Effects of Diet and/or Exercise in Enhancing Spinal Cord
Sensorimotor Learning
M. Selvan Joseph1, Zhe Ying1, Yumei Zhuang3, Hui Zhong5, Aiguo Wu1, Harsharan S. Bhatia1,
Rusvelda Cruz1, Niranjala J. K. Tillakaratne1,5, Roland R. Roy1,5, V. Reggie Edgerton1,2,3,5,Fernando Gomez-Pinilla1,3,4,5*
1 Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, California, United States of America, 2 Department of Neurobiology,
University of California Los Angeles, Los Angeles, California, United States of America, 3 Department of Neurosurgery, University of California Los Angeles, Los Angeles,
California, United States of America, 4 UCLA Brain Injury Research Center, University of California Los Angeles, Los Angeles, California, United States of America, 5 Brain
Research Institute, University of California Los Angeles, Los Angeles, California, United States of America

Abstract
Given that the spinal cord is capable of learning sensorimotor tasks and that dietary interventions can influence learning
involving supraspinal centers, we asked whether the presence of omega-3 fatty acid docosahexaenoic acid (DHA) and the
curry spice curcumin (Cur) by themselves or in combination with voluntary exercise could affect spinal cord learning in adultspinal mice. Using an instrumental learning paradigm to assess spinal learning we observed that mice fed a diet containing
DHA/Cur performed better in the spinal learning paradigm than mice fed a diet deficient in DHA/Cur. The enhanced
performance was accompanied by increases in the mRNA levels of molecular markers of learning, i.e., BDNF, CREB, CaMKII,
and syntaxin 3. Concurrent exposure to exercise was complementary to the dietary treatment effects on spinal learning. The
diet containing DHA/Cur resulted in higher levels of DHA and lower levels of omega-6 fatty acid arachidonic acid (AA) in the
spinal cord than the diet deficient in DHA/Cur. The level of spinal learning was inversely related to the ratio of AA:DHA.
These results emphasize the capacity of select dietary factors and exercise to foster spinal cord learning. Given the noninvasiveness and safety of the modulation of diet and exercise, these interventions should be considered in light of theirpotential to enhance relearning of sensorimotor tasks during rehabilitative training paradigms after a spinal cord injury.
Citation: Joseph MS, Ying Z, Zhuang Y, Zhong H, Wu A, et al. (2012) Effects of Diet and/or Exercise in Enhancing Spinal Cord Sensorimotor Learning. PLoS
ONE 7(7): e41288. doi:10.1371/journal.pone.0041288
Editor: Cesario V. Borlongan, University of South Florida, United States of America
Received March 26, 2012; Accepted June 19, 2012; Published July 20, 2012
Copyright: ß 2012 Joseph et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the National Institutes of Health R01 NS056413, Diversity Supplement NS056413, and R01 NS050465. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: fgomezpi@ucla.edu

cognitive [8,9] and locomotor deficits [10,11] via normalizing
levels of BDNF in rodents with brain trauma.
Exercise alone also can improve cognitive learning andlocomotor function in intact rats. Volunteer wheel running
improved the performance of rats in the Morris water maze that
correlated positively with BDNF mRNA levels in the hippocampus[12]. Similarly, long-term locomotor treadmill exercise of intact
rats increased BDNF mRNA levels in the lumbar spinal cord
compared to sedentary rats [13]. Voluntary wheel running of micewith a spinal cord contusion injury for 3–7 weeks improved theiropen field locomotor scores [14] and treadmill stepping ability[15] compared to sedentary mice.
Given that BDNF levels are modulated with DHA, curcumin,and exercise, we have focused on molecular systems related to theaction of BDNF on synaptic plasticity, such as calcium/calmodulin
activated protein kinase II (CaMKII), the gene transcription factor
cAMP-response element binding protein (CREB), and syntaxin 3.
BDNF is a powerful modulator of neuronal excitability andsynaptic transmission [16,17] and learning and memory in the
hippocampus [18–20]. BDNF facilitates monosynaptic excitatory
post-synaptic potentials (EPSPs) in motoneurons [21]. BDNF
mediated activation of CaMKII is a key intermediate in thefacilitation of the early phase of long-term potentiation (LTP)

Introduction
Recent evidence indicates that select dietary factors such as
docosahexaenoic acid (DHA) can improve learning in the brain byacting on molecular systems involved with synaptic plasticity [1,2].
DHA belongs to the omega-3 fatty acid family and has importantroles in cell communication, synaptic plasticity, and hippocampal
learning [3,4]. DHA has been shown to normalize levels of brainderived neurotrophic factor (BDNF), reduce oxidative damage,and counteract learning disabilities in animal models of brain
trauma [3]. DHA also has been shown to improve locomotor
recovery and decrease cellular damage after spinal cord injury. For
example, intravenous acute DHA injection combined with dietary
supplementation of DHA after a spinal cord compression injury inadult rats decreased the amount of apoptotic cell death and whitematter degradation [5]. In addition, the DHA-fed animals showeda significant improvement in their open field locomotor ability at 4
weeks compared to rats receiving either a diet with no DHA or a
DHA injection alone. The curry spice curcumin, used in India formany years for several medical purposes, has shown important
effects in models of learning and plasticity in animals [6,7]. For
example, curcumin supplementation has been shown to reduce

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Diet and Exercise Affect Spinal Cord Learning

higher in the DHA/Cur+Ex than DHA/Cur+Sed group, again
reflecting a complementary effect of exercise and the DHA/Cur
diet. No differences were observed in the CtrlDiet+Sed and
CtrlDiet+Ex groups. Response duration across groups was
positively and significantly correlated with the mRNA levels ofeach molecule (Fig. 2B, D, F and H). Thus it appears that there is
a complementary effect of adding DHA/Cur to the diet andexercise in promoting spinal learning.

leading to a strengthening of synaptic efficacy [22,23]. CREB is
one of the best described stimulus-induced transcription factors
involved in gene transcription [24] and memory formation [18].
Syntaxin 3 protein is a pre-synaptic membrane bound protein thatparticipates in vesicular docking that is up-regulated during
synaptic plasticity [25–27].
With sensorimotor training, the spinal cord can learn toperform both simple and complex motor tasks such as stepping[28–32], standing [33], avoiding obstacles [34], and paw
withdrawal [35–37], even in the absence of supraspinal input[38]. The motor task that is practiced is learned, e.g., if a spinal cat
is trained to step, stepping performance is improved [29], whereas
if the spinal cat is trained to stand the ability to stand improves[33]. In addition, learning to avoid shock of the paw in an animal
with a complete thoracic spinal cord transection has been
demonstrated in a variety of species over a period of decades[35,37,39]. This spinal learning is mediated via the proprioceptive
input to the spinal circuitry, which has the capacity to interpretwith considerable detail the patterns of sensory input associatedwith a given motor task [40]. For example, the spinal cord caninterpret sensory changes in load [41] and in treadmill speed[42,43] and direction [44,45].
The purpose of the present study was to determine the potential
therapeutic role of a combination of DHA and curcumin with andwithout voluntary exercise in improving spinal learning using thepaw withdrawal (PaWL) instrumental learning paradigm in mice[37]. In this paradigm, mice whose spinal cords are completely
severed at a mid-thoracic level learn to dorsiflex the paw above a
set vertical threshold position to reduce the net shock exposure in
response to a mild electric shock applied to the tibialis anterior(TA) muscle. BDNF mRNA levels have been shown to increase
with a similar instrumental learning paradigm in spinal rats [36].
Furthermore, delivery of BDNF into the injured spinal cord
improves [36], whereas blocking BDNF action hinders the
instrumental learning in spinal rats [46,47]. This PaWL model,
therefore, provides the opportunity to evaluate the effects of
dietary factors with and without exercise on BDNF-mediatedspinal cord learning. The potential benefits of diet and exercise on
spinal cord learning have a strong translational potential based on
the high efficacy and low invasive profile of these interventions.

DHA and AA levels in the spinal cord of each group
The spinal cord levels of DHA (Fig. 3A) were higher and thoseof AA (Fig. 3C) lower in the DHA/Cur+Sed and DHA/Cur+Ex
groups than in both CtrlDiet groups. These differences resulted ina lower AA:DHA ratio in the DHA/Cur+Sed and DHA/Cur+Ex
groups than in the two CtrlDiet groups (Fig. 3E). Across groups,
there was a positive and significant correlation between mean
response time and the levels of DHA (Fig. 3B) and a negative andsignificant correlation between response duration and both thelevels of AA (Fig. 3D) and the AA:DHA ratio (Fig. 3F) in the spinal
cord. Thus the addition of DHA/Cur to the diet results in achange in the fatty acid profile that might promote spinal learning.

Effects of blocking BDNF function on spinal learning andthe levels of molecules associated with learning andplasticity
The lumbar region of the spinal cord of a separate group of
spinal mice fed the DHA/Cur+Sed diet was injected with either
TrkB IgG or vehicle (saline) 24 hr prior to the PaWL test. The
mean response duration in the mice injected with TrkB IgG was
significantly lower than in the mice injected with saline beginningat the 13 min time bin and at all time points thereafter (Fig. 4A).
Similarly, the mean total response duration (Fig. 4B) and themRNA levels of BDNF (Fig. 4C), CaMKII (Fig. 4D), and CREB
(Fig. 4E), but not syntaxin 3 (Fig. 4F), were lower in the mice
injected with TrkB IgG than in the mice injected with saline. Thusan acute decrease in BDNF protein attenuates spinal learning anddecreases the levels of plasticity-related markers in the spinal cord.

Discussion
The principal findings were 1) a diet supplemented with DHA/
Cur enhances spinal learning and increases the levels of several
learning-related cellular markers, and 2) diet supplementation with
DHA/Cur and exercise have complementary effects on spinal
learning. Molecular evidence suggests that the spinal learning wasmediated through the BDNF pathway as the selective blocking of
BDNF signaling with TrkB IgG chimera resulted in decreased
learning. In addition, the diet containing DHA/Cur increased
DHA and decreased AA levels in the spinal cord: the resulting
decrease in the ratio of AA:DHA was correlated with a greater
spinal learning ability.

Results
The effects of DHA/Cur diet and exercise on spinal
learning
The PaWL paradigm was used to evaluate spinal learning in
mice. In general, the mean response duration was longer in the
DHA/Cur and DHA/Cur/Ex groups than in both CtrlDiet
groups, with these differences being significant at the 12 min timebin and at almost all time points thereafter (Fig. 1A). Total
response duration was longer in the DHA/Cur and DHA/Cur/
Ex groups than in both CtrlDiet groups (Fig. 1B). In addition, total
response time was longer in the DHA/Cur/Ex than DHA/Cur
group, reflecting the complementary effect of exercise with the
DHA/Cur.

Can diet modulate spinal learning?
The dietary effects on spinal learning were accompanied bysignificant increases in the levels of BDNF, CaMKII, CREB, andsyntaxin 3 mRNAs. These results are consistent with previous
studies in the brain showing the capacity of DHA [48–55] andcurcumin [10,11,56] to counteract the effects of brain injury on
hippocampal-dependent spatial learning performance [11,55,57–
59]. DHA also has shown some beneficial effects when injected in
the tail vein 30 min after a spinal cord injury, as indicated by anincrease in the survival of neurons and an improvement inlocomotor performance after a spinal cord hemisection [60] orcompression injury [5].

The effects of DHA/Cur diet and exercise on the levels ofmolecules associated with learning
The mRNA levels of BDNF (Fig. 2A), CaMKII (Fig. 2C),
CREB (Fig. 2E), and syntaxin 3 (Fig. 2G) were higher in bothgroups receiving DHA/Cur than in both CtrlDiet groups exceptthat there was no difference in the levels of CaMKII, CREB, orsyntaxin 3 mRNA in the CtrlDiet+Ex and DHA/Cur+Sed groups.
In addition, the levels of all molecules, except for syntaxin 3, were
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Diet and Exercise Affect Spinal Cord Learning

Figure 1. Spinal cord learning evaluated using the paw withdrawal learning paradigm. (A) The groups receiving DHA/Cur with andwithout Ex had longer mean response durations than both CtrlDiet groups, initially at the 12 min time bin and at almost all time points thereafter
during the 30-min PaWL sessions. (B) Mean total response duration in the groups receiving DHA/Cur with and without Ex had longer total response
durations than in both CtrlDiet groups. The DHA/Cur+Ex group had a longer mean total response duration than the DHA/Cur+Sed group, reflecting
the complementary effect of exercise with the DHA/Cur diet. Values are mean 6 SEM (n = 13/group). One-way ANOVA followed by Tukey’s post-hoc
test at P,0.05. *, {, {: Significantly different from CtrlDiet+Sed, CtrlDiet+Ex, and DHA/Cur+Sed, respectively.
doi:10.1371/journal.pone.0041288.g001

stabilizing membrane homeostasis and reducing cognitive decay[7,10]. In addition, curcumin has been shown to reduce cell
damage in models of toxicity in culture [9,74]. The combination of
curcumin and DHA in the current study may be particularly
effective for fostering plasticity as they act on similar molecularsystems. For example, both DHA and curcumin can affect BDNFrelated synaptic plasticity [52,55] and also may influence themetabolic actions of BDNF on glucose and molecules that are
crucial for the production of ATP necessary for learning [75,76].

These results were associated with differential levels of DHA in
the plasma membrane. DHA is critical for maintaining membrane
fluidity, which is required for proper neuronal function andtransmission of information [50,61]. A reduction in the levels of
DHA can lower membrane fluidity leading to dysfunction oftransmembrane receptors, potentially affecting the induction of
LTP [48] and subsequent learning [49,62]. Membrane instability
and loss of function is suggested by our results showing that thediet with added DHA was associated with an increase in syntaxin
3, a pre-synaptic membrane vesicular transport marker that isregulated by DHA [63]. The levels of both DHA [64] andcurcumin [7] can differentially regulate syntaxin 3 in the brain. A
deficiency in DHA in the cerebral cortex has been shown to
reduce phosphorylation of the BDNF TrkB receptors [52] that, inturn, may reduce the levels of learning-related factors such as
CREB [3,65]. DHA also may influence neurotrophic signaling byactivating the PI3-K/Akt pathway that phosphorylates CREB
[66]. The increased CREB activation then can influence theexpression of other genes including BDNF [4,18,67]. In addition,
DHA has been implicated in enhancing neurogenesis andplasticity. For example, dietary administration of DHA significantly increased the number of 5-bromo-2(BrdU) 9-deoxyuridine
positive newborn neurons in the granule cell layer of the dentate
gyrus in adult rats [68,69]. Exercise also increases hippocampal
neurogenesis [70]. Whether the DHA/curcumin diet, exercise, ora combination of both interventions contribute to neurogenesis in
the spinal cord was not addressed in the present study. Further
investigation using BrdU labeling will be needed to establish
whether neurogenesis occurs in the spinal cord as a result of thediet and/or exercise interventions. In the hippocampus, it has
been reported that the binding of DHA to GRP40 may provide afast way to impact plasticity through second messenger pathways
such as PLC, PIP3, PKC, and CREB and the c-fos system [71–73]. Whether DHA acts through a similar GPR40-mediatedmechanism in the spinal cord is unknown and future studies will be
designed to address this possibility.

Role of exercise and BDNF on spinal learning
The combination of exercise and DHA/Cur promoted asignificant enhancement in spinal learning compared to either
intervention alone. Exercise positively influences hippocampaldependent learning via a BDNF-mediated mechanism [12]. In
addition, exercise such as wheel running or stepping on a treadmill
can improve locomotor recovery after a spinal cord injury [14,15],
an effect mediated at least in part via the BDNF pathway[12,77,78]. To confirm that the DHA/Cur diet works through the
BDNF pathway, we blocked BDNF function using TrkB IgG in
DHA/Cur fed mice. The TrkB IgG injection diminished thespinal learning ability by 3-fold, and reduced the levels of BDNF,
CaMKII, and CREB mRNAs in the lumbar segments of the spinal
cord. These and other observations [36,47] support the possibility
that the BDNF system is an important intermediate for spinal cordlearning. The present results demonstrate that the DHA/Cur dietinfluenced spinal learning through BDNF-, CaMKII-, and CREBrelated pathways. The actions of DHA and curcumin in the brain
have been associated with BDNF and TrkB signaling pathways[52,55], findings consistent with BDNF being an important pointof convergence for the beneficial actions of our dietary paradigm
on spinal plasticity and learning.
Interestingly, we observed that a diet deficient of DHA/Cur
completely suppressed any effect of exercise on spinal learning or
molecular markers associated with spinal learning (BDNF, CREB,
CaMKII, and syntaxin 3). These results suggest that some DHA
must be available in the diet for spinal learning to occur. The
results, however, do not indicate how much DHA must be in thediet before an exercise-mediated effect can be detected. We haveobserved previously that exercise can compensate for the
detrimental effects of a diet rich in saturated fat and sugar on
the hippocampal levels of BDNF and spatial learning [79,80].

Role of curcumin in spinal plasticity and learning
We combined curcumin with DHA based on its action inmaintaining metabolic homeostasis that can be critical for
supporting learning and memory [1,6,7]. For example, thebeneficial action of curcumin on brain trauma is associated with
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Figure 2. Dietary and exercise effects on the levels of BDNF, CaMKII, CREB, and syntaxin 3 mRNA in the lumbar region of the spinal
cord. (A) Mean BDNF mRNA levels were higher in the groups receiving DHA/Cur with and without Ex than in both CtrlDiet groups. Exercise in the
DHA/Cur treated group enhanced this response. (C) CaMKII mRNA levels were higher in the DHA/Cur treated groups than in CtrlDiet+Sed group.
Exercise in the DHA/Cur treated group enhanced this response. (E) CREB mRNA levels were higher in both groups receiving DHA/Cur than in the
CtrlDiet+Sed group. Exercise enhanced the effect in the DHA/Cur group. (G) Syntaxin 3 mRNA levels were higher in both groups receiving DHA/Cur
than in the CtrlDiet+Sed group and higher in the DHA/Cur+Ex than in the CtrlDiet+Ex group. There was a significant positive correlation between
response duration (spinal learning) and BDNF (B), CaMKII (D), CREB (F), and syntaxin 3 (H) mRNA levels across groups. Values are mean 6 SEM (n = 13/
group). One-way ANOVA followed by Tukey’s post-hoc test and Pearson product correlations between PaWL and mRNA levels. *, {, {: significantly

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Diet and Exercise Affect Spinal Cord Learning

different from CtrlDiet+Sed, CtrlDiet+Ex, and DHA/Cur+Sed, respectively, at P,0.05.
doi:10.1371/journal.pone.0041288.g002

recent study, deficiency of DHA during brain maturation was
associated with reduced TrkB signaling in the brain as well as withincreased risk for anxiety-like behavior during adulthood in rats[52]. Overall the evidence seems to indicate that some critical levelof DHA and curcumin must play an important role in engaging
mechanisms of synaptic plasticity and learning in the spinal cord.

Role of the fatty acid profile in the spinal cord on spinal
learning
DHA is an essential fatty acid and a structural component ofplasma membranes, synaptic vesicles, and membranes of otheressential organelles important for brain function and, based on thepresent results, the spinal cord as well. We observed a decrease in
DHA and an increase in the omega-6 fatty acid (AA) in the spinal
cord of mice fed a DHA and curcumin deficient diet. The
possibility that excessive levels of AA can result in reduction of
TrkB signaling is suggested by our results showing that the ratio of
AA:DHA negatively correlated with PaWL performance. In a

Conclusions
Our results indicate that a dietary combination of DHA andcurcumin facilitates spinal cord learning via a BDNF-related
mechanism. The concurrent exposure of exercise during the

Figure 3. Levels of DHA and AA fatty acids in the spinal cord. The levels of DHA were higher (A) and those of AA lower (C) in the two DHA/Cur
groups compared to the two CtrlDiet groups. Consequently, the AA:DHA ratio was lower (E) in the two groups treated with DHA/Cur compared tothe two CtrlDiet groups. There was a significant positive correlation between response duration (spinal learning) and DHA levels in the spinal cord (B)and a significant negative correlation between response duration and AA levels in the spinal cord (D) and the AA:DHA ratio (F) across groups. Values
are mean 6 SEM (n = 13/group). One-way ANOVA followed by Tukey’s post-hoc test and Pearson product correlations between PaWL and mRNAlevels. *, {, {: significantly different from CtrlDiet+Sed, CtrlDiet+Ex, and DHA/Cur+Sed, respectively, at P,0.05.
doi:10.1371/journal.pone.0041288.g003

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Diet and Exercise Affect Spinal Cord Learning

Figure 4. The effect of TrkB IgG on PaWL and the levels of BDNF, CaMKII, CREB, and syntaxin 3 mRNA in the spinal cord. (A)Sequestering of BDNF protein with TrkB IgG significantly decreased spinal learning in mice that were on a DHA/Cur diet. Mean response durations
were lower at 13 min and thereafter during the 30-min PaWL sessions in the group receiving TrkB IgG compared to the saline injected group. (B)Mean total response duration was lower in the group injected with TrkB IgG than in the saline injected group. BDNF (C), CaMKII (D), and CREB (E), but
not syntaxin 3 (F), mRNA levels were lower in the group receiving TrkB IgG compared to the saline injected group. Values are mean 6 SEM (n = 9/group). Mann-Whitney t-test. *: significant difference between Saline vs. TrkB IgG groups, at P,0.05.
doi:10.1371/journal.pone.0041288.g004

sedentary or a voluntary exercise condition, with subgroups fromeach condition exposed to either a Ctrl or DHA/Cur (1.25%
DHA; Nordic Naturals, Inc. Watsonville, CA+500 ppm Curcumin: Sigma Aldrich) diet for 21 days. The dose of DHA andcurcumin used in this study were the same as used previously inrats [8,52]. The diets were provided ad libitum and administered in
powder form.
The two custom diets (CtrlDiet and DHA/Cur) used werebased on the composition of the American Institute of Nutrition
diet and prepared commercially (Dyets, Bethlehem, PA) as
described previously [52] (Table 1). Both diets had the same
macronutrients, vitamins, minerals, and basal fats (hydrogenatedcoconut and safflower oils). The only difference between the Ctrl
and DHA/Cur diets was the presence of n-3 fatty acids in the
DHA/Cur diet (0.48 and 1.2 g/100 g diet of flaxseed oil and
DHA, respectively).
The running wheel (diameter, 12 cm; width, 5 cm) used for thevoluntary exercise rotated freely and was attached to a receiverthat monitored the number of revolutions (VitalViewer Data
Acquisition System software, Mini Mitter, Sunriver, OR, USA).
The mice were allowed to exercise ad libitum in individual cageswith unlimited access to the running wheel [82]. The amount of
exercise wheel running was not significantly different for the
CtrlDiet+Ex and DHA/Cur+Ex groups. After 21 days of exercise
and/or the respective diets, all mice underwent a spinal cordsurgery and PaWL testing.

dietary treatment resulted in an additional improvement in spinal
learning. The observation that the spinal learning was proportional to the levels of spinal cord DHA and the DHA-related
synaptic marker syntaxin 3, suggest that the action of DHA on theplasma membrane is an important factor for spinal learning as it isin learning tasks involving supraspinal networks [81]. Given thatspinal learning can occur in the sensorimotor circuits of the spinal
cord and that these circuits can be modulated by dietarymodulation of DHA and curcumin, the potential of theseinterventions for improving recovery after a spinal cord injury
should be examined.

Materials and Methods
The experiments were performed in accordance with theUnited States National Institutes of Health Guide for the Care andUse of Laboratory Animals. The UCLA Chancellor’s Animal
Research Committee approved all procedures used in this study.

Diet and exercise procedures
Adult male C57BL6 mice (Jackson Lab), approximately 10
weeks of age, were housed in standard polyethylene cages in anenvironmentally controlled room (22–24uC) with a 12 h light/
dark cycle. The mice were divided randomly into 4 groups(n = 13/group): (1) Control Diet plus Sedentary (CtrlDiet+Sed);
this group served as a control for all comparisons, (2) CtrlDiet plus
exercise (CtrlDiet+Ex), (3) DHA plus curcumin plus Sedentary(DHA/Cur+Sed), and (4) DHA/Cur+Ex. After acclimatization for
1 week on standard mice chow, the mice were exposed to either a

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Table 1. Dietary Content for CtrlDiet and DHA/Cur diets.

Ingredient

Amount (g/100 g diet)
CtrlDiet

DHA/Cur

Alacid 710, acid casein

20

20

Cornstarch

15

15

Sucrose10

10

Dextrose

19.9

19.9

Maltose-dextrin

15

15

Cellulose

5.0

5.0

Salt-mineral mix

3.5

3.5

Vitamin mix

1.0

1.0

L-cystine

0.3

0.3

Choline bitartrate

0.25

0.25

TBHQ

0.002

0.002

Fat sources
Hydrogenated coconut oil

8.1

7.45

Safflower oil

1.9

1.77

Flaxseed oil

none

0.48

DHA

none

1.2

Curcuminnone

500 ppm

doi:10.1371/journal.pone.0041288.t001

from the midline and at a 0.32 mm depth [85,86]. A 10 ml
Hamilton syringe fitted with a 100 mm (tip) glass pulled needle wasused to inject 0.4 ml of 5 mg/ml of TrkB IgG in saline solution at
both sites (total of 0.80 ml) or the same amount of saline as avehicle control [36].

Surgical procedures
The spinal cord was completelytransected at the T7–T8 vertebral level in all mice as described
previously [83]. Briefly, under 2% isoflurane anesthesia, a dorsal
midline skin incision was made from T6 to T9 and themusculature covering the dorsal vertebral column was retracted
to expose the spinal laminae. A partial laminectomy of the T7 andT8 vertebrae was performed to expose the spinal cord. The spinal
cord, including the dura, was transected completely using
microscissors. The completeness of the lesion was verified byseparating the cut ends of the spinal cord with small cotton pellets
and by passing a fine glass probe through the lesion site. The skinincision was closed using small surgical staples.
After surgery, the wound sites were treated with triple antibiotic
ointment (Bacitracin) and the mice were given lactated Ringer’s
solution (1.5 ml/30 g body weight, s.c.). Because preventing leg
extension during recovery has been shown to facilitate subsequent
learning in rats [84], both hindlegs were bound with the knee andankle joints fully flexed until PaWL testing. The mice recovered in
an incubator maintained at 37uC until fully awake and then werereturned to their home cages. The mice were allowed to recoverfor 24 hr before PaWL testing. To minimize bias, the surgeons
and testers were blind to the diet and exercise conditions during
surgery and PaWL testing.
TrkB IgG Injection. A separate cohort of mice were fed the
DHA/Cur diet for 21 days and subdivided into two groups (n = 9/group): the spinal cord of one group was injected withphysiological saline and the other group with TrkB IgG (R&D
Research, rhTrkB/Fc Chimera Cat. # 688). Immediately after the
spinal cord transection surgery, the mouse was transferred to astereotaxic apparatus (David Kopf Instruments, Tujunga, CA).
The spinal processes rostral and caudal to the injection site (L2–L3
spinal level) were secured using tissue clamps connected to thestereotaxic apparatus. Injections were made bilaterally at 0.4 mm
Spinal cord transection.

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PaWL testing
PaWL is a unique paradigm used to demonstrate that the spinal
cord can learn a motor task. In this paradigm, a mouse that has
undergone a complete mid-thoracic spinal cord transection learns
to dorsiflex the paw above a pre-determined threshold when amild electric shock is applied to the TA muscle. A learned response
is determined by the amount of time the paw remains dorsiflexed
above the threshold during a 30-min testing period [37].
The PaWL test was conducted 24 h after the spinal cord
transection surgery on mice that had received 3 weeks of DHA/
Cur+Sed, DHA/Cur+Ex, CtrlDiet+Sed, or CtrlDiet+Ex. The
details of the PaWL testing for mice have been described
previously [37]. Briefly, during testing the mice were restrainedin a closed cloth harness with two slots cut at the end of the harness
to allow for both hindlegs to hang freely. Two fine-wire hookelectrodes were constructed by removing ,1 mm of insulation at
the end of nylon-coated single strand stainless steel wires(California Fine Wire Co., Grover City, CA). Each wire waspassed through a 32-gauge needle: one electrode was inserted
intramuscularly into the left TA muscle and the second electrodewas inserted subcutaneously at the base of the lateral malleolus on
the same side to serve as a ground. The electrodes were attached
to a stimulator (S88, Grass Product Group; W. Warwick, RI),
through a stimulus isolation unit (SIU5; Grass Product Group) anda constant current isolation unit (CCU1; Grass Product Group). A
stimulus duration of 50 msec followed by a 10 msec delay between
consecutive pulses was used throughout the PaWL test session aspreviously described [37].
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The stimulation intensity used for each mouse was determinedfrom the current:force curve relationship: we determined the
maximum force and then extrapolated to the current required to
produce 2/3 of the maximum force. This stimulation intensity
then was used for the PaWL testing. We previously have shown
this to be the optimal force level needed to elicit instrumental paw
withdrawal learning [35,37]. To determine the optimal force, one
end of a silk thread was tied firmly around the distal end of themetatarsals just proximal to the metarsophalangeal joint and theother end was attached to a force transducer (Dual Mode Muscle
Lever 300BLR, Aurora Scientific Inc., Aurora, Ontario, Canada).
A sequence of stimuli (0 to 1.0 mA at increments of 0.1 mA andwith a 30 sec delay between each stimulus) was administered andthe force was recorded [37].
To begin the PaWL testing, we positioned the mouse such thatits foot was in view of the camera. To identify the resting positionof the paw, three rapid priming stimuli were applied at thedetermined stimulation intensity. The paw position was tracked
continuously using a video-based point tracking system (CMUCam2; Carnegie Mellon University). The video information thenwas converted to two-dimensional axial components for kinematics analysis [37].
A stringent vertical threshold for the paw was set at 1.5 mmabove resting position. We initially tested vertical thresholds at 1,
1.5, and 2.0 mm in a pilot study using three mice in each of the
CtrlDiet+Sed and DHA/Cur+Sed groups. At a 1 mm threshold
both groups learned, indicating that this threshold was notdiscriminating. At 2 mm neither group learned, indicating that
this threshold was too difficult. At the 1.5 mm threshold there wasa group difference in the level of learning.
The duration of the PaWL test session was 30 min and the levelof learning was assessed by the response duration. The response
duration reflects the time that the paw is above the threshold
during the PaWL test and incorporates the number of times thefoot drops below the threshold resulting in a shock. The response
duration is calculated as follows:syntaxin 3: forward (59- GCTGGAAGAGATGTTGGA GAG 39); reverse (59- TGCTTGGAAATCTGGGAGTC -39); CREB:
forward (59- ACAGA TTGCCACATTAGCCC -39); reverse (59GAGACTGGATAACTGATGGCTG -39); CaMKII: forward(59- CTTTCAGCCAGAGATCACCAG -39); reverse (59- ACCAGTAA CCAGATCGAAGATAAG -39). GAPDH: forward(59- CTTTGTCAAGCTCATTTCCTGG -39); reverse (59TCTTGCTCAGTGTCCTTGC -39). The mRNAs for BDNF,
syntaxin 3, CaMKII, and CREB were measured using the CFX96
Real-Time PCR Detection System (Bio-Rad). GAPDH gene wasused as an endogenous control to standardize the amount ofsample loading. The amplification cycle at which the firstsignificant increase of fluorescence occurred was designated as
the threshold cycle (CT). The CT value of each sample then wascompared with those of the internal standard (GAPDH). The
resulting corrected values were used to make comparisons acrossthe different experimental groups. The mean mRNA levels werecomputed for the four groups. To compare mRNA levels between
all experimental groups, we expressed the mRNA levels as apercent of the CtrlDiet+Sed group: the mean mRNA level in eachgroup was divided by the mean of the mRNA level in the
CtrlDiet+Sed group and then multiplied by 100 to express thevalues as a percent (Fig. 2A, C, E, and G). To compare the mRNAlevels between the experimental and saline group, the normalization was done similarly and values were expressed as a percent of
the saline group (Fig. 4C–F). Error bars for the CtrlDiet+Sed orsaline group represent the variation for the individual percent
values in that group (Fig. 4C–F).

Gas Chromatography
Total lipids from entire cervical region of the mouse spinal cord
(both sides) were extracted according to the method of Bligh andDyer [87]. Briefly, the tissues were homogenized with 2 ml lysis
solution of chloroform-methanol (2:1 vol:vol ) including 0.005%
butylated hydroxytoluene. C23:0Me (tricosanoic acid methylester)
was added as an internal control. After centrifugation, the liquid
was mixed with 0.5 ml 9% NaCl. The chloroform layer containing
lipids was collected and dried under nitrogen. Lipids weretransmethylated using the BF3/methanol reagent (14 wt/v%
Boron Trifluoride) at 90u for 1 hr as described previously[52,55]. Fatty acid composition was analyzed by gas chromatograph (Clarus 500, PerkinElmer, Waltham, MA) equipped with an
Elite-WAX column (60 m, 0.32 mm internal diameter, PerkinElmer). The injector and detector temperature was held at 250uC
and 300uC, respectively. Hydrogen was used as the carrier gas
with a split ratio of 100:1. Peaks were identified by comparison
with fatty acid standards (GLC Reference standard 682, Nu-ChekPrep, Elysian, MN, and 37-component FAME mix, sigmaAldrich, Carlsbad).

Response Duration~(time (sec) the paw is above the threshold)=(# of shocksz1)

Data collected include the vertical and horizontal paw position,
the threshold, and the time when the mice were shocked during all
PaWL trials. After completion of the test, the data were postprocessed using custom scripts written in MATLAB (The Math
Works, Inc., Natick, MA, USA). Data are reported as the response
duration for each min binned over the 30-min test (e.g., Fig. 1A) oras the sum of the response duration at each min of the 30-min test
(total response duration) (e.g., Fig. 1B). Thirty min after thecompletion of the PaWL test, the spinal cord was quickly dissected
and fresh frozen on dry ice for lipids and mRNA measurements.

Statistics
Data are reported as the mean values 6 standard error of themean (SEM). For comparisons including four groups, a one-way
analysis of variance (ANOVA) and Tukey’s post-hoc tests wereused to determine overall and individual group differences,respectively. Repeated measures ANOVAs were used for the
response duration across time bin analyses. Pearson productcorrelations were used to determine the relationships between
response duration (learning) vs. all plasticity markers and the levelsof fatty acids in the spinal cord. For comparisons including two
groups (BDNF blocking experiments, Fig. 4), unpaired MannWhitney t-tests were used. All analyses were performed using
GraphPad (GraphPad Software Inc. San Diego, CA). The level ofsignificance was chosen as P,0.05 for all comparisons.

RT-PCR measurements
Total RNA was isolated using the RNA STAT-60 kit (TELTEST, Inc., Friendswood, TX, USA) as per the manufacturer’s
protocol. Total RNA (100 ng) was converted to cDNA using
iScript cDNA Synthesis kit (Bio-Rad). The SsoFast EvaGreen
Supermix kit was used for qPCR and the cycling conditions wereaccording to the manufacturer’s protocol (Bio-Rad). The sequences of the primers were designed using the Integrated DNA
Technologies (IDT) online software (‘‘IDT SciTools RealTime
PCR’’). BDNF: forward (59- TTACCTTCCTGCATCTGTTGG-39); reverse (59- AACATT GTGGCTTTGCTGTCCTGG -39);
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8

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Diet and Exercise Affect Spinal Cord Learning

the data: MSJ ZY YMZ. Contributed reagents/materials/analysis tools:
MSJ ZY NT. Wrote the paper: MSJ FGP ZY VRE RRR.

Author Contributions
Conceived and designed the experiments: FGP VRE MSJ ZY NT.
Performed the experiments: MSJ ZY YMZ HSB AW RC HZ. Analyzed

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