatment reduced

    BDNF expression in the primary hippocampal culture.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    14/44

    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 phospho-

    AMPK F2, 9 = 165.42, p < 0.01). Such effects were suppressed by prior exposure to

    Compound C (Fig. 6B; for BDNF F4, 15 = 88.11, p < 0.01; for phospho-AMPK F4, 15 = 106.84,

    p < 0.01).

    3.5 AICAR treatment induced hippocampal BDNF and phospho-AMPK expression,

    enhanced alternation in the Y-maze test, and reduced escape latency.

    Since hippocampal AMPK activity mediated the induction of mature BDNF expression in

    vitro, the ensuing experiments explored whether AICAR would exert a similar effect on

    memory function in vivo (Fig. 7A). Seven consecutive days of AICAR treatment enhanced

    the alternation rate in the Y-maze test up to 2 weeks after the last treatment (Fig. 7Ac; t14 = -

    3.68, p < 0.05). Similarly, AICAR treatment up regulated the expression of hippocampal

    mature BDNF and phospho-AMPK (Fig. 7Ab; t6= -11.71, p < 0.01). Also, in water maze test,

    the escape latency decreased by ALCAR treatment on day 4-5 (Fig. 7B; the interaction effect

    of group x day F4, 72 = 1.74, p > 0.05; the main effect of group F1, 18 = 18092.12, p < 0.01; the

    main effect of day F4, 72 = 158.97, p < 0.01). 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; the interaction effect

    of group x day F12, 144 = 0.93, p > 0.05; the main effect of group F1, 36 = 40774.08, p < 0.01;

    the main effect of day F4, 144 = 333.89, p < 0.01).

    3.6 Compound C treatment during exercise intervention blocked exercise-induced

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    15/44

    14

    memory improvement in mice subjected to restraint.

    We investigated whether AMPK inactivation by treatment with Compound C blocked

    exercise-induced memory improvement in stressed mice. Whilst exercise reduced the escape

    latency of the water maze task in stressed mice, this effect was blocked by Compound C

    treatment (Fig. 9; the interaction effect of group x day F12,144 = 9.48, p < 0.01; the main effect

    of group F1, 36 = 35766.26, p < 0.01; the main effect of day F4, 144 = 245.29, p < 0.01).

    3.7 Compound C treatment during exercise intervention suppressed exercise-produced

    hippocampal neurogenesis enhancement in mice subjected to restraint.

    We investigated whether AMPK inactivation by treatment with Compound C suppressed

    exercise-produced enhancement of Ki-67-and doublecortin-positive cells in chronically

    stressed mice. Compound C treatment reduced exercise-induced increase in hippocampal Ki-

    67- and doublecortin-positive cells in chronically restrained mice (Fig. 10; for Ki-67 F3, 28 =

    16.86, p < 0.01; for doublecortin F3, 28 = 16.86, p < 0.01).

    3.8 Compound C treatment during exercise intervention blocked exercise-induced

    memory improvement in mice subjected to restraint.

    We investigated whether AMPK inactivation by treatment with Compound C suppressed

    exercise-produced enhancement of BDNF, synaptophysin, and PSD-95 mRNA in chronically

    stressed hippocampus (Fig. 11). Compound C treatment reduced exercise-induced increase in

    hippocampal BDNF, synaptophysin, and PSD-95 mRNA levels in chronically restrained mice

    (Fig. 7; for BDNF F3, 16 = 62.97, p < 0.01; for synaptophysin F3, 16 = 63.21, p < 0.01; for PSD-

    95 F3, 16 = 57.61, p < 0.01).

    4 DISCUSSION

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    16/44

    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).

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    17/44

    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 PSD-

    95 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

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    18/44

    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 hippocampal energy metabolism 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 stress-

    induced 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 hypothalamic-

    pituitary-adrenal (HPX) axis activation, which the persisted increase in CORT may affect

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    19/44

    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.,

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    20/44

    19

    2011; Xu et al., 2013). AMPK activation suppresses 3-hydroxy-3-methylgutaryl coenzyme A

    reductase (HMG-CoA reductase) and acetyl-CoA carboxylase, thereby restoring ATP levels

    by reducing fatty acid and cholesterol synthesis (Hardie, 2003). More recently, a study

    demonstrated that statin, a selective inhibitor for HMG-CoA reductase, facilitates CREB-

    mediated BDNF induction via its binding to PPAR, and therefore causes an improvement in

    memory function (Roy et al., 2015). These articles support the results of this study,

    suggesting an important role for hippocampal AMPK-mediated BDNF induction in the

    improvement of memory function. This suggests that regular and prolonged exercise induces

    hippocampal AMPK activation and exerts a neurotrophic effect on surrounding tissue,

    thereby promoting memory function in chronically stressed mice. To further explore the role

    of hippocampal AMPK with regard to exercise-induced changes in memory function, mice

    were intraperitoneally administrated with Compound C, an AMPK antagonist during

    treadmill running in chronically stressed mice (Fig. 9A). Enhanced memory in exercised mice

    relative to control mice was reversed by Compound C treatment (Fig. 9B). Furthermore, to

    investigate the role of AMPK in hippocampal neurogenesis as well as BDNF, synaptophysin,

    and PSD-95 mRNA levels in chronically stressed mice, we measured hippocampal

    neurogenesis (Ki-67: a proliferating marker, doublecortin: a differentiating marker) and the

    mRNA levels of BDNF, synaptophysin, and PSD-95 in hippocampus (Fig. 10-11). Chronic

    stress-produced decrease in Ki-67- and doublecortin-positive cells, BDNF, synaptophysin,

    and PSD-95 mRNA levels were reversed by exercise intervention. However, exercise-exerted

    these changes were suppressed by Compound C treatment. This result suggests that AMPK

    activity may contribute to hippocampal neurogenesis as well as BDNF, synaptophysin, PSD-

    95 expression in hippocampus of chronically restrained mice, which exercise-modulated

    hippocampal AMPK activity may play crucial role in hippocampal neurogenesis and the

    expression of BDNF and synaptic proteins. Collectively, pharmacological manipulation of

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    21/44

    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

    The authors declare no financial and non-financial competing interests.

    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

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    22/44

    21

    (2014) AMPK activation prevents prenatal stress-induced cognitive impairment: Modulation

    of mitochondrial content and oxidative stress. Free Rad Biol Med 75:156-166.

    Conner JM, Lauterborn JC, Yan Q, Gall CM, Varon S (1997) Distribution of brain-derived

    neurotrophic factor (BDNF) protein and mRNA in the normal adult rat CNS: evidence for

    anterograde axonal transport. J Neurosci 17: 22952313.

    Culmsee C, Monnig J, Kemp BE, Mattson MP (2001) AMP-activated protein kinase is highly

    expressed in neurons in the developing rat brain and promotes neuronal survival following

    glucose deprivation. J Mol Neurosci 17:45-58.

    Dagon Y, Avraham Y, Magen I, Gertler A, Ben-Hur T, Berry EM (2005) Nutritional status,

    cognition, and survival: A new role for leptin and AMP kinase. J Biol Chem 280(51):42142-

    42148.

    Duman RS, Monteggia LM (2006) A neurotrophic model for stress-related mood disorders.

    Biol Psychiatry 59:11161127.

    Eadie BD, Redila VA, Christie BR (2005) Voluntary exercise alters the cytoarchitecture of the

    adult dentate gyrus by increasing cellular proliferation, dendritic complexity, and spine

    density. J Comp Neurol 486:39-47.

    El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS (2000) PSD-95

    involvement in maturation of exciatory synapses. Science 290:134-1368.

    Gomez-Pinilla F, Vaynmas S, Ying Z (2008) Brain-derived neurotrophic factor functions as a

    metabotrophin to mediate the effects of exercise on cognition. Eur J Neurosci 28:2278-2287.

    Hadad SM, Fleming S, Thompson AM (2008) Targeting AMPK: a new therapheutic

    opportunity in breast cancer. Crit Rev Oncol Hematol 67:1-7.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    23/44

    22

    Han TK, Lee JK, Leem YH (2015) Chronic exercise prevents repeated restraint stress-

    provoked 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 455:894-

    902.

    KwonD, 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

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    24/44

    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.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    25/44

    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 PPARto 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,

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    26/44

    25

    Carling D (2005) Ca2+/calmodulin-dependent protein kinase-beta acts upstream of AMP-

    activated 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

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    27/44

    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, t 12= -2.82, p 0.05).

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    28/44

    27

    Data are presented as the mean SEM.

    Data are presented as the mean SEM. * and ** denote differences at p < 0.05 and p < 0.01,

    respectively.

    Figure 3. Regular and prolonged treadmill running ameliorated stress-induced learning

    and memory impairment.

    A. Experimental design

    B. Quantitative analysis of Y-maze and water maze test data

    a. Quantitative analysis of Y-maze test data.

    b. Quantitative analysis of the escape latency of water maze test.

    c. Photomicrograph showing swimming patterns on the final day of water maze testing.

    Data are presented as the mean SEM. ** denote differences at p < 0.01, and denote

    difference at p < 0.01 from CON.

    Figure 4. Regular and prolonged treadmill running reversed chronic stress-elicited

    reduction of hippocampal phospho-AMPK, BDNF, synaptophysin and PSD-95 levels.

    A. Quantitative analysis of hippocampal phospho-AMPK and BDNF.

    a. Photomicrograph showing western blot data for AMPK and BDNF.

    b. Quantitative analysis of phospho-AMPK and BDNF.

    B. Quantitative analysis of hippocampal phospho-AMPK and BDNF.

    a. Photomicrograph showing western blot data for synaptophysin and PSD-95.

    b. Quantitative analysis for synaptophysin and PSD-95.

    Data are presented as the mean SEM. ** denote differences at p < 0.01.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    29/44

    28

    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.

    Figure 6. AICAR treatment upregulated phospho-AMPK and BDNF, which was

    reversed by Compound C in hippocampal primary culture.

    A. Quantitative analysis of hippocampal phospho-AMPK and BDNF in hippocampal primary

    culture treated with AICAR.

    a. Photomicrograph showing western blot data for AMPK and BDNF.

    b. Quantitative analysis of phospho-AMPK and BDNF.

    B. Quantitative analysis of hippocampal phospho-AMPK and BDNF in hippocampal primary

    culture treated with AICAR or/and Compound C.

    a. Photomicrograph showing western blot data for AMPK and BDNF.

    b. Quantitative analysis of phospho-AMPK and BDNF.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    30/44

    29

    Data are presented as the mean SEM. * and ** denote differences at p < 0.05 and p < 0.01,

    respectively.

    Figure 7. Intraperitoneal injection of AICAR enhanced alternation in the Y-maze test,

    concomitant with hippocampal phospho-AMPK and BDNF levels.

    A. Quantitative analysis of hippocampal phospho-AMPK and BDNF levels, and Y-maze test

    data

    a. Experimental design

    b. Photomicrograph showing western blot data and quantitative analysis for hippocampal

    phospho-AMPK and BDNF in mice treated with AICAR.

    c. Quantitative analysis of Y-maze test data.

    B. Quantitative analysis of water maze data

    a. Experimental design

    b. Quantitative analysis of water maze test

    Data are presented as the mean SEM. * and ** denote differences at p < 0.05 and p < 0.01,

    respectively. denote difference from 0 mg/kg at p < 0.01 from 0 mg/kg.

    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.

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    31/44

    30

    Data are presented as the mean SEM. ** denote differences from 0 mg/ kg at p < 0.01.

    Figure 9. Compound C treatment during exercise intervention blocked exercise-induced

    memory improvement in mice subjected to restraint.

    A. Experimental design

    B. Quantitative analysis of water maze test data

    a. Quantitative analysis of escape latency of water maze test.

    b. Photomicrograph showing swimming patterns on the final day of water maze testing.

    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 exercise-

    induced 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 exercise-

    induced enhancement of BDNF, synaptophysin, and PSD-95 mRNA in mice subjected to

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    32/44

  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    33/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032289&guid=ccc2e5e2-9fec-4b1e-b5b9-0180559ed8fc&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    34/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032290&guid=93abb7ce-c869-45c3-a1e1-ec07eaa3994f&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    35/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032291&guid=8755aa32-40ef-45e7-98aa-5cfcfd295b28&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    36/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032292&guid=df315305-a343-4f92-9f0c-18f1a72acea1&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    37/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032293&guid=73ce3484-d615-42b6-9b20-d9d3bc482b61&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    38/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032294&guid=e1d9509b-0f77-4f0d-b3e4-4c2681558687&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    39/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032295&guid=1ad10424-6e99-4fd5-b8f4-256344fbdd7c&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    40/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032296&guid=fd156011-d608-407c-8f11-37697b88fcfe&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    41/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032297&guid=84404582-3c46-41da-8655-50730c010fbd&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    42/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032298&guid=08122e49-a1a8-4f3b-b423-17d825026908&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    43/44

    http://ees.elsevier.com/nsc/download.aspx?id=1032299&guid=4dc40e17-1366-4464-bf8b-469a3273f9be&scheme=1
  • 7/26/2019 Chronic Stress-Induced Memory Deficits is Reversed by Regular Exercise via AMPK-mediated BDNF Induction

    44/44

    32

    RESEARCH HIGHLIGHTS

    Chronic stress causes hippocampal-dependent memory deficit.

    Exercise improves memory in chronically stressed mice.

    Chronic stress reduces hippocampal AMPK activity, BDNF, and neurogenesis.

    Exercise enhances hippocampal AMPK activity in chronically stressed mice.

    AMPK is required for exercise-exerted memory and neurogenesis enhancement.