JOURNAL OF NEUROTRAUMA 28:299–309 (February 2011)ª Mary Ann Liebert, Inc.
DOI: 10.1089/neu.2010.1594

Activity-Dependent Increase in Neurotrophic Factors
Is Associated with an Enhanced Modulationof Spinal Reflexes after Spinal Cord Injury
Marie-Pascale Côté, Gregory A. Azzam, Michel A. Lemay, Victoria Zhukareva, and John D. Houlé

Abstract

Activity-based therapies such as passive bicycling and step-training on a treadmill contribute to motor recovery
after spinal cord injury (SCI), leading to a greater number of steps performed, improved gait kinematics,
recovery of phase-dependent modulation of spinal reflexes, and prevention of decrease in muscle mass. Bothtasks consist of alternating movements that rhythmically stretch and shorten hindlimb muscles. However, theparalyzed hindlimbs are passively moved by a motorized apparatus during bike-training, whereas locomotor
movements during step-training are generated by spinal networks triggered by afferent feedback. Our objective
was to compare the task-dependent effect of bike- and step-training after SCI on physiological measures of spinal
cord plasticity in relation to changes in levels of neurotrophic factors. Thirty adult female Sprague-Dawley rats
underwent complete spinal transection at a low thoracic level (T12). The rats were assigned to one of threegroups: bike-training, step-training, or no training. The exercise regimen consisted of 15 min/d, 5 days/week, for
4 weeks, beginning 5 days after SCI. During a terminal experiment, H-reflexes were recorded from interosseus
foot muscles following stimulation of the tibial nerve at 0.3, 5, or 10 Hz. The animals were sacrificed and thespinal cords were harvested for Western blot analysis of the expression of neurotrophic factors in the lumbar
spinal cord. We provide evidence that bike- and step-training significantly increase the levels of brain-derived
neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4 in the lumbar enlargement of SCI rats, whereas
only step-training increased glial cell-derived neurotrophic factor (GDNF) levels. An increase in neurotrophic
factor protein levels that positively correlated with the recovery of H-reflex frequency-dependent depression
suggests a role for neurotrophic factors in reflex normalization.
Key words: complete transection; cycling; exercise; locomotor training; spinal cord injury

Introduction

S

pinal cord injury (SCI) unequivocally reduces theamount of activity performed by an individual (van den
Berg-Emons et al., 2008). Rehabilitative strategies routinely
integrate activity-based therapies to induce repetitive activation of the neuromuscular system, to limit further structural/anatomical loss and facilitate functional recovery. Activitydependent processes triggered by exercise affect multiple
neurotrophic factors by increasing the level of brain-derived
neurotrophic factor (BDNF; Dupont-Versteegden et al., 2004;
Gomez-Pinilla et al., 2004; Hutchinson et al., 2004), glial cellderived neurotrophic factor (GDNF; Dupont-Versteegden
et al., 2004), neurotrophin-3 (NT-3; Gomez-Pinilla et al., 2004;
Hutchinson et al., 2004), and neurotrophin-4 (NT-4; Skupet al., 2002) in the spinal cord and hindlimb muscles. Neuro-

trophic factors are powerful regulators of neuronal survival,
maintenance, and synaptic strength. After SCI, exogenous
neurotrophic factors can rescue neurons (Novikova et al.,2000, 2002), promote regeneration (Bregman et al., 2002;
Dolbeare and Houlé, 2003; Ma et al., 2010; Ye and Houlé,1997), and facilitate functional recovery (Boyce et al., 2007;
Fortun et al., 2009; Jakeman et al., 1998). Neurotrophic factors
also have significant effects on the development of motor
pathways (Seebach et al., 1999), and synaptic transmission(Mendell and Arvanian, 2002; Mendell et al., 2001).
Studies in which BDNF action was blocked using TrkB-IgG
chimera showed that BDNF is required for the success of instrumental spinal learning (Gomez-Pinilla et al., 2007)
and motor recovery (Ying et al., 2008). High levels of BDNF
within the lumbar spinal cord are associated with an increased effect of descending drive on the tibial motoneuronal

Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania.

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pool, normalization of motoneuronal properties, resolution ofallodynia, and improved locomotor recovery (Beaumont et al.,2008; Hutchinson et al., 2004; Ying et al., 2008). Recent findings indicate that BDNF serum levels are increased as well in
SCI athletes (Rojas Vega et al., 2008).
After SCI, the loss of supraspinal control and the subsequent increase in the gain of afferent feedback causes the excitability of spinal reflexes to be exacerbated. Therefore, theability to modulate spinal reflexes is decreased, resulting in
long-term undesirable effects such as inappropriate timing ofactivation of muscles during movement (Knikou, 2010; Nielsen et al., 2007). Hyperreflexia is also manifest by the incapacity to decrease the transmission of the H-reflex, theelectrical analog of the stretch reflex, when successive volleys
are transmitted from group Ia afferents to motoneurons(Field-Fote et al., 2006; Grey et al., 2008; Lee et al., 2005, 2007;
Lloyd, 1957; Thompson et al., 1992). Impaired depression of
the spinal reflexes with repetitive stimulation has been correlated with spasticity, and passive exercise helps recover Hreflex depression and decrease spasticity in animals (Reeseet al., 2006; Yates et al., 2008b), and humans (Kiser et al., 2005),
after SCI. Spastic patients also responded better to biketraining than did non-spastic individuals (Phadke et al., 2009).
Task-specific training has emerged as a crucial concept
when designing rehabilitation strategies (de Leon et al., 1998;
Edgerton et al., 1997). Although motor skills achieved bypracticing one task are not transferable to another skill, otherfunctional gains such as prevention of muscle mass loss anddecreased spasticity result from several types of exercise. Bothbike- and step-training consist of alternating movements thatrhythmically stretch and shorten hindlimb muscles; however,step-training entails an active loading component that does
not appear to be achieved with passive bicycling.
The objectives of this study were threefold: (1) characterizespinal plasticity evoked by bike- and step-training after acomplete SCI at the thoracic level as measured by changes inneurotrophic factor levels and H-reflex excitability; (2) compare spinal plasticity with different training paradigms, passive bicycling versus step-training on a treadmill; and (3)investigate the relationship between neurotrophic factor
protein levels and recovery of H-reflex frequency-dependent
depression.
Methods
All procedures were performed in accordance with protocols approved by the Drexel University College of Medicine
Institutional Animal Care and Use Committee, and followed
National Institutes of Health guidelines for the care and use oflaboratory animals.
Surgical procedures and postoperative care
Thirty adult female Sprague-Dawley rats underwent acomplete spinal transection at a low thoracic level (T12)under aseptic conditions. Briefly, the rats were anesthetizedwith isoflurane (1–2%) in oxygen. A laminectomy was performed at the T10–T11 vertebral level and the dura wascarefully opened. A 2-mm cavity was created by gentle aspiration, and saline-soaked surgical foam was used toachieve hemostasis. The complete removal of spinal cord
tissue was confirmed by the retraction of the rostral andcaudal portions of the cord, by examining the ventral floor ofCÔTÉ ET AL.
the spinal canal, and by subsequent histology. The dura wasclosed with 10-0 sutures. Overlying muscles were sutured
and the skin incision was closed with wound clips. The animals were given dextrose in saline (5 mL SC), buprenorphine (0.05 mg/kg IM) for 3 days as an analgesic, andampicillin (100 mg/kg SC) for 7 days to prevent infection.
The bladders were expressed manually twice a day until thevoiding reflex returned (*14 days).
Bicycle and locomotor training
The rats were randomly assigned to one of three groups:
passive cycling (n ¼ 8, bike-trained group), step-training on atreadmill with perineal stimulation and manual assistance(n ¼ 14, step-trained group), or no training (n ¼ 8, untrainedgroup). Beginning on day 5, the exercised groups received
15 min of daily exercise, 5 days a week, for 4 weeks. Briefly,
animals undergoing passive bicycling were secured in a
support harness with the flaccid hindlimbs hanging. The feetwere secured to the pedals with surgical tape. This custom
motor-driven apparatus moved the hindlimbs through acomplete range of motion at a rate of 45 rpm, as described
previously (Houlé et al., 1999; Ollivier-Lanvin et al., 2010;
Skinner et al., 1996).
Animals receiving step-training were placed over a motorized treadmill belt (*9–11 cm/sec) with the forelimbs
resting on an acrylic glass platform. Perineal stimulation was
continuously provided to induce and maintain bipedal locomotor movements (Etlin et al., 2010). Weight support of thehindlimbs was provided by the experimenter and adjusted toprevent collapsing of the hindlimbs; the animals typically
performed weight-bearing stepping with plantar placementof the foot 5–10 days after the onset of the training regimen(unpublished observations). Weight support was then solely
provided in between stepping episodes when no perineal
stimulation was provided. Step-trained animals provided
with perineal stimulation could perform >20 consecutive
steps (see Supplementary Video 1; see online supplementarymaterial at http://www.liebertonline.com).
H-reflex recordings and analysis
In a terminal experiment, the rats were anaesthetized withan IP injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). The tibial nerve was dissected free, mounted on a bipolar
hook electrode for stimulation, and skin flaps were used toform a pool of mineral oil to prevent desiccation. Bipolar wire
electrodes (Cooner Wire, Chatsworth, CA) were inserted into
the interosseus muscles for EMG recordings and the groundelectrode was inserted into the skin of the leg.
H-reflexes were evoked via an isolated pulse stimulator (AM Systems, Carlsborg, WA), delivering single bipolar pulses(100 msec) to the tibial nerve. H- and M-waves in the interosseus muscle were recorded in response to a range of increasing stimulus intensities to determine the threshold andthe maximal response amplitude for both the M- and H-wave(Mmax and Hmax). The stimulation intensity that elicited Hmax
response was then used for three series of 17 consecutive
stimulation pulses delivered at different frequencies (0.3, 5,
and 10 Hz). Before termination, the 0.3-Hz series was repeatedto verify that the M-wave was still within 95% of the initial
trial, otherwise the trial was discarded. The EMG recordings
were amplified (1000; A-M Systems), and bandpass filtered

NEUROTROPHIC FACTOR VERSUS SPINAL REFLEX MODULATION(10–5 kHz). The signal was digitized (10 kHz) and fed tocustom software.
Response latency (onset of response), peak latency (delay toreach peak), and peak-to-peak amplitude were measured for
the H and M responses evoked by single pulses. The recruitment curve was plotted by expressing the amplitude of the
response as a function of stimulus intensity. Mmax, the response of all motor units with supramaximal stimulation ofaxons of the tibial nerve, and Hmax, the maximal H-reflex
amplitude, were determined. The Hmax:Mmax ratio was calculated to assess the relative proportion of motoneurons recruited through the monosynaptic reflex loop as compared tothe activation of the entire motor pool. This measure isthought to give an estimate of motoneuronal excitability. The
Hmax:M ratio was also calculated to estimate the relative activation of the motor pool required to reach maximal reflex
amplitude. The slope of the recruitment curve was evaluatedfrom a linear regression line fitted to the recruitment curve, asreported by others (Thompson et al., 1992).
For the analysis of frequency-dependent depression of the
H-reflex, the first five responses to a train of stimulation werediscarded to allow reflex stabilization. The 12 remaining
responses were then averaged for every train frequency (0.3,5, and 10 Hz). Peak-to-peak amplitude of the M and H
responses were then measured. H-reflex amplitude wasnormalized to Mmax, and the change in H-reflex response at
5 Hz and 10 Hz was calculated as a percentage of the
response measured at 0.3 Hz.
Western blots
Thirty days after spinal cord transection, the rats were
overdosed with Euthasol (390 mg/kg sodium pentobarbital
and 50 mg/kg phenytoin IP). The T10–L6 spinal cord was
rapidly harvested, the spinal roots and meninges were stripped off, and four separate spinal segments (T10–T11, T12–L1,
L2–L3, and L4–L6) were prepared and placed in cold RIPA
buffer (50 mM Tris buffer [pH 7.6], 0.1% SDS, 0.25% deoxycholate, and 150 mM NaCl), in the presence of protease andphosphatase inhibitors (Roche Diagnostics, Indianapolis, IN),
2 mM phenyl-methyl sulfonyl-fluoride (PMSF), and 1 mM
sodium fluoride.
Tissue blocks were sonicated and centrifuged at 14,000g for
40 min at 48C. The supernatants were collected and aliquots
were stored at 808C. For Western blot analysis, the samples
were boiled in Laemmli sample buffer for 5 min, and equalamounts of total protein were separated on 10% SDS-PAGE
gels and transferred onto polyvinylidene difluoride (PVDF)
membranes (BioRad, Hercules, CA). Each nitrocellulose replica was blocked with 5% nonfat milk in Tris-buffered salinewith 0.1% Tween-20 (TBS-T), probed with primary rabbit
polyclonal antibodies against BDNF (1:400; Abcam, Cambridge, MA), GDNF (1:200; Santa Cruz Biotechnology, Santa
Cruz, CA), NT-3 (1:200; Abcam), or NT-4 (1:200; Santa Cruz
Biotechnology), followed by incubation with the horseradish
peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (IgG; Jackson ImmunoResearch Laboratories, West
Grove, PA). Blots for each sample were run two or three times
for each primary antibody to ensure replication of the results.
To confirm equal loading of protein in each lane, the blots
were stripped using buffer containing 65 mM Tris buffer (pH6.8), 2% SDS, and 1% b-mercaptoethanol for 30 min, and re-301

probed with mouse monoclonal anti-actin antibody (1:8000;
Sigma-Aldrich, St. Louis, MO). Immunoreactivity was detectedusing an enhanced chemiluminescence kit (ECL; Amersham
Biosciences, Piscataway, NJ), and optical densities of the protein bands were assessed using GeneTool Analysis software(Syngene, Frederick, MD). Values for each sample were normalized to actin and combined for each group. Final data(mean  standard error of the mean [SEM]) are presented as aratio of the untrained group, which was assigned a value of 1.0.
Statistical analysis
A one-way analysis of variance (ANOVA) followed by theHolm-Sidak post-hoc test were used to determine significant
differences across groups for all data unless stated otherwise.
If the sample variables did not fit a normal distribution orwere not equally variant, a one-way ANOVA on ranks followed by Dunn’s post-hoc test was performed.
A two-way ANOVA followed by the Holm-Sidak post-hoc
test was used to assess whether stimulation frequency andtreatment group had a significant effect on the amplitude of
the H-reflex, and to evaluate if the interaction of these factors
affected the variable. All data are reported as mean  SEM.
The Fisher’s exact test evaluating the frequency distribution
was used to identify differences between groups for the occurrence of H-reflexes with a threshold below the motor
threshold. Linear regression analysis was used to correlate
neurotrophic factor levels to the rate of H-reflex depression.
Statistical analysis was performed using Sigma Plot software
11.0 and PASW Statistics 18. For all statistical tests, the significance level was set to p < 0.05.
Results
Bike-training and step-training increaseneurotrophic factor levels in the spinal cord
Figure 1 illustrates representative Western blots with positive
bands for BDNF (*18 kDa), NT-4 (*27 kDa), NT-3 (20 kDa),
and GDNF (*37 kDa) protein, for two untrained, two biketrained, and two step-trained animals. Protein expression levels
were averaged and compared across groups for separate spinallevels rostral (T10–T11) and caudal (T12–L1, L2–L3, and L4–L6)
to the lesion site. Bike- and step-training increased the expression of BDNF in the L2–L6 lumbar cord compared to untrained
animals (Fig. 1A and A’). This increase was significantly larger
in the bike-trained groups than in the step-trained group in theupper lumbar segments (L1–L3), but not in the lumbar enlargement (L4–L6). NT-4 (Fig. 1B and B’) and NT-3 protein
levels (Fig. 1C and C’) were amplified in all caudal segments of
the lumbar spinal cord after bike-training or step-training. The
GDNF protein level was increased in the L4–L6 region after
step-training, but not after bike-training (Fig. 1D and D’). Only
NT-3 and GDNF levels were increased in spinal segments
rostral to the transection injury (T10–T11) following either exercise regimen. We observed that bike-training tended to have alarger effect than step-training at L1–L3 for BDNF, NT-3, andNT-4 (significant only for BDNF), but in the L4–L6 region they
were very close in expression level.
Step-training modifies spinal excitability
The H-reflex is frequently used to assess motoneuronal
excitability after an injury to the spinal cord with or without

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FIG. 1. Western blots showing the expression of brainderived neurotrophic factor (BDNF) (A), neurotrophin-4 (NT4) (B), NT-3 (C), and glial cell-derived neurotrophic factor(GDNF) (D) rostral to the injury site (T10–T11), at the lesion
site (T12–L1), and caudal to the injury site (L2–L3 and L4–L6),
in untrained, bike-trained, and step-trained animals (n ¼ 8 pergroup). Overall, bike- and step-training increased the expression of BDNF (A’), NT-3 (B’), NT-4 (C’), and GDNF (D’). Also,
step-training increased GDNF protein levels in the lumbar
enlargement more than bike-training (D’). The blots were reprobed with anti-actin to provide an indication of total protein
loaded per lane (E). BDNF, NT-3, NT-4, and GDNF proteins
were detected and quantified by Western blots using actin asa standard control (*p < 0.05 versus untrained; **p < 0.001 versus untrained; #p < 0.05 versus bike-trained; ##p < 0.001 versus
bike-trained).
treatment. It provides an estimation of neurotransmission
from Ia afferents to target motoneurons. A single stimulus
evokes two subsequent EMG responses: the initial M-wave,
which results from the direct activation of motor axons, andthe delayed H-wave, evoked by the synaptic activation of
motoneurons by group Ia muscle afferents. Therefore, the H-

CÔTÉ ET AL.
reflex yields information about the properties of the monosynaptic reflex. Table 1 describes the features of the H-reflex in
the different groups of animals, indicating that there were no
significant differences between untrained and trained groups
with regard to latency, amplitude, and threshold of M- and Hwaves. For example, the H-reflex response latency and peaklatency were 8.49  0.20 msec and 9.31  0.23 msec, respectively, in untrained animals, and this was not modified
by bike-training or step-training. Exercise did not further
modify M-wave response latency and peak latency compared
to untrained animals (2.30  015 msec and 3.24  0.12 msec,
respectively).
Motor threshold (MT), which is the lowest stimulation intensity evoking an M-wave, was similar across groups (Table
1). On average, the H-reflex appeared slightly above MT in
untrained animals (1.02  0.09 MT), whereas it required aweaker stimulation intensity in exercised animals (biketrained: 0.99  0.06 MT; step-trained: 0.96  0.02 MT), but thisdifference was not significant. Further analysis showed thatthe H-reflex appeared at stimulation intensities lower than
MT in 14% of untrained, 50% of bike-trained, and 79% of steptrained animals. Fisher’s exact test revealed an increased occurrence of an H-response below MT in step-trained animals
versus the other groups ( p < 0.001).
Recruitment curves were constructed by plotting the Hreflex amplitude against a series of stimulus intensities, represented here as a multiple of the motor threshold. Figure 2A
illustrates a recruitment curve with an initial linear component(*1–1.3 MT), followed by an asymptotic portion (*1.5 T), with
an M-wave reaching maximal amplitude around 2 MT in untrained animals, as has been reported previously (Thompson
et al., 1992). We observed differences in the shape of the recruitment curves after bike-training and step-training. The
Hmax tended to occur closer to MT than in untrained animals(Fig. 2A and B), and consequently occurred when the M-wave
was of much smaller amplitude, close to MT, in the ascending
part of the recruitment curve. This is expressed as an increase in
the Hmax:M ratio (Fig. 2C), which represents the fraction of
motoneurons activated to reach a maximal H-reflex.
The Hmax:Mmax ratio estimates the fraction of motoneurons
recruited via the H-reflex relative to the activation of the entire
motor pool. There was a tendency toward an increased ratio
in exercised animals, with values of 0.546 for bike-trained
animals, and 0.614 for step-trained animals, compared to
0.367 in the untrained group (Fig. 2D). Statistical tests were
below the desired power because of the wide range of values
displayed in the exercised groups. As illustrated in Figure 2A,
exercised animals displayed inconsistent recruitment
curves, with some of the step-trained animals even showing
H-reflex amplitude larger than the M-wave amplitude (i.e.,
Hmax:Mmax > 1).
The slope of the recruitment curve is one of the determinants of the gain of the reflex pathway. It has been shown to
be markedly reduced at 28 days after SCI, with values around
3.35, versus 6.79 for intact animals (Thompson et al., 1992). We
observed an increase in slope after exercise, from 3.217 in
untrained animals to 7.990 in bike-trained and 11.064 in steptrained animals. Only step-training significantly increased
slope compared to untrained animals ( p < 0.046). Together,
the decrease in H-reflex initiation threshold and steeper slopeof the recruitment curve in the step-trained group suggest an
increase in reflex gain.

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303

Table 1. Properties of the M-Wave and H-Reflex: Latency, Amplitude, and Threshold

Untrained (n ¼ 8)
Bike-trained (n ¼ 8)
Step-trained (n ¼ 12)

Motor
M-waveM-wave peak
H-reflex
H-reflex peak
H-reflex
threshold (mA) latency (msec) latency (msec) latency (msec) latency (msec) threshold (xMT)

Hmax
(xMT)

0.028  0.007
0.040  0.005
0.037  0.008

1.5  0.2
1.4  0.2
1.2  0.1

2.30  0.15
2.19  0.11
2.33  0.11

3.24  0.12
3.00  0.16
3.07  0.11

8.49  0.20
8.06  0.20
8.07  0.18

9.31  0.23
8.93  0.20
8.94  0.19

1.02  0.09
0.99  0.06
0.96  0.02

Values are mean  SEM.
MT, motor threshold.

The modulation of the H-reflex
is recovered after exercise
Figure 3 shows representative recordings of the H-reflex at
0.3 Hz and 10 Hz from individual untrained (A), bike-trained(B), and step-trained (C) animals. These recordings demonstrate that increasing the frequency of stimulation leads to asmaller decrease in H-reflex amplitude in untrained animals(78%), compared to bike-trained (18%) or step-trained (4%)
animals. Figure 3D illustrates the H-reflex amplitude (normalized to Mmax) expressed as a percentage of the baseline
value obtained at a frequency of 0.3 Hz. Therefore, the lower
the value on the y axis, the greater the reflex depression,
whereas higher y values indicate less depression of the Hreflex. As a group, exercised animals displayed a better
modulation of the H-reflex both at 5 Hz and 10 Hz comparedto the untrained group (Fig. 3D), going from 71% (untrained)
at 5 Hz, to 39% in bike-trained and 29% in step-trained
animals, and from 62% (untrained) at 10 Hz, to 29% in
bike-trained and 18% in step-trained animals. A two-way
ANOVA revealed statistically significant differences acrossstimulation frequency ( p < 0.001), across experimentalgroups ( p < 0.001), and for the interaction between frequency and group ( p < 0.001). Post-hoc comparisons showedthat 5-Hz and 10-Hz values were different than 0.3-Hz values in all groups, and that bike-trained and step-trained
values were different from untrained animals, both at 5 Hz
and 10 Hz, suggesting that exercise leads to restoration of Hreflex depression.
Relationship between neurotrophic
factor levels and H-reflex depression
To determine if there was a relationship between neurotrophic factor levels and recovery of the H-reflex, BDNF, NT3, NT-4, and GDNF protein levels for each animal wereplotted against the individual amplitude of its H-reflex at
10 Hz (expressed as a percentage of the amplitude at 0.3 Hz).
Linear regression analysis yielded a significant relationshipbetween H-reflex frequency-dependent depression recovery
and BDNF ( p < 0.025, R2 ¼ 0.262), NT-3 ( p < 0.011, R2 ¼
0.307), and NT-4 protein levels ( p < 0.002, R2 ¼ 0.492), in the

FIG. 2. (A) Representative recruitment curves for individual animals in the untrained, bike-trained, and step-trained groups.
The untrained animals display Hmax *1.5 MT, and Mmax *2 MT. Bike- and step-training shifted the Hmax value toward 1 MT.
(B) Hmax occurrence expressed as a multiple of MT. (C) The Hmax:M ratio tends to increase with step-training, suggesting that
Hmax occurs when M is of smaller amplitude than in untrained animals. (D) The Hmax:Mmax ratio tends to increase with exercise.
(E) The slope of the recruitment curve is significantly steeper in step-trained animals than in untrained animals (MT, motor
threshold; Mmax, maximal response amplitude for the M-wave; Hmax, maximal response amplitude for the H-wave).

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lumbar enlargement (Fig. 4). In addition, the untrained animals (gray boxes in Fig. 4) were linearly separable from theexercised groups, being segregated in the upper left corner(i.e., untrained animals showed low neurotrophic factor levels
and little depression of the H-reflex). GDNF protein levels
were not correlated with the depth of modulation of the Hreflex (Fig. 4D; p ¼ 0.455, R2 ¼ 0.0313). Frequency-dependent
depression was low in untrained animals (high on the y axis),
compared to exercised animals (low on the y axis), but theCÔTÉ ET AL.
GDNF protein level was similar across all animals, (i.e., thevalues were clustered around 1 on the x axis).
Discussion
Local reflexes are considered to play an important role after
SCI (Pearson, 2001). The results of the present study demonstrate significant changes in a simple monosynaptic reflex
pathway of the lumbar spinal cord induced by step- or biketraining after SCI. Different measures of H-reflex excitability
were analyzed in animals with complete spinal cord transection that were either sedentary (untrained), step-trained, or
bike-trained: reflex threshold, slope of the recruitment curve,
maximal H-reflex:maximal M-response (Hmax:Mmax ratio),and frequency-dependent depression. We showed that steptraining decreased the H-reflex threshold and facilitatedthe recruitment of the motoneuronal pool in response toafferent input. Also, both step- and bike-training restored thefrequency-dependent depression of the H-reflex, andincreased neurotrophic factor levels in the spinal cord. This
indicates both a facilitation of the transmission in this reflex
pathway, and a better modulation when repetitive stimuli
were applied. More importantly, the increased level of neurotrophic factors in the lumbar enlargement was predictive of
the recovery of spinal reflex modulation. Effects of exercise
were not limited to regions of the spinal cord directly associated with hindlimb activity (i.e., a significant increase of
neurotrophic factors was observed in the lower thoracic cord,and even rostral to the complete transection lesion).
A primary objective of activity-based rehabilitation is toprovide optimal sensorimotor cues to activate spinal networks. Sensory information is critical to motor learning, as thepattern and timing of information that is provided plays acritical role in functional recovery (de Leon et al., 1998, 1999).
Stepping and biking are repetitive and rhythmical movements that involve alternation between flexion and extensionof the hip, knee, and ankle. Passive biking provides a steadyand timed afferent flow of information to the spinal cord, anddoes not necessitate high levels of postural control, whereas
stepping is incontestably a more complex task, that involves
weight-bearing, foot placement, and constant control of posture and position of limb joints. Our passive biking model
does not involve cycle-to-cycle variation in the kinematics andsensorimotor feedback as is the case for stepping. Interest-

FIG. 3. H-reflex frequency-dependent depression is recovered after 30 days of step-training and bike-training in spinal
cord injury (SCI) animals. H-reflexes were evoked by thestimulation of the tibial nerve and recorded from the interosseus muscles. Representative averages of H-reflex recordings following a train of stimulation at 0.3 Hz (black), and
10 Hz (gray), in untrained (A), bike-trained (B), and steptrained (C) animals. There was a modest decrease in the
average amplitude of the H-reflex at 10 Hz compared to
0.3 Hz in untrained animals, whereas this decrease wasconsiderable in bike-trained and step-trained animals.
Overall, the animals in the exercised groups showed amarked reduction of the H-reflex amplitude as the stimulation frequency increased (i.e., increased depression of thereflex), compared to the untrained group (D). There was asignificant decrease in H-reflex amplitude in bike-trained
and step-trained animals compared to untrained animals at
both 5 Hz and 10 Hz (D; *p < 0.05; **p < 0.001).

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305

FIG. 4. Linear regression analysis between H-reflex depression and brain-derived neurotrophic factor (BDNF) (A),neurotrophin-4 (NT-4) (B), NT-3 (C), and glial cell-derived neurotrophic factor (GDNF) (D) protein levels in the L4–L6 spinal
segments. There was a significant correlation between BDNF, NT-3, and NT-4 levels, and the depth of the H-reflex depression
at 10 Hz (expressed as a percentage of 0.3 Hz). This correlation was not observed with GDNF. Untrained animals aresegregated in the upper left quadrant, and are linearly separable from the exercised groups (gray boxes), for BDNF, NT-3, andNT-4, expressed as a function of H-reflex depression, but GDNF is not.

ingly, some variation in the pattern has been shown to be of
crucial importance for the control and recovery of repetitive
movements (Cai et al., 2006; Hausdorff, 2005; Ziegler et al.,2010).
Step-training facilitates transmission
in the H-reflex pathway
The H-reflex is an objective measure of the physiologicalchanges occurring after SCI. The threshold for H-reflex initiation has been shown to occur below MT in some animals andabove MT in others (Hosoido et al., 2009). We showed that the
proportion of animals in which an H-reflex was elicited below
the motor threshold was increased in the step-trained group.
In addition, step-training increased the initial slope of therecruitment curve. The relatively steep slope of the recruitment curve indicated that once the minimum reflex excitability level was reached, additional units were readily
recruited with only a minimal increase in the relative amplitude of the afferent volley. The excitatory drive to a motor
pool required to initiate a reflex discharge and the subsequent
progression of recruitment depends on the distribution of
excitability among the motoneurons of a given pool (Hunt,
1955; Kernell and Hultborn, 1990).

Because the amplitude of the H-reflex is proportional to thenumber of motoneurons activated, an increase in Hmax:Mmax
ratio after exercise would likely reflect an amplified transmission from Ia afferents to motoneurons. We observed a tendency
toward an increase in Hmax:Mmax ratio after bike-training or
step-training, but it did not reach statistical significance, mainly
because some of the animals displayed an Hmax of higher
amplitude than Mmax, yielding great variability in the ratio.
Variability in electrophysiological features in step-trained animals has been reported previously, and has been shown tocorrelate with success of recovery (Petruska et al., 2007). Together, these results suggest that transmission in this reflex
pathway was facilitated, at least in a subset of animals, and thatexercise impacts individuals to varying degrees.
Our observations indicate that step-training increased the
lumbar cord reflex excitability, and this suggests an increasein the gain of this reflex pathway. An enhancement of group Ia
afferent-evoked monosynaptic excitatory postsynaptic potentials (EPSPs) has been reported previously in step-trainedtransected rats. An EPSP of large amplitude was associatedwith successful step-training, whereas poor steppers displayed EPSPs of smaller amplitude (Petruska et al., 2007).
Evidence suggests that the recovery of locomotor function isfacilitated by an increase in hindlimb reflex activity, produced

306
in part by enhanced segmental synaptic input via collateralsprouting of primary afferent terminals (Murray and Goldberger, 1974).
Restoration of frequency-dependent
depression is not load-dependent
When the spinal cord is intact, the amplitude of the H-reflex
decreases considerably with increasing stimulus frequency(Lee et al., 2005; Ollivier-Lanvin et al., 2010; Thompson et al.,1992). Since biological stimuli are usually transmitted as repetitive signals, the mechanism that modulates motor output
relative to repetitive input is thought to shape reflex excitability (Koerber and Mendell, 1991). This ability to attenuate
the response is impaired after complete or incomplete SCI inanimals and humans (Boulenguez et al., 2010; Grey et al.,2008; Ollivier-Lanvin et al., 2010; Thompson et al., 1992).
Previous studies have shown that passive exercise (i.e., cycling) helps to recover the frequency-dependent depression of
the H-reflex in animals (Reese et al., 2006; Skinner et al., 1996)
and humans (Kiser et al., 2005; Phadke et al., 2009) after SCI.
Our results suggest that both passive bicycling and steptraining induce robust effects on the modulation of reflex
pathways independently of the loading component, timing ofmuscle activation, and cycle-to-cycle variation. The rhythmic
motion elicited by exercise provides recurrent signaling thatmodifies hyper-reflexive circuitry to restore frequencydependent depression of the H-reflex.
The load experienced by the hindlimbs during cycling andstepping was not quantified in this study. It is unknown how
much load was experienced during passive cycling in our
animals; however, the animal was suspended in a harness toprevent weight-bearing on the hindlimb muscles. Previous
reports have shown that the soleus muscle is activated duringflexion of the foot (Houlé et al., 1999). In humans, the soleus
muscle, which is also activated during flexion when pedaling,has been shown to be much smaller during passive thanduring active cycling, which requires force generation(Boorman et al., 1992). In these conditions, it is unlikely thatmuch loading occurs during our bicycle training paradigm.
We cannot rule out a contribution of perineal stimulation incausing changes on our two key outcome measures. However, we believe it is unlikely that it was responsible for the
improvement in H-reflex depression and increase in neurotrophin levels, because similar results were observed in biketrained animals, which did not receive perineal stimulation.
This suggests that exercise triggers these changes and notafferent feedback induced by perineal stimulation.
Mechanisms underlying frequency-dependent
depression
Frequency-dependent depression provides an estimationof the inhibitory influence that contributes to changes in reflex
excitability during movement. Hence, under dynamic conditions, the H-reflex undergoes cyclic changes with a variety ofrhythmic behaviors, such as stepping (Crenna and Frigo,1987), running (Capaday and Stein, 1987), and pedaling(Brooke et al., 1992). With reflex modulation being phasedependent and task-dependent (reviewed in Rossignol et al.,2006), future studies need to address and compare the modulation of spinal reflexes during the actual motor task (i.e.,
bicycling or stepping).

CÔTÉ ET AL.
The similarity between the H-reflex recorded from the interosseous muscle, and the monosynaptic reflex depression
recorded from the tibial nerve, indicates that hyperreflexia
after SCI is likely due to mechanisms of central origin rather
than an impairment of the neuromuscular junction (Thompson et al., 1992). Several explanations were investigated, including increased electrical coupling mediated by gap
junctions (Yates et al., 2008a), reciprocal facilitation of antagonistic muscles (Xia and Rymer, 2005), decreased presynaptic
inhibition (Hultborn et al., 1996; Reese et al., 2006; SchindlerIvens and Shields, 2000; Thompson et al., 1992), and changes
in the persistent inward currents (PICs; Bennett et al., 2001;
Hornby et al., 2006). PICs are thought to be involved in thedevelopment of spasticity after SCI. Interestingly, the recovery of the H-reflex depression is associated with a decrease inspasticity (Kiser et al., 2005; Phadke et al., 2009).
Neurotrophin level is predictiveof the depth of modulation of the H-reflex
Postsynaptic factors can also regulate the frequency oftransmission in reflex pathways (Koerber and Mendell, 1991).
SCI involves changes in cellular properties and synaptic relationships in spinal neurons (Mendell, 1984), which aremodulated by exercise (Beaumont et al., 2004, 2008; Petruska
et al., 2007). Individually, animals that displayed higher levelsof BDNF, NT-3, and NT-4, also exhibited better modulation of
spinal reflexes.
In an elegant series of experiments, Boulenguez and colleagues proposed a new mechanism that could explain thedisinhibition caused by the lack of descending influence after
SCI, in spite of the increased levels of inhibitory transmittersin the spinal cord (Boulenguez et al., 2010). Their results
suggest a role for KCC2, a potassium chloride co-transporter,in the depth of modulation of the frequency-dependent depression of the H-reflex (Boulenguez et al., 2010). Acute exogenous administration of BDNF increases KCC2 expression
and restores frequency-dependent depression (Boulenguez
et al., 2010). Hence, signaling pathways relating BDNF, TrkB
receptors, and KCC2, have been described (Rivera et al., 2002,
2004). It is well established that exercise increases BDNF
protein and mRNA levels in the spinal cord after SCI (Yinget al., 2005, 2008), and that blocking BDNF prevents favorable training effects (Gomez-Pinilla et al., 2007). Activitydependent processes triggered by exercise include an increasein the expression of neurotrophic factors in the spinal cordthat are associated with spinal learning and motor recovery
after SCI. The correlation between BDNF, NT-3, and NT-4
protein levels, and the amount of recovery of the H-reflex
depression evoked by repetitive stimuli, suggests a possible
common pathway in support of the Vinay hypothesis.
Unlike BDNF, NT-3, and NT-4, which showed a general
upregulation in the lumbar cord with exercise, GDNF was
upregulated primarily in spinal segments rostral to the injury.
This increase could influence trunk stability, and also stimulate regeneration above the injury level. In addition, only
step-training upregulated GDNF protein levels in the lumbar
enlargement. After SCI, the GDNF protein level is downregulated in the segment caudal to the lesion, suggesting a
potential role in physiological and pathological conditions(Zhou et al., 2008). Delivery of exogenous GDNF can promote
locomotor recovery (Cao et al., 2004; Hashimoto et al., 2005).

NEUROTROPHIC FACTOR VERSUS SPINAL REFLEX MODULATION
A transient increase in GDNF expression at the lumbar level
was previously described following passive exercise, but returned to untrained values within 30 days (Keeler et al., 2009),
suggesting that the time course of GDNF modulation may bedifferent for bike-training and step-training.
Conclusions
Our results suggest that both step-training and biketraining induce remarkable spinal cord plasticity that includes
an increase in neurotrophic factor levels and a concomitant
improvement in H-reflex depression. Neurotrophic factors
induce spinal plasticity through synaptic rearrangement, butalso act as chemo-attractants for elongating axons. Further
studies are needed to test whether this local increase in neurotrophic factors can promote axonal outgrowth across a
spinal lesion, thereby increasing the formation of new synaptic contacts with host neurons to further improve modulation of spinal reflexes. Future studies should also address
the long-term effect of combined activity-based therapies andtheir association with functional recovery.
Acknowledgments
We thank Kassi Miller for performing bike-training, Drs.
Jed Shumsky and Corey Hart for help with statistical analysis,
and Dr. Jeffrey Petruska for suggestions made during the data
analysis process. This study was supported by National Institutes of Health grant NS 055976 (to J.D.H.).
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:John D. Houlé, Ph.D.
Department of Neurobiology and Anatomy
Drexel University College of Medicine
Philadelphia, PA 19129
E-mail: jhoule@drexelmed.edu