J Appl Physiol 113: 1260–1266, 2012.
First published August 30, 2012; doi:10.1152/japplphysiol.00869.2012.
Voluntary resistance running with short distance enhances spatial memory
related to hippocampal BDNF signaling
Min Chul Lee,1,2 Masahiro Okamoto,1,2 Yu Fan Liu,1 Koshiro Inoue,1 Takashi Matsui,1 Haruo Nogami,3
and Hideaki Soya1
1
Laboratory of Exercise Biochemistry and Neuroendocrinology, Institute for Health and Sports Science, University of
Tsukuba, Tsukuba, Japan, 2Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan; and
3
Laboratory of Neuroendocrinology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan
Submitted 13 July 2012; accepted in final form 24 August 2012
resistance exercise; energy expenditure; cognitive functions; brainderived neurotrophic factor; hippocampus
effects on our brains and
brawn, not only with regard to muscular metabolic efficiency
but also with regard to our brain and mental functions. In
particular, a number of studies have shown that voluntary
exercise enhances neuronal plasticity (46), reduces depression
and anxiety (4), and improves cognitive functions (45). Voluntary exercise-enhanced cognitive functions have been shown
to be associated with synaptic plasticity and altered gene
expression in the hippocampus (13, 47), which suggests that
exercise-induced synaptic plasticity is crucial for hippocampaldependent learning and memory. Although previous studies
PHYSICAL EXERCISE HAS BENEFICIAL
Address for reprint requests and other correspondence: H. Soya, Laboratory
of Exercise Biochemistry & Neuroendocrinology, Inst. for Health & Sports
Sciences, Univ. of Tsukuba, Tennodai, Tsukuba 305-8574, Ibaraki, Japan
(e-mail: [email protected]).
1260
have indicated that hippocampal plasticity changes with voluntary exercise, the optimal voluntary exercise condition for
generating beneficial effects in cognitive function is still poorly
understood.
Indeed, voluntary wheel running is commonly used to enhance hippocampal plasticity, but with this type of exercise,
only the running distance can be evaluated. Previous studies
have shown that hippocampal brain-derived neurotrophic factor (BDNF) levels are positively correlated with wheel running
distance (22, 32). In contrast, other studies reveal no such
correlations between running distance and BDNF expression
(17, 37). It has been suggested that these differences are
probably related to exercise-induced stress levels. Physical
exercise is known to activate the hypothalamus-pituitary-adrenal (HPA) axis depending on intensity, duration, and wheel
running distance (12, 14, 33, 39). Also, severe stress suppresses hippocampal BDNF expression (40, 42). Thus it is
necessary to find a more suitable determinant for exerciseenhanced hippocampal plasticity than running distance.
Voluntary resistance wheel running (RWR) allows for a
given load on a running wheel and increases work levels,
which results in muscular adaptation in fast-twitch muscle
without using physical and psychological stressors such as
electrical shock and additional weight (3, 21, 26). Therefore,
RWR is a suitable model that integrates the effects of exercise
on both muscular and hippocampal adaptations through increased neuronal activation. However, whether voluntary
wheel running with additional resistance has potential effects
on hippocampal-dependent cognitive functions with synaptic
plasticity-related molecules has never been tested.
The mechanisms that underlie exercise-enhanced cognitive
functions are regulated by several types of molecular signaling,
such as BDNF (47) and insulin-like growth factor 1 (IGF-1)
(5). Particularly, BDNF is a key protein supporting the growth,
development, and survival of neurons (25, 35), and it also plays
an important role in mediating cognitive functions (47) and
synaptic plasticity (36). Physical exercise increases BDNF
mRNA and protein (32, 43), and BDNF engages its highaffinity receptor tyrosine-related kinase B (TrkB) (1) to activate its signaling-related molecules such as protein kinase A
(PKA) (8), protein kinase C (PKC) (30), mitogen-activated
protein kinase (MAPK) (48), and transcriptional regulator
cAMP response element-binding protein (CREB) (16). In addition, activation of BDNF signaling may mediate the postsynaptic N-methyl-D-asparate receptor (NMDA-R) (44). This evidence suggests that exercise may mediate the potentiation of
synaptic transmission and synaptic plasticity by employing
BDNF signaling. Particularly, alteration in BDNF signaling is
8750-7587/12 Copyright © 2012 the American Physiological Society
http://www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
Lee MC, Okamoto M, Liu YF, Inoue K, Matsui T, Nogami H,
Soya H. Voluntary resistance running with short distance enhances
spatial memory related to hippocampal BDNF signaling. J Appl
Physiol 113: 1260 –1266, 2012. First published August 30, 2012;
doi:10.1152/japplphysiol.00869.2012.—Although voluntary running
has beneficial effects on hippocampal cognitive functions if done
abundantly, it is still uncertain whether resistance running would be
the same. For this purpose, voluntary resistance wheel running (RWR)
with a load is a suitable model, since it allows increased work levels
and resultant muscular adaptation in fast-twitch muscle. Here, we
examined whether RWR would have potential effects on hippocampal
cognitive functions with enhanced hippocampal brain-derived neurotrophic factor (BDNF), as does wheel running without a load (WR).
Ten-week-old male Wistar rats were assigned randomly to sedentary
(Sed), WR, and RWR (to a maximum load of 30% of body weight)
groups for 4 wk. We found that in RWR, work levels increased with
load, but running distance decreased by about half, which elicited
muscular adaptation for fast-twitch plantaris muscle without causing
any negative stress effects. Both RWR and WR led to improved
spatial learning and memory as well as gene expressions of hippocampal BDNF signaling-related molecules. RWR increased hippocampal
BDNF, tyrosine-related kinase B (TrkB), and cAMP response element-binding (CREB) protein levels, whereas WR increased only
BDNF. With both exercise groups, there were correlations between
spatial memory and BDNF protein (r ⫽ 0.41), p-CREB protein (r ⫽
0.44), and work levels (r ⫽ 0.77). These results suggest that RWR
plays a beneficial role in hippocampus-related cognitive functions
associated with hippocampal BDNF signaling, even with short distances, and that work levels rather than running distance are more
determinant of exercise-induced beneficial effects in wheel running
with and without a load.
Resistance Exercise-Enhanced Cognitions With BDNF Signaling
apparently necessary for the effects of exercise on hippocampal plasticity in rodents, since blocking BDNF signaling
inhibits exercise-enhanced learning and memory (47, 48),
whereas blocking IGF-1 signaling does not (11). These
findings lead us to postulate that BDNF signaling, rather
than other molecules, may play a role as a significant
regulator in exercise-enhanced cognitive function.
In the present study, we investigated whether RWR has
potential effects on hippocampal cognitive functions similar to
those of WR and associated such effects with the possible
mechanisms underlying hippocampal BDNF signaling.
MATERIALS AND METHODS
Lee MC et al.
1261
interference, began. Each rat was given four trials per day for 4
consecutive days. At the start of a trial, the rats were placed into the
tank facing the wall from one of four equally spaced start locations,
and each release point was randomly altered every trial. Each trial
lasted until the rat found the platform or for a maximum duration of
60 s. If a rat did not find the platform within 60 s, it was gently guided
to the platform. At the end of each trial, animals were allowed to rest
on the platform for 10 s. Escape latency (s) and path length (cm) for
each trial were recorded. For the spatial probe test, which was
performed 1 day after the last acquisition trial, the platform was
removed, and the rats were allowed to swim for 60 s. The percentage
of time spent in each quadrant was recorded by a video tracking
system (SMART, Polyvalent video-tracking system, Panlab, Barcelona, Spain).
Isolation of RNA and quantitative real-time RT-PCR. Between
0800 and 1200 on the day after the final day of exercise, the animals
were immediately decapitated using a guillotine, and their hippocampi
were rapidly dissected, snap frozen in liquid nitrogen, and stored at
⫺80°C before use. Total RNA was extracted by the method described
previously (7), and RNA concentration was determined by spectrophotometry at 260 nm. The cDNA synthesis was carried out using
murine leukemia virus reverse transcriptase (Applied Biosystems,
Foster City, CA) and random hexamer primer sets (Takara Shuzo,
Shiga, Japan) following the manufacturer’s protocol. Gene expression
levels of BDNF signaling-related molecules were determined by
quantitative real-time PCR (PE-ABI Prism 7300, Applied Biosystems) using a fast-start universal SYBR green master mix (Roche
Applied Science, Mannheim, Germany) following the protocol provided by the manufacturer. Primer 3 software (38) was used to design
reaction primers, and the sequences were as follows: BDNF forward,
5=-gcggcagataaaaagactgc-3=; BDNF reverse, 5=-gccagccaattctctttttg-3=
(NCBI accession no. NM-012513); CREB forward, 5=-tcagccgggtactaccattc-3=; CREB reverse, 5=-cctctctctttcgtgctgct-3= (NCBI accession
no. X14788.1); TrkB forward, 5=-gacctgatcctgacgggtaa-3=; TrkB reverse, 5=-tggtcacagacttcccttcc-3= (NCBI accession no. NM-001163169);
NMDA-R forward, 5=-tcattgtcctcagctgttgc-3=; NMDA-R reverse, 5=tgcacgaggtgaagtcgtag-3= (NCBI accession no. S61973.1); PKA forward,
5=-ttcttcgctgaccagcctat-3=; PKA reverse, 5=-tgaccccgttcttgagattc-3= (NCBI
accession no. NM-001100922); PKC forward, 5=-tccctgatcccaaaagtgag-3=; PKC reverse, 5=-aacttgaaccagccatccac-3= (NCBI accession no.
NM-012713); MAPK forward, 5=-cctacggcatggtttgttct-3=; MAPK reverse, 5=-tgccgatgatgttctcatgt -3= (NCBI accession no. NM-053842);
␤-actin forward, 5=-aaccctaaggccaaccgtga-3=; ␤-actin reverse, 5=cagggacaacacagcctgga-3= (NCBI accession no. NM-031144). In brief,
after 10 min of incubation at 95°C, the PCR reaction was carried out
for 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. As
a validated endogenous control, ␤-actin was amplified in a separate
reaction for normalization. Each sample was processed in duplicate,
and melting curve analysis was performed on all samples.
Western blot analysis. The hippocampi were homogenized in RIPA
buffer [50 mM Tris·HCl buffer (pH 7.6); 150 mM NaCl; 0.5% sodium
deoxy cholate; 1% Nonidet P40; 0.1% SDS; and protease inhibitors
cocktails]. Homogenates were centrifuged to remove insoluble material (10,000 g for 10 min at 4°C), and total protein concentration was
determined as per the BCA procedure (Pierce, Rockford, IL). The
proteins (20 ␮g) were heated to 70°C for 10 min in the presence of
dithiothreitol, rapidly cooled on ice for 1 min, and then separated by
electrophoresis on a 12.5% polyacrylamide gel for 110 min. The gel
was then transferred to a PVDF membrane (Immuno-Blot, Amersham
Pharmacia Biotech, Piscataway, NJ) for 1 h at a constant voltage of 15
V. The membranes were blocked with 5% nonfat skim milk for 1 h
and then separately incubated overnight at room temperature with one
of the following primary antibodies: anti-BDNF (sc-546) 1:1,000
(Santa Cruz Biotechnology, Santa Cruz, CA); anti-CREB (sc-186)
and anti-p-CREB (sc-7978) 1:250 (Santa Cruz Biotechnology). The
membranes were then washed with washing buffer, after which they
were incubated with secondary antibodies, horseradish peroxidase
J Appl Physiol • doi:10.1152/japplphysiol.00869.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
Animals. Ten-week-old male Wistar rats (320 –340 g; SEAS,
Saitama, Japan) were randomly allocated to three groups: 1) housed in
standard cages and used as nonactive controls (Sed), 2) wheel running
with no resistance (WR), and 3) housed in cages with resistance
running wheels with an adjustable resistance (RWR). The rats of each
group were designated for analysis of exercise performance, muscle
adaptations, and gene expression of mRNA levels (n ⫽ 7 rats/group)
or for a behavioral test of cognitive function and analysis of protein
levels (n ⫽ 7–10 rats/group). All rats were individually housed, kept
in a controlled environment with a 12-h light-dark cycle (lights on at
8:00 AM), and given ad libitum access to food and water. All the
experiments were performed in accordance with protocols approved
by the University of Tsukuba Animal Experiment Committee based
on the NIH Guidelines for the Care and Use of Laboratory Animals
(NIH publication, 1996).
Running-wheel and loading protocol. Sedentary rats were housed
individually in standard polyethylene cages without access to a
running wheel. The rats of both running wheel groups (WR and RWR)
were housed individually and had free access to a specially designed
running wheel apparatus (diameter ⫽ 31.8 cm, width ⫽ 10 cm; Rat
Analyzer KI-103, Aptec, Kyoto, Japan). The resistance attached to the
wheel could be changed arbitrarily with a range of 0 –200 g. The
resistance necessary to overcome the inertia of the wheel at its
minimum load was 4.5 g. This RWR apparatus and its setup for
resistance running were similar to those previously described (21). In
the present study, the rats were allowed to run voluntarily in the wheel
24 h/day. The rats in the RWR group were exercised with minimum
resistance (i.e., 4.5 g) for the first week, and then the resistance was
progressively increased to reach 30% of their body weight during the
4 wk of exercise. A resistance of 30% of body weight in wheel
running is sufficient to produce muscular adaptation similar to that of
other exercises performed at 60% of body weight (23). Daily work
levels (J) as total energy expenditure during exercise were calculated
and expressed relative to body weight and day as follows: work ⫽
force (N) ⫻ distance (m)/body wt (kg)/day, where force is the
resistance of the wheel, and distance is the number of revolutions
times the circumference of the wheel.
Morris water maze task. To evaluate the effects of resistance wheel
exercise on spatial learning and memory, rats were tested on the
Morris water maze (MWM) (31). The circular swimming pool
(150-cm diameter, 45-cm height) was filled with water to a depth of
22.5 cm and divided into four equal quadrants. A hidden escape
platform (12-cm diameter, 21-cm height) was fixed in a permanent
position 1.5 cm below the surface of the water. The water was kept at
a steady 25 ⫾ 1°C and was made opaque with nontoxic white poster
color to prevent the rats from seeing the platform. Spatial cues of
different patterns and sizes were affixed to each of the testing room
walls to provide sufficient spatial information for the animals to learn
the location of the escape platform.
The animals were acclimatized to the experimental room for at
least 1 h before the experimental procedures, which were carried out
between 1300 and 1600 to avoid circadian variations and stress
•
1262
Resistance Exercise-Enhanced Cognitions With BDNF Signaling
•
Lee MC et al.
Fig. 1. Effects of resistance wheel running (RWR) on exercise performance. A: average daily running distance is shown for rats in wheel running without a load
(WR) and RWR groups. B: the average running distance of the RWR group significantly decreased compared with the WR group. C: average daily work levels
are shown for rats in WR and RWR groups. D: the average work levels significantly increased by ⬃8.5-fold in the RWR group compared with the WR group.
All data are presented as means ⫾ SE (n ⫽ 7 rats/group). Significant difference compared with WR group after repeated-measures, two-way ANOVA,
Bonferroni’s multiple comparison tests, or Student’s t-test: *P ⬍ 0.05; ***P ⬍ 0.0001.
parison tests for post hoc analysis. Water maze test escape latency and
path-length data were analyzed with a repeated-measures, two-way
ANOVA, followed by Bonferroni’s multiple comparison tests for post
hoc analysis, whereas muscle mass, CS activity, stress-related factors,
water maze probe test, real-time RT-PCR, and Western blot data were
analyzed with a one-way ANOVA followed by Tukey’s multiple
comparison tests for post hoc analysis. Relationships between spatial
memory performance and work levels, running distance, BDNF protein, and p-CREB protein were examined using Pearson’s correlation
analysis. Statistical significant difference was evaluated at P ⬍ 0.05.
RESULTS
Effects of RWR on exercise performance and muscular
adaptation. Average running distance of the RWR group
(1,024 m/day) decreased to ⬃55% of that of the WR group
(1,846 m/day) (P ⬍ 0.05; Fig. 1, A and B), whereas average
work levels significantly increased by ⬃8.5-fold in the RWR
group (1,805 N·m·kg body wt⫺1·day⫺1) compared with the
WR group (210.8 N·m·kg body wt⫺1·day⫺1) (P ⬍ 0.0001; Fig.
1, C and D). Hindlimb skeletal muscle mass and CS activity
were analyzed to determine the effect of RWR on muscular
adaptation. Relative plantaris muscle wet mass had increased
in both the WR and RWR groups compared with the Sed group
[F(2,18) ⫽ 11.98, P ⬍ 0.001; Table 1]. The soleus muscle CS
activity significantly increased in WR and RWR groups compared with the Sed group [F(2,18) ⫽ 5.668, P ⬍ 0.05; Table
1], whereas the plantaris muscle CS activity significantly
Table 1. Effects of RWR on muscular adaptation and stress-related parameters
Group
Body weight, g
Muscle mass and CS activity
Relative soleus wet mass to BW, mg/100 g
Relative plantaris wet mass to BW, mg/100 g
Soleus CS activity, ␮mol · ml⫺1 · min⫺1
Plantaris CS activity, ␮mol · ml⫺1 · min⫺1
Stress-related factors
Relative adrenals Wt to BW, mg/100 g
Relative thymus Wt to BW, mg/100 g
Plasma corticosterone, ng/ml
Sed
WR
RWR
499.4 ⫾ 9.6
447.1 ⫾ 5.6**
444.9 ⫾ 10.7**
41.6 ⫾ 1.4
92.6 ⫾ 2.5
1.6 ⫾ 0.5
1.7 ⫾ 0.5
46.0 ⫾ 1.9
100.4 ⫾ 2.4*
3.0 ⫾ 0.1*
2.7 ⫾ 0.4
45.6 ⫾ 1.5
104.3 ⫾ 1.0**
2.8 ⫾ 0.1*
3.5 ⫾ 0.2*
13.9 ⫾ 0.6
106.4 ⫾ 6.0
23.9 ⫾ 9.5
13.8 ⫾ 0.5
120.5 ⫾ 11.4
38.5 ⫾ 6.6
14.1 ⫾ 0.3
110.3 ⫾ 7.4
23.1 ⫾ 5.6
Data are means ⫾ SE (n ⫽ 7 rats/group). BW, body weight; CS, citrate synthase; Wt, weight; Sed, sedentary; WR, wheel running with no resistance; RWR,
resistance wheel running. Significant difference compared with sedentary after an ANOVA with Tukey’s multiple comparison tests: *P ⬍ 0.05; **P ⬍ 0.01.
J Appl Physiol • doi:10.1152/japplphysiol.00869.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
(HRP)-conjugated goat anti-rabbit for BDNF and CREB (dilution:
1:5,000). HRP-conjugated cat anti-goat was used for p-CREB (dilution: 1:5,000). HRP-conjugated goat anti-mouse was used for ␤-actin
(dilution: 1:10,000). Immunocomplexes were made visible using
chemiluminescence with the ECL kit (Nakalai Tesque, Kyoto, Japan)
following the manufacturer’s instructions. The signals were digitally
scanned and then quantified using image analysis software (GE
Healthcare, Bio-Science). The band intensity of the protein of interest
was normalized to the band intensity of ␤-actin and total CREB,
respectively.
Measurement of skeletal muscle aerobic capacity. An increase in
citrate synthase (CS) activity, which reflects an improved aerobic
metabolism in mitochondria of skeletal muscle, is commonly used to
confirm exercise-training effects. Therefore, the CS activity in the
plantaris and soleus muscles was measured using a method described
previously (41). The CS activity in muscles was determined in
duplicate using the CS Assay Kit (Sigma, St. Louis, MS) following
the manufacturer’s instructions.
Radioimmunoassay. The plasma corticosterone levels were determined by the RIA method (28) by using [3H]corticosterone as a tracer.
In brief, plasma was separated from trunk blood by centrifugation at
3,000 rpm for 10 min. Plasma samples were then stored at ⫺80°C
before analysis. Samples and corticosterone standards were thawed at
room temperature and added to antibody-coated tubes in triplicate.
Then, 1.0 ml of [3H]-labeled corticosterone was added and mixed in
each tube before incubating for 2 h at room temperature. Samples
were then decanted and counted using a gamma counter.
Statistical analysis. The average running distance and work levels
for each of the experimental days were analyzed with repeatedmeasures, two-way ANOVA, followed by Bonferroni’s multiple com-
Resistance Exercise-Enhanced Cognitions With BDNF Signaling
•
Lee MC et al.
1263
Fig. 2. Effects of RWR on spatial learning and memory. Both WR and RWR groups presented faster escape latency (A) and shorter path-length values (B) in
the Morris water maze (MWM) task, demonstrating improved spatial learning performance compared with the sedentary (Sed) group (*P ⬍ 0.05, Sed vs. RWR;
#P ⬍ 0.05, Sed vs. WR; repeated-measures two-way ANOVA, Bonferroni’s multiple comparison tests). C: representative samples of trials traveled during the
probe test. RWR increased memory retention for the MWM task as indicated by the finding that RWR group rats spent significantly more time in the platform
(P) quadrant than did Sed group rats. All data are presented as means ⫾ SE (n ⫽ 8 –10 / group). *Significant difference compared with sedentary after an ANOVA
with Tukey’s multiple comparison tests (P ⬍ 0.05).
preference for the platform quadrant compared with the Sed
group.
Effects of RWR on the gene expression of BDNF signalingrelated molecules. To investigate the possible mechanisms
underlying spatial learning and memory, the gene expression
of hippocampal BDNF signaling-related molecules were evaluated. Both RWR and WR groups significantly increased both
BDNF mRNA expression [F(2,18) ⫽ 3.77, P ⬍ 0.05; Fig. 3A]
and BDNF protein [F(2,23) ⫽ 5.841, P ⬍ 0.01; Fig. 4A]
compared with the Sed group. Also, TrkB mRNA was significantly increased in the RWR and WR groups [F(2,18) ⫽
5.027, P ⬍ 0.05; Fig. 3B]. TrkB protein was only increased in
the RWR group [F(2,21) ⫽ 4.842, P ⬍ 0.05; Fig. 4B]. CREB
mRNA expression [F(2,18) ⫽ 3.731, P ⬍ 0.05; Fig. 3A] and
p-CREB protein [F(2,23) ⫽ 5.666, P ⬍ 0.01; Fig. 4C] were
only increased in the RWR group. The WR group had a strong
trend toward increase in p-CREB protein levels, although there
were no significant differences among groups (P ⫽ 0.09; Fig.
4C). In addition, RWR and WR significantly increased the
mRNA levels of NMDA-R [F(2,18) ⫽ 9.945, P ⬍ 0.01; Fig.
3B] and PKC [F(2,18) ⫽ 11.63, P ⬍ 0.001; Fig. 3C], respectively, compared with the Sed group. PKA [F(2,18) ⫽ 2.783,
P ⫽ 0.08; Fig. 3C] and MAPK [F(2,18) ⫽ 2.44, P ⫽ 0.11; Fig.
3C] expression were only increased in the RWR group.
Relationship between spatial memory and BDNF, p-CREB,
and exercise parameters. We found that the percentage of time
spent in the platform quadrant during the probe test was
Fig. 3. Effects of RWR on the gene expression of signaling-related molecules. Both WR and RWR groups increased the BDNF (A), TrkB, and NMDA-R mRNA
(B), PKC mRNA expression (C). CREB (A), PKA, and MAPK (C) expression only increased in the RWR group. D: proposed mechanism of RWR-enhanced
hippocampal cognitive functions via BDNF signaling. Results are expressed as percentages of sedentary control levels and all data are presented as means ⫾
SE (n ⫽ 7 / group). *Significant difference compared with sedentary after an ANOVA with Tukey’s multiple comparison tests (P ⬍ 0.05).
J Appl Physiol • doi:10.1152/japplphysiol.00869.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
increased only in the RWR group [F(2,18) ⫽ 4.613, P ⬍ 0.05;
Table 1].
Effects of RWR on stress-related factors. Exercise affects
stress levels, which in turn could influence spatial learning,
memory, and BDNF signaling. Therefore, after completion of
exercise training, stress-related factors were measured. Our
results revealed no significant change in the relative weights of
adrenals [F(2,18) ⫽ 0.118, P ⫽ 0.88], thymus [F(2,18) ⫽
0.717, P ⫽ 0.50], and plasma corticosterone levels [F(2,18) ⫽
1.344, P ⫽ 0.28] among the three groups (Table 1).
Effects of RWR on spatial learning and memory. To access
spatial learning and memory, animals were tested in the Morris
water maze. Repeated measures with ANOVA showed significant group [F(2,101) ⫽ 10.5, P ⬍ 0.0001] and day [F(3,303) ⫽
54.4, P ⬍ 0.0001] effects on escape latency (Fig. 2A). The post
hoc analysis indicated that WR (day 3, P ⬍ 0.05) and RWR
(day 4, P ⬍ 0.05) groups had faster escape latency values
compared with the Sed group. The path length data analysis
also revealed significance for group [F(2,101) ⫽ 12.44, P ⬍
0.0001] and day [F(3,303) ⫽ 45.21, P ⬍ 0.0001] effects (Fig.
2B). The post hoc analysis indicated that the WR group had
shorter path length values compared with the Sed group at day
3 (P ⬍ 0.01). To test memory retention for the task, the
platform was removed for a 60-s probe test 1 day after the last
trial on day 4. The RWR group spent more time in the platform
quadrant than the Sed group [F(2,23) ⫽ 5.429, P ⬍ 0.05; Fig.
2C]. On the other hand, WR did not show a significant
1264
Resistance Exercise-Enhanced Cognitions With BDNF Signaling
•
Lee MC et al.
Fig. 4. Effects of RWR on the protein levels of BDNF, TrkB, and p-CREB. A: both WR and RWR increased BDNF protein levels. However, TrkB (B) and
p-CREB (C) protein were only increased in the RWR group. D: representative Western blot gels for group data shown in A, B, and C. Band intensity of each
protein was normalized for common ␤-actin levels. Results are expressed as percentages of sedentary control levels and all data are presented as means ⫾ SE
(n ⫽ 7–10 rats/group). *Significant difference compared with sedentary after an ANOVA with Tukey’s multiple comparison tests (P ⬍ 0.05).
DISCUSSION
To our knowledge, this is the first study to test the hypothesis
that voluntary resistance wheel running (RWR) has potential
effects on hippocampal cognitive functions and is associated with
the possible mechanisms underlying hippocampal BDNF signaling. Here, we found that RWR increased work levels with load but
decreased running distance by about half, which elicited muscular
adaptation for fast-twitch plantaris muscles without causing any
negative stress effects. Both RWR and WR led to improve spatial
learning and memory as well as increased gene expressions of
hippocampal BDNF signaling-related molecules. In addition, in
both exercise groups, there were positive correlations between
spatial memory and BDNF protein (r ⫽ 0.41), p-CREB protein (r
⫽ 0.44), and work levels (r ⫽ 0.77). These findings support the
hypothesis that voluntary resistance wheel running, even with
short distances, is beneficial for promoting hippocampal plasticity
associated with BDNF signaling and present a new concept that
work levels rather than running distance are more determinant for
inducing exercise-enhancing cognitive function with hippocampal
plastic changes in voluntary running with and without a load.
As expected, the performance of 4 wk of RWR elicited distinct
activity patterns that were affected by applying a load equal to
30% of body mass. Compared with the WR group, the average
daily running distance significantly decreased with resistance in
the RWR group (Fig. 1, A and B); however, the average work
levels of the RWR group were 8.5 times higher than those of the
WR group (Fig. 1, C and D). This is consistent with previous
studies with the same and similar loading systems (3, 21, 24),
suggesting that there are fast-type-specific muscular adaptations.
Indeed, we confirmed that the plantaris citrate synthase (CS)
activity, a functional indication of muscular hyperactivity, was
increased only in the RWR group, although the relative plantaris
muscle wet mass was increased in both running groups (Table 1).
This supports the previous studies in demonstrating that WR has
more pronounced effects on the slow-twitch soleus muscle (26).
Thus it may be that RWR is a distinct exercise model that has a
relatively higher recruitment of fast-twitch rather than slow-twitch
muscles with loaded running (20, 21, 26), which is clearly different from the traditional voluntary wheel of distance precedency.
However, intense exercise regimens have often been shown
to induce stress adaptation, which dampens hippocampal
BDNF expression (40, 42); physical exercise activates the
hypothalamus-pituitary-adrenal (HPA) axis depending on intensity, duration (14, 33, 39), and wheel-running distance (12).
Our results show that the RWR group, having a load of 30% of
body weight, showed no changes in plasma corticosterone
levels. This together with the adrenals and thymus weight
Fig. 5. Relationship between spatial memory and BDNF, p-CREB protein levels, and exercise parameters. A positive correlation between the percentage of time
spent in the platform (P) quadrant and work levels (A; Pearson’s correlation, r ⫽ 0.77, P ⬍ 0.01), BDNF protein (C; r ⫽ 0.41, P ⬍ 0.05), and p-CREB protein
(D; r ⫽ 0.44, P ⬍ 0.05). However, there is no significant correlation between the percentage of time spent in platform quadrant and average running distance
(B; r ⫽ 0.01, P ⫽ 0.94). Each point represents an individual rat.
J Appl Physiol • doi:10.1152/japplphysiol.00869.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
positively correlated to work levels (Pearson’s correlation, r ⫽
0.77, P ⬍ 0.01; Fig. 5A), BDNF protein (r ⫽ 0.41, P ⬍ 0.05;
Fig. 5C), and p-CREB protein (r ⫽ 0.44, P ⬍ 0.05; Fig. 5D).
However, there is no significant correlation between the percentage of time spent in platform quadrant and average running
distance (r ⫽ 0.01, P ⫽ 0.94; Fig. 5B).
Resistance Exercise-Enhanced Cognitions With BDNF Signaling
Lee MC et al.
1265
The present results show that the potential roles of RWR in
cognitive function may be associated with the action of BDNF
signaling by integrating mechanisms of synaptic plasticity.
However, several other molecules may also be important. For
example, circulating insulin-like growth factor-1 (IGF-1) levels increased following resistance exercise (6) and caused
increased BDNF mRNA levels in the hippocampus (5). Other
neurochemical factors such as corticosteroids (40), androgen
(34), or estrogen (2) also affect hippocampal plasticity and may
mediate changes in neurotrophins after exercise. RWR and its
involvement in the mechanisms of hippocampal plasticity are
worthy to be explored in future studies.
In conclusion, this study demonstrates for the first time that
voluntary resistance exercise represents a successful model to
enhance spatial learning and memory associated with increased
hippocampal BDNF signaling, even with short running distances.
Resistance exercise produces short distances but high work levels,
which may suggest that “less is more” for effective running
distances leading to hippocampal plasticity since excessive running distances would be adverse to the improvement of spatial
learning (37). Thus we must consider an alternative concept that
a proper combination of load and distance is more beneficial for
promoting cognitive functions in wheel running, although the
optimum combination remains to be determined. In addition, this
study provides a new hypothesis that work levels, rather than
running distances, are more determinant in inducing exerciseenhancing cognitive function with hippocampal plastic changes in
voluntary running with and without a load. Future study to
uncover the specific mechanisms may shed more light on the
mechanisms behind strength exercise conditions and improved
hippocampal cognitive functions.
ACKNOWLEDGMENTS
We appreciate Prof. Akihiko Ishihara (University of Kyoto) and Prof.
Norikatsu Kasuga (Aichi University of Education) for kind instruction for
establishing RWR. Part of this study was introduced in the New York Times
on January 19, 2011.
GRANTS
This research was supported in part by the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) for the Body and Mind Integrated
Sports Sciences Project of Japan (2011). M. C. Lee and M. Okamoto are both
Research Fellows of JSPS.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.C.L. and H.S. conception and design of research;
M.C.L., M.O., K.I., and T.M. performed experiments; M.C.L., M.O., and H.S.
analyzed data; M.C.L., M.O., Y.F.L., H.N., and H.S. interpreted results of
experiments; M.C.L. prepared figures; M.C.L. and H.S. drafted manuscript;
M.C.L. and H.S. edited and revised manuscript; M.C.L., M.O., Y.F.L., K.I.,
T.M., H.N., and H.S. approved final version of manuscript.
REFERENCES
1. Barbacid M. The Trk family of neurotrophin receptors. J Neurobiol 25:
1386 –1403, 1994.
2. Berchtold NC, Kesslak JP, Pike CJ, Adlard PA, Cotman CW. Estrogen and exercise interact to regulate brain-derived neurotrophic factor
mRNA and protein expression in the hippocampus. Eur J Neurosci 14:
1992–2002, 2001.
3. Call JA, McKeehen JN, Novotny SA, Lowe DA. Progressive resistance
voluntary wheel running in the mdx mouse. Muscle Nerve 42: 871–880, 2010.
J Appl Physiol • doi:10.1152/japplphysiol.00869.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
indicate that 4 wk of RWR does not produce any negative
stress effect (Table 1), which indicates that it is a suitable
exercise model for inducing hippocampal plasticity.
Indeed, we found that both WR and RWR groups presented
faster escape latency and shorter path length values in the water
maze task, demonstrating improved spatial learning performance compared with the Sed group (Fig. 2, A and B), and the
RWR group spent significantly more time in the escapeplatform quadrant compared with the Sed group (Fig. 2C).
Although it is unclear why this WR group showed no robust
effects with short-term memory (by probe test), RWR produced better performance in the spatial learning and memory of
rodents after voluntary physical activity as with previous studies (11, 29, 45) with long distances. In addition, the gene
expression of BDNF signaling-related molecules increased in
both WR and RWR groups (Fig. 3, A–C). The mechanisms that
underlie exercise-enhanced cognitive functions are controlled
by several types of molecular signaling. Particularly, BDNF
signaling plays an important role in hippocampal plasticity as
a key molecule mediating cellular functions including cell
growth, proliferation differentiation, motility, survival, as well
as cognitive functions (9, 19, 27, 47); BDNF regulates synaptic
transmission through postsynaptic actions involving TrkB (1)
and its signaling-related molecules such as PKA (8), PKC (30),
MAPK (48), and CREB (16). In addition, NMDA-R enhances
receptor binding and synaptic transmission via the release of
glutamate, which is modulated by hippocampal BDNF (44). This
evidence indicates that exercise may mediate synaptic plasticity
by using BDNF signaling. The importance of BDNF signaling has
also been demonstrated since inhibition of its pathway impairs
hippocampal LTP and associative learning (18) and an intrahippocampal injection of TrkB antibody abolishes the beneficial
effects of exercise on hippocampal-dependent spatial learning and
memory (47, 48). These results, supporting previous studies,
suggest that RWR has robust effects with enhanced spatial memory and increased hippocampal BDNF signaling.
In addition, we found that RWR increased both hippocampal
BDNF, TrkB, and p-CREB protein levels (Fig. 4, A–C). WR
increased BDNF protein levels and had a strong trend toward
increased p-CREB protein levels, although there were no
significant differences among groups (P ⫽ 0.09; Fig. 4C).
Previous studies have also shown that the effect of exercise on
cognitive function is associated with increased levels of
BDNF, and this in turn is associated with increased levels of
CREB (29, 47), a transcriptional regulator that has been linked
to long-term potentiation (LTP) and spatial learning and memory (10, 15). Our findings that showed moderate correlation
between spatial memory and BDNF (r ⫽ 0.41, P ⬍ 0.05) and
p-CREB (r ⫽ 0.44, P ⬍ 0.05) protein levels also support this
notion (Fig. 5, C and D). Furthermore, there was a positive
correlation between spatial memory and work levels (r ⫽ 0.77,
P ⬍ 0.01). In contrast, there was no significant correlation
between spatial memory and running distance (r ⫽ 0.01, P ⫽
0.94) (Fig. 5, A and B). These results are consistent with
previous studies (29, 47), suggesting that BDNF and p-CREB
have crucial functional roles in the exercise-enhanced hippocampal spatial memory and present a new concept that work
levels, rather than running distance, are more determinant for
inducing exercise-enhanced cognitive function with hippocampal plastic changes in voluntary running with and without load.
•
1266
Resistance Exercise-Enhanced Cognitions With BDNF Signaling
Lee MC et al.
27. Liu YF, Chen HI, Wu CL, Kuo YM, Yu L, Huang AM, Wu FS,
Chuang JI, Jen CJ. Differential effects of treadmill running and wheel
running on spatial or aversive learning and memory: roles of amygdalar
brain-derived neurotrophic factor and synaptotagmin I. J Physiol 587:
3221–3231, 2009.
28. Maldonado R, Dauge V, Callebert J, Villette JM, Fournie-Zaluski
MC, Feger J, Roques BP. Comparison of selective and complete inhibitors of enkephalin-degrading enzymes on morphine withdrawal syndrome. Eur J Pharmacol 165: 199 –207, 1989.
29. Molteni R, Wu A, Vaynman S, Ying Z, Barnard RJ, Gomez-Pinilla F.
Exercise reverses the harmful effects of consumption of a high-fat diet on
synaptic and behavioral plasticity associated to the action of brain-derived
neurotrophic factor. Neuroscience 123: 429 –440, 2004.
30. Molteni R, Ying Z, Gomez-Pinilla F. Differential effects of acute and
chronic exercise on plasticity-related genes in the rat hippocampus revealed by microarray. Eur J Neurosci 16: 1107–1116, 2002.
31. Morris RG, Garrud P, Rawlins JN, O’Keefe J. Place navigation
impaired in rats with hippocampal lesions. Nature 297: 681–683, 1982.
32. Neeper SA, Gomez-Pinilla F, Choi J, Cotman C. Exercise and brain
neurotrophins. Nature 373: 109, 1995.
33. Ohiwa N, Chang H, Saito T, Onaka T, Fujikawa T, Soya H. Possible
inhibitory role of prolactin-releasing peptide for ACTH release associated
with running stress. Am J Physiol Regul Integr Comp Physiol 292:
R497–R504, 2007.
34. Okamoto M, Hojo Y, Inoue K, Matsui T, Kawato S, McEwen BS, Soya
H. Mild exercise increases dihydrotestosterone in hippocampus providing
evidence for androgenic mediation of neurogenesis. Proc Natl Acad Sci
USA 109: 13100 –13105, 2012.
35. Pencea V, Bingaman KD, Wiegand SJ, Luskin MB. Infusion of brainderived neurotrophic factor into the lateral ventricle of the adult rat leads
to new neurons in the parenchyma of the striatum, septum, thalamus, and
hypothalamus. J Neurosci 21: 6706 –6717, 2001.
36. Poo MM. Neurotrophins as synaptic modulators. Nat Rev Neurosci 2:
24 –32, 2001.
37. Rhodes JS, van Praag H, Jeffrey S, Girard I, Mitchell GS, Garland T
Jr, Gage FH. Exercise increases hippocampal neurogenesis to high levels
but does not improve spatial learning in mice bred for increased voluntary
wheel running. Behav Neurosci 117: 1006 –1016, 2003.
38. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for
biologist programmers. Methods Mol Biol 132: 365–386, 2000.
39. Saito T, Soya H. Delineation of responsive AVP-containing neurons to
running stress in the hypothalamus. Am J Physiol Regul Integr Comp
Physiol 286: R484 –R490, 2004.
40. Schaaf MJ, de Jong J, de Kloet ER, Vreugdenhil E. Downregulation of
BDNF mRNA and protein in the rat hippocampus by corticosterone. Brain
Res 813: 112–120, 1998.
41. Shatalin K, Lebreton S, Rault-Leonardon M, Velot C, Srere PA.
Electrostatic channeling of oxaloacetate in a fusion protein of porcine
citrate synthase and porcine mitochondrial malate dehydrogenase. Biochemistry (Mosc) 38: 881–889, 1999.
42. Smith MA, Makino S, Kvetnansky R, Post RM. Stress and glucocorticoids
affect the expression of brain-derived neurotrophic factor and neurotrophin-3
mRNAs in the hippocampus. J Neurosci 15: 1768 –1777, 1995.
43. Soya H, Nakamura T, Deocaris CC, Kimpara A, Iimura M, Fujikawa
T, Chang H, McEwen BS, Nishijima T. BDNF induction with mild
exercise in the rat hippocampus. Biochem Biophys Res Commun 358:
961–967, 2007.
44. Suen PC, Wu K, Levine ES, Mount HT, Xu JL, Lin SY, Black IB.
Brain-derived neurotrophic factor rapidly enhances phosphorylation of the
postsynaptic N-methyl-D-aspartate receptor subunit 1. Proc Natl Acad Sci
USA 94: 8191–8195, 1997.
45. van Praag H, Christie BR, Sejnowski TJ, Gage FH. Running enhances
neurogenesis, learning, and long-term potentiation in mice. Proc Natl
Acad Sci USA 96: 13427–13431, 1999.
46. van Praag H, Kempermann G, Gage FH. Running increases cell
proliferation and neurogenesis in the adult mouse dentate gyrus. Nat
Neurosci 2: 266 –270, 1999.
47. Vaynman S, Ying Z, Gomez-Pinilla F. Hippocampal BDNF mediates the
efficacy of exercise on synaptic plasticity and cognition. Eur J Neurosci
20: 2580 –2590, 2004.
48. Vaynman S, Ying Z, Gomez-Pinilla F. Interplay between brain-derived
neurotrophic factor and signal transduction modulators in the regulation of the
effects of exercise on synaptic-plasticity. Neuroscience 122: 647–657, 2003.
J Appl Physiol • doi:10.1152/japplphysiol.00869.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 16, 2017
4. Carek PJ, Laibstain SE, Carek SM. Exercise for the treatment of
depression and anxiety. Int J Psychiatry Med 41: 15–28, 2011.
5. Carro E, Nunez A, Busiguina S, Torres-Aleman I. Circulating insulinlike growth factor I mediates effects of exercise on the brain. J Neurosci
20: 2926 –2933, 2000.
6. Cassilhas RC, Lee KS, Fernandes J, Oliveira MG, Tufik S, Meeusen
R, de Mello MT. Spatial memory is improved by aerobic and resistance
exercise through divergent molecular mechanisms. Neuroscience 202:
309 –317, 2012.
7. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem
162: 156 –159, 1987.
8. Chytrova G, Ying Z, Gomez-Pinilla F. Exercise normalizes levels of
MAG and Nogo-A growth inhibitors after brain trauma. Eur J Neurosci
27: 1–11, 2008.
9. Cotman CW, Berchtold NC. Exercise: a behavioral intervention to
enhance brain health and plasticity. Trends Neurosci 25: 295–301, 2002.
10. Dash PK, Hochner B, Kandel ER. Injection of the cAMP-responsive
element into the nucleus of Aplysia sensory neurons blocks long-term
facilitation. Nature 345: 718 –721, 1990.
11. Ding Q, Vaynman S, Akhavan M, Ying Z, Gomez-Pinilla F. Insulinlike growth factor I interfaces with brain-derived neurotrophic factormediated synaptic plasticity to modulate aspects of exercise-induced
cognitive function. Neuroscience 140: 823–833, 2006.
12. Droste SK, Gesing A, Ulbricht S, Muller MB, Linthorst AC, Reul JM.
Effects of long-term voluntary exercise on the mouse hypothalamicpituitary-adrenocortical axis. Endocrinology 144: 3012–3023, 2003.
13. Farmer J, Zhao X, van Praag H, Wodtke K, Gage FH, Christie BR.
Effects of voluntary exercise on synaptic plasticity and gene expression in
the dentate gyrus of adult male Sprague-Dawley rats in vivo. Neuroscience
124: 71–79, 2004.
14. Farrell PA, Garthwaite TL, Gustafson AB. Plasma adrenocorticotropin
and cortisol responses to submaximal and exhaustive exercise. J Appl
Physiol 55: 1441–1444, 1983.
15. Finkbeiner S. CREB couples neurotrophin signals to survival messages.
Neuron 25: 11–14, 2000.
16. Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM,
Greenberg ME. CREB: a major mediator of neuronal neurotrophin
responses. Neuron 19: 1031–1047, 1997.
17. Groves-Chapman JL, Murray PS, Stevens KL, Monroe DC, Koch
LG, Britton SL, Holmes PV, Dishman RK. Changes in mRNA levels for
brain-derived neurotrophic factor after wheel running in rats selectively
bred for high- and low-aerobic capacity. Brain Res 1425: 90 –97, 2011.
18. Gruart A, Sciarretta C, Valenzuela-Harrington M, Delgado-Garcia
JM, Minichiello L. Mutation at the TrkB PLCgamma-docking site affects
hippocampal LTP and associative learning in conscious mice. Learn
Memory 14: 54 –62, 2007.
19. Hu Y, Russek SJ. BDNF and the diseased nervous system: a delicate
balance between adaptive and pathological processes of gene regulation. J
Neurochem 105: 1–17, 2008.
20. Ishihara A, Hirofuji C, Nakatani T, Itoh K, Itoh M, Katsuta S. Effects
of running exercise with increasing loads on tibialis anterior muscle fibres
in mice. Exp Physiol 87: 113–116, 2002.
21. Ishihara A, Roy RR, Ohira Y, Ibata Y, Edgerton VR. Hypertrophy of
rat plantaris muscle fibers after voluntary running with increasing loads. J
Appl Physiol 84: 2183–2189, 1998.
22. Johnson RA, Mitchell GS. Exercise-induced changes in hippocampal
brain-derived neurotrophic factor and neurotrophin-3: effects of rat strain.
Brain Res 983: 108 –114, 2003.
23. Kasuga N, Yamashita S, Ogasawara H, Suzuki H, Tsuzimoto H,
Ishihara A. Various in running pattern and skeletal muscle adaptations in
voluntary running rats at different load. Jpn J Phys Fit Sport 48: 99 –110,
1999.
24. Konhilas JP, Widegren U, Allen DL, Paul AC, Cleary A, Leinwand
LA. Loaded wheel running and muscle adaptation in the mouse. Am J
Physiol Heart Circ Physiol 289: H455–H465, 2005.
25. Lee J, Duan W, Mattson MP. Evidence that brain-derived neurotrophic
factor is required for basal neurogenesis and mediates, in part, the
enhancement of neurogenesis by dietary restriction in the hippocampus of
adult mice. J Neurochem 82: 1367–1375, 2002.
26. Legerlotz K, Elliott B, Guillemin B, Smith HK. Voluntary resistance
running wheel activity pattern and skeletal muscle growth in rats. Exp
Physiol 93: 754 –762, 2008.
•