doi:10.1093/brain/awt265

Brain 2013: 136; 3362–3377| 3362BRAIN
A JOURNAL OF NEUROLOGYUse of quadrupedal step training to re-engage
spinal interneuronal networks and improve
locomotor function after spinal cord injury
Prithvi K. Shah,1 Guillermo Garcia-Alias,1 Jaehoon Choe,2 Parag Gad,3 Yury Gerasimenko,1,4Niranjala Tillakaratne,1,5 Hui Zhong,1 Roland R. Roy1,5 and V. Reggie Edgerton1,5,6,7

Correspondence to: V. Reggie Edgerton, PhD,
Department of Integrative Biology and Physiology,
University of California,
Los Angeles,
610 Charles E. Young Drive,
Los Angeles,
CA 90095-1527,
USA
E-mail: vre@ucla.edu
Correspondence may also be addressed to: Prithvi K. Shah, PhD, E-mail: pshahucla@gmail.com

Can lower limb motor function be improved after a spinal cord lesion by re-engaging functional activity of the upper limbs? We
addressed this issue by training the forelimbs in conjunction with the hindlimbs after a thoracic spinal cord hemisection in adultrats. The spinal circuitries were more excitable, and behavioural and electrophysiological analyses showed improved hindlimb
function when the forelimbs were engaged simultaneously with the hindlimbs during treadmill step-training as opposed totraining only the hindlimbs. Neuronal retrograde labelling demonstrated a greater number of propriospinal labelled neurons
above and below the thoracic lesion site in quadrupedally versus bipedally trained rats. The results provide strong evidence that
actively engaging the forelimbs improves hindlimb function and that one likely mechanism underlying these effects is thereorganization and re-engagement of rostrocaudal spinal interneuronal networks. For the first time, we provide evidence thatthe spinal interneuronal networks linking the forelimbs and hindlimbs are amenable to a rehabilitation training paradigm.
Identification of this phenomenon provides a strong rationale for proceeding toward preclinical studies for determining whether
training paradigms involving upper arm training in concert with lower extremity training can enhance locomotor recovery after
neurological damage.

Keywords: spinal cord hemisection; rats; motor coordination; propriospinal system; quadrupedal locomotion

Received April 30, 2013. Revised August 26, 2013. Accepted September 1, 2013. Advance Access publication October 7, 2013
ß The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oup.com

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1 Departments of Integrative Biology and Physiology, University of California, Los Angeles, California 90095, USA
2 Neuroscience, University of California, Los Angeles, California 90095, USA
3 Biomedical Engineering, University of California, Los Angeles, California 90095, USA
4 Pavlov Institute of Physiology, St. Petersburg, 199034, Russia
5 Brain Research Institute, University of California, Los Angeles, California 90095, USA
6 Neurobiology, University of California, Los Angeles, California 90095, USA
7 Neurosurgery, University of California, Los Angeles, California 90095, USA

Forelimb activity enhances locomotor control

Introduction

Materials and methods
All experimental procedures were performed in compliance with the
University of California Los Angeles Chancellor’s Animal Research| 3363

Committee and complied with the guidelines of the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals (National Research Council, 2011).

Experimental groups, procedures andtimeline
The experimental groups and timeline for the study are shown in
Fig. 1. All surgeries were performed under aseptic conditions withthe rats deeply anaesthetized with isoflurane gas (1.0 to 2.5%). The
basic surgical procedures for the hemisection injury at T10 spinal segment and post-surgical care are as described previously (Shah et al.,2011). Forelimb (biceps brachii and triceps brachii) and hindlimb (tibialis anterior and medial gastrocnemius) muscles were implanted bilaterally with bipolar intramuscular EMG recording electrodes as
described previously (Roy et al., 1991; Shah et al., 2011). Epiduralelectrodes were implanted at spinal cord segment S1 at the sametime as the spinal cord hemisection (right side) surgery as described
previously (Shah et al., 2012). To identify the propriospinal neurons,
the retrograde tracer dye tetramethyl rhodamine dextran amine (TMD;
molecular weight of 3000; 10% dissolved in distilled water, Invitrogenmolecular probe D3308) was injected (by surgeons blinded to thestudy) below the hemisection into the right L2 segment of the spinal
cord. Injections at L2 were chosen to target the long propriospinal
terminal connections in the L2 intermediate grey matter that descendfrom the cervical enlargement at C5–C8 (Reed et al., 2006).
Rhodamine tracer dye injections (four sites, 0.1 ml per site) weremade stereotaxically into the right hemicord 0.67 mm lateral to themidline and at multiple depths starting at a depth of 1.2 mm andinjecting every 0.3 mm while retracting towards the cord dorsum
using a 100 mm glass micropipette mounted over a 10-ml Hamilton
syringe. A total of 3 min was allowed between each injection. Two
weeks after the tracer injections, the rats were anaesthetized deeply
with sodium pentobarbital (450 mg/kg intraperitoneally), perfusedtranscardially with 150 ml of Sorenson’s buffer solution containing
sodium phosphate and NaOH, followed by 400 ml of 4% phosphatebuffered paraformaldehyde, pH 7.4. The spinal cords were removed,
post-fixed for 1–3 h, and stored in 30% sucrose in 0.1 M phosphatebuffer.

Locomotor training
For quadrupedal step-training, the rats were placed on the treadmill
surface to walk with full weight bearing on their forelimbs and hindlimbs. For the first 3–5 days of training, the trainer supported the rat’s
trunk to facilitate ground clearance of the right hindlimb during stepping. No such support was necessary thereafter. For bipedal step-training, an upper body harness was used to position the rats over thetreadmill belt to enable bipedal locomotion (Shah et al., 2012). Rats
were trained 7 days/week, at 21 cm/s, 35 min/session for 2.5 weeks(total of 17 training sessions) starting 5 days after hemisection surgery.
Animal groups randomly went through testing sessions on the first day
and subsequent testing sessions were performed around the same timefor each animal.

Kinematics and electrophysiologicalanalyses of locomotor ability
Quadrupedal stepping ability on a treadmill at 21 cm/s was assessed
before and 4, 8, and 21 days post-hemisection. Data were collectedfrom 10–15 consecutive step cycles for the hindlimbs and forelimbs

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Abundant anatomical and electrophysiological evidence demonstrates that spinal neuronal networks are exquisitely linked andmodulated through ascending and descending propriospinal pathways (Jankowska et al., 1974; Juvin et al., 2005). Functionally, thepropriospinal intersegmental linkage can mediate and/or facilitate
motor coordination and regulate postural adjustments of the limb
and trunk muscles during a variety of motor tasks in rodents (Juvinet al., 2005, 2012), cats (Jankowska et al., 2000; Gerasimenko
et al., 2009), and humans (Dietz and Michel, 2009; Mezzarane
et al., 2011). Descending propriospinal neurons receive input fromthe long descending supraspinal system (Kazennikov et al., 1991)
as well as from direct afferent feedback from the limbs and trunkthat can excite motor neuronal pools independently of supraspinalcontrol (Akay et al., 2006). Furthermore, passive forelimb movements alone can facilitate hindlimb stepping in decerebrated catsthrough direct activation of propriospinal neurons (Gerasimenko
et al., 2009). Additionally, several studies have demonstrated
that the spared spinal pathways after a spinal cord injury undergo
extensive reorganization to increase recruitment of propriospinal
pathways for motor recovery, even in the absence of therapeuticinterventions (Bareyre et al., 2004; Darlot et al., 2012).
Collectively, these observations suggest that these interneuronal
pathways could be used in rehabilitation paradigms by attempting
to re-engage them after a severe spinal cord injury.
Activating interneuronal networks to enhance locomotor coordination has major implications for designing strategies that seek to
use interlimb networks in gait rehabilitation after neurological injury(Behrman and Harkema, 2000; Ferris et al., 2006). There is someevidence showing that in individuals with spinal cord injury, rhythmic
movements of the upper limbs enhance lower limb muscle recruitment during stepping (Visintin and Barbeau, 1994) or locomotor-like
activity (Ferris et al., 2006; Kawashima et al., 2008). Clinical evidence demonstrating the direct impact of upper extremity training
on lower extremity function, however, is preliminary and inconclusive (Behrman and Harkema, 2000; Tester et al., 2011). Moreover,animal studies using locomotor training paradigms to promote ipsilesional hindlimb locomotor recovery have not exploited the conceptof actively ‘training’ the forelimbs (Battistuzzo et al., 2012).
We hypothesized that active involvement of the forelimbs throughan intense quadrupedal step-training paradigm can facilitate therecovery of hindlimb function after a spinal cord injury. We demonstrate that training both the forelimbs and hindlimbs is more effective
than training only the hindlimbs for the recovery of coordinatedhindlimb function after a thoracic lateral hemisection in adult rats.
We also hypothesized that the rostrocaudal interneuronal networks
are re-engaged after quadrupedal training to enhance the locomotor
recovery: this proposition is supported by the greater excitability oflumbar neuronal networks and greater neuronal labelling of propriospinal interneurons bilaterally above and below the lesion site.

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Figure 1 Timeline of experimental procedures and injury extent. (A) After initial handling and pretraining, rats (n = 23) underwent EMG
electrode implantation in select forelimb and hindlimb muscles, implantation of spinal cord epidural electrodes, and a right lateral thoracic
hemisection injury (Hx) at T10. Rats were either non-trained (NT, n = 7) or trained to step (30 min/day) quadrupedally (QT, n = 8) or
bipedally (BT, n = 8) for 17 days beginning 4 days (4 d) post-hemisection. Data were collected pre-hemisection and at multiple time points
post-hemisection. All accompanying data are presented from six animals per group after eliminating animals with an incomplete hemisection or dye diffusion. Retrograde tracers were injected into the spinal cord and the rats were later perfused. An uninjured group of rats

(continued)

Forelimb activity enhances locomotor control

Forelimb–hindlimb coordination
Forelimb–hindlimb coordination was assessed as the ratio of thenumber of forelimb steps for every 10 hindlimb steps before andpost-hemisection. Forelimb/hindlimb ratios other than 1 indicate an
uncoordinated gait. The mean horizontal distance between the righthindlimb and left forelimb and between the left hindlimb and rightforelimb throughout a step cycle was computed for all steps analysed.
Coordination between the forelimb and hindlimb was further quantified by computing the mean time lag between the contact of the toes

| 3365on the treadmill surface for the right hindlimb and left forelimb and forthe left hindlimb and right forelimb (Shah et al., 2012).

Inter-hindlimb coordination
Coordination between the two hindlimbs was determined kinematically by joint probability distribution plots obtained from the vertical (y)positions of the left and right metatarsophalangeal markers during 10
consecutive steps (Shah et al., 2012).

Intralimb coordination
To determine intralimb coordination, joint probability distribution plots
were obtained from the rectified EMG activity of the tibialis anterior
and medial gastrocnemius from the same hindlimb for 10 consecutive
steps. A data point in these joint probability distribution plots represents the tibialis anterior and medial gastrocnemius activity in the same
hindlimb at a given time in a single step and the overall pattern of data
distribution qualitatively represents the intralimb coordination. Hipknee and ankle-knee angle-angle kinematics plots were generated
for the left and right hindlimbs to further examine intralimb coordination during stepping. The directionality of the plot trajectories wasdetermined algebraically from differences in the vector-to-vector angle
within specific phases of the step cycle and then the angles weresummed to obtain the total change in angle for the trajectory(Fig. 6C and insets). The triangles described by angle-angle plots(Fig. 6C) were used to generate measures (see below) that were
examined statistically to compare the shapes of each trajectory between steps, animals, and experimental groups (Fig. 6D). Maximum
rotation of the shape trajectory was measured by comparing angles ofsegment AC with the x (hip) axis per step. Accordingly, a large anticlockwise rotation of the trajectory results in large angles of segment
AC that indicates a change in the angular phase relationships between
joints within the hindlimb during stepping and hence reflects compromised intralimb coordination of the hindlimb.

Quantification of propriospinal neurons
Spinal cords were dissected, post-fixed in 4% paraformaldehyde for
1 h, and cryoprotected in 30% solution of phosphate-buffered (pH7.4) sucrose at 4 C. Before sectioning, the cords were segmented
into five blocks using the dorsal roots as landmarks as follows:
C1–C5, C6–T1, T2–T6, T7–T10, T11–L1 and L2–L5. Spinal cord
blocks were embedded in O.C.T. compound embedding medium(Tissue-Tek) in small peel-away histomolds, frozen over dry ice, andstored at 80 C until sectioning. Each block was cut transversely (30 mm thick sections), collected as free floating sections, serially mounted,
and coverslipped with VectashieldÕ mounting media containing DAPI.
A subset of sections adjacent to those used for tracer analysis wasprocessed for immunofluorescent detection of the rhodamine tracer
using an anti-rabbit tetramethylrhodamine antibody (Invitrogen).
The spinal cord sections were analysed using a rhodamine filter on a
fluorescence microscope (Axio Imager 2, Carl Zeiss Microscopy, LLC).
Digital images were captured using a camera and Image-Pro Plus

Figure 1 Continued(n = 8) was used for tracer dye injections out of which data from six animals are reported. (B) Histological reconstruction of spinal cordlesions: schematics of histological sections at the maximum extent of the spinal lesion (L = left; R = right) for six animals in the quadrupedal, bipedal, and non-trained groups show the extent of the hemisection lesion. Grey areas represent spinal grey matter, black areas scartissue, and stippled areas cystic cavitations. (C) The spinal lesions quantified as the percentage of spared white matter (SWM) at the lesion
epicentre normalized to intact white matter above the lesion site show no differences in the lesion areas among groups. (D) No differenceswere detected in the lesion extent of the contralateral (left) ventral white matter among groups.

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bilaterally. EMG signals (2 kHz) were amplified and filtered(10–1000 Hz bandpass). Activation patterns (muscle waveforms andtiming) for each muscle were obtained by taking an average of 10
filtered, rectified, and normalized (to the step cycle) EMG bursts fromeach muscle. Mean peak EMG amplitudes, integrals, and durations ofidentified EMG bursts from each muscle were computed for each rat
during each testing session (Shah et al., 2012).
Kinematics data were collected using 3D video recordings. SIMI
motion capture system (SIMI Reality Motion Systems) was used toobtain 3D coordinates of limb markers (at the iliac crest, hip, knee,
ankle, metatarsophalangeal joint, and toe). A range of kinematics gaitparameters including cycle period, stance phase duration, paw dragduration, and joint angle measurements were computed for eachgait cycle. The quality of stepping and coordination within a limb
(intralimb) and between the two hindlimbs and between the forelimbs
and hindlimbs (interlimb) were assessed via both kinematics and electrophysiological measures as described below. Spatial consistencies of
the hindlimb endpoint trajectories from the x–y coordinates of theankle and metatarsophalangeal joint markers were measured as theamount of variance explained by the first principal component(Courtine et al., 2009). Placement of the foot on the treadmill
during mid-stance, reflecting the vertical postural alignment and ability
to support body weight at mid-stance, was measured as the verticaldistance between the pelvis and knee joint markers and the treadmill
surface.
At 21 days post-hemisection, epidurally-evoked motor responses
were recorded during bipedal standing. An upper body harness wasused to position the rats over the treadmill belt and the amount ofbody weight supported by the rat was maintained at 20% such that
there was complete foot contact while the rat stood on the treadmill
surface. Early and middle responses were evoked in the tibialis anterior
muscles bilaterally via epidural stimulation (0.3 Hz, rectangular pulses,
0.5 ms duration, 10 kHz sampling frequency) at S1 (Lavrov et al.,2006) to determine the stimulus intensity-response amplitude curve.
The responses of the tibialis anterior motor neuronal pool were measured as the peak-to-peak amplitude of averaged (n = 10 at eachstimulation intensity) epidurally evoked potentials at stimulation intensities ranging from 0.5 to 5 V in increments of 0.5 V. Tibialis anterior
middle responses were normalized to the mean maximum early responses recorded at the higher stimulation voltages (4, 4.5 and 5 V
for each animal), as described previously (Lavrov et al., 2006).

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P. K. Shah et al.
dorsal white matter (Fig. 1B). The rostrocaudal extension of thehemisection (2.95  0.08 mm in quadrupedal step-trained
rats, 3.03  0.11 mm in bipedal step-trained rats, and2.76  0.15 mm in non-trained rats), area of spared whitematter (Fig. 1C), and lesion area of contralateral ventral whitematter (Fig. 1D) did not differ among groups. Moreover,there was no correlation between the severity of injury and behavioural or electrophysiological outcomes in all animals.

Assessment of hemisection lesion anddye injection site

Spontaneous motor recovery after a
mid-thoracic lateral hemisection

The lesion size was determined by processing 30 -mm spinal cord sections from segments T8–T11 using Cresyl Violet staining. The slides
were dehydrated stepwise through alcohol, cleared in xylene, andcover-slipped with Permount (Fisher Scientific). The spinal cord sections were photographed and the lesion extent was quantified using
ImageJ software (National Institutes of Health). Firstly, the total areaof the white matter was measured in an uninjured rostral portion (T8
spinal segment). The area of the white matter bridge at the lesion
epicentre (the minimum white matter area) was measured and theratio between these two measures (  100) was defined as the percentage of spared white matter for each animal. Secondly, the rostrocaudal extension (length) of the lesion was quantified as the distance
between the most rostral and the most caudal transverse tissue sections in which evidence of tissue damage appeared with Cresyl Violet
staining. Lastly, the area of contralateral ventral white matter lesion
was quantified as a percentage of damaged ventral white matter at
the lesion epicentre to the uninjured rostral white matter. Only the
animals with a complete right spinal cord hemisection were included in
the analyses (2 of 23 animals were excluded).
The area of tracer spread and dorsoventral and mediolateral spreadof the dye (Fig. 7B) was determined from photomicrographs of theinjection site and calibrated using a scale bar taken at the same magnification (Ballermann and Fouad, 2006). Animals in which the tracer
injection site was not confined to the ipsilateral cord were excluded
from further evaluation for histology (n = 2 for uninjured and 3 for the
injured groups).

Immediately after the thoracic hemisection, adult rats developed
motor paralysis of the ipsilateral hindlimb that partially recovered
during the first three weeks post-hemisection. Figure 2A–H showsdetailed kinematics analyses of the right (lesioned side) forelimband hindlimb of a non-trained rat before (pre-hemisection) and at
4, 8, and 21 days post-hemisection. Right hindlimb paralysis persisted for at least 4 days post-hemisection (Fig. 2B). Partial motor
recovery was evident at 8 days post-hemisection: rats recoveredsome ability in the right hindlimb for weight support and someindependent stepping (Fig. 2C). By 21 days post-hemisection, agross pattern of normal locomotion was recovered, but all rats
continued to show persistent motor deficits in the right hindlimbin stepping kinematics and most measures of stepping ability (seebelow and Supplementary Figs 1–4).
The kinematics and EMG data post-hemisection (Figs 2–6)
demonstrate that the degree of locomotor recovery more closely
resembled those recorded pre-hemisection (Supplementary
Video 1) in rats that engaged both the forelimbs and hindlimbs(quadrupedal step-trained) than in rats that used only the hindlimbs during the treadmill training sessions (bipedal step-trained)
or were not trained at all (non-trained, non-trained rats).

Statistical analyses
All data are reported as mean  SEM. Overall significant differenceswere determined using one-way or two-way ANOVA or repeatedmeasures ANOVA. Bonferroni post hoc tests were used to identify
significant differences among individual groups. Normality of distribution was assessed by the Shapiro-Wilk test. Bootstrapping analyses (resampling model, 10 000 iterations with replacement) (Efron andTibshirani, 1991) were used to determine statistical differencesamong groups for interlimb coordination. Differences among groups
were considered statistically significant at P 5 0.05. All statistical analyses were performed using MATLAB (Mathworks) and STATISTICA(Statsoft Inc).

Results
Spinal cord lesions
In all animals, complete lateral hemisection of the right hemicord
was accompanied with minimal damage to the left ventral andQuadrupedal step-training facilitateshindlimb stepping performance
After 17 sessions of training, quadrupedal step-trained rats had ashorter step cycle duration (Fig. 2I) and a lower per cent dragduration of the right hindlimb in a given step cycle (Fig. 2J)
than bipedal step-trained rats and non-trained rats. Generally at
4 and 8 days post-hemisection, the forelimbs propelled the bodyforward by taking approximately twice the number of steps takenby the hindlimbs (Fig. 2B and C). This forelimb compensation had
not returned to normal even at 21 days post-hemisection, although the forelimb-hindlimb step ratio was closer to pre-hemisection levels in quadrupedal step-trained rats compared withbipedal step-trained and non-trained rats (Fig. 2K). Quadrupedal
step-trained rats showed a consistency in the ankle(Supplementary Fig. 2A) and toe (Supplementary Fig. 2B) trajectories similar to pre-hemisection animals, whereas these values inbipedal step-trained rats and non-trained rats were less consistentthan in both the pre-hemisection and quadrupedal step-trained
rats. The vertical postural trunk alignment of the body during
stepping (as determined by knee and pelvis heights from thetreadmill surface) showed similar values for pre-hemisection,

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software (Media Cybernetics Inc.). Spinal cord segmental levels wereverified by the shape of the spinal cord matter and Rexed’s laminae as
a guide (Watson, 2009). The numbers of retrogradely labelled neurons
were counted through the microscope eyepiece by a tester blind to theexperimental design from every eighth section in each spinal block.
Only neurons with a nucleus and proximal dendritic processes thatwere visible upon focusing up and down through the thickness ofthe section were included in the cell counts. Cell counts are reported
from each spinal block as group means.

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Figure 2 Spontaneous partial locomotor recovery post-hemisection (Hx). (A–D) Stick diagrams for a representative non-trained rat (NT)during the swing phase of a single step cycle for the right (lesion side) forelimb and hindlimb during quadrupedal stepping pre-hemisection(A) and at 4 (B), 8 (C), and 21 days (D) post-hemisection. Traces below the stick diagram show the foot (metatarsophalangeal (MTP)
marker) and wrist joint trajectories for 10 consecutive steps (thin grey lines) and the corresponding mean values for the foot (thick greenline) and wrist (thick red line). The red vertical lines indicate the vertical displacement of the knee from the treadmill (knee height). (E–H)
Individual (thin grey lines) and mean knee (thick red line) and ankle (thick green line) angle movements for the same 10 steps as in A–D.
(continued)

3368| Brain 2013: 136; 3362–3377

quadrupedal step-trained and bipedal step-trained rats, but lower
in non-trained rats (Supplementary Fig. 2C and D).

Quadrupedal step-training increases therecruitment and excitability of hindlimb
motor neuronal pools

lumbosacral motor neurons (Fig. 3F–H). This could be becauseof changes in the intrinsic properties of the motor neurons. The
ratio of the mean threshold of excitation for the early and middle
responses was between 1–1.2 for all groups, indicating that thedorsally located axons (dorsal root fibres) could have evoked the
middle responses.

Quadrupedal step-training improvesinterlimb and intralimb coordination
during stepping
Forelimb–hindlimb coordination
The interlimb phase difference between the forelimb and diagonal
hindlimb for consecutive steps was altered dramatically(Fig. 4A and B). Although a positive time lag was observed forthe first two steps, a negative time lag persisted for the remaining
eight steps during the test period at all time points in all rats,
indicating that the hindlimb lagged behind the forelimb duringconsecutive steps in all groups (Fig. 4B). Accordingly, with larger
time lags, there was less forelimb-hindlimb coordination.
Quadrupedal step-trained rats had an 80% decrease in timelags for the right hindlimb–left forelimb at 21 days comparedwith 8 days post-hemisection. Additionally, the horizontal distance
between both forelimb-hindlimb combinations was longest at 4days post-hemisection (Fig. 4C). Although this distance did not
return to pre-hemisection values, step length distances between
each forelimb-hindlimb combination was shortest in quadrupedal
step-trained rats at 21 days post-hemisection (Fig. 4C), whichenabled this group to step faster (Fig. 2I) and with greater consistency than bipedal step-trained and non-trained rats
(Supplementary Fig. 2A and B).

Inter-hindlimb coordination
By 21 days post-hemisection, quadrupedal step-trained rats
showed interlimb stepping coordination similar to pre-hemisection
as reflected by: (i) the ‘L-shaped’ pattern in the joint probability
distribution plots of vertical step heights for the right versus left
hindlimbs (Fig. 5); and (ii) the time lag between the two hindlimbs(Supplementary Fig. 3A). Electrophysiological recordings also indicated greater reciprocity between hindlimbs in quadrupedal steptrained rats compared to the non-trained rats, but not bipedal
step-trained rats (Supplementary Fig. 3B–D).

Figure 2 Continued
Dotted horizontal lines indicate the mean maximum and minimum joint angles at each joint and the dotted vertical lines indicate
the transition between the stance and swing phases. Step cycle phases across groups are normalized to 100% of the step cycle.
(I) Stepping at faster treadmill speeds in the quadrupedal step-trained rats (QT) than in the bipedal step-trained (BT) and non-trained
rats at 21 days post-hemisection is indicated by shorter step cycle durations. (J) Mean percentage of foot drag during stepping is shorter inthe quadrupedal step-trained rats than bipedal step-trained and non-trained rats. (K) The ratio of the number of forelimb steps for every
10 hindlimb steps (forelimb/hindlimb) is lower in the quadrupedal step-trained rats than in the bipedal step-trained and non-trained rats.
Values are mean  SEM for 10 steps for each group (n = 6/group). The first set of horizontal lines immediately above the bars indicate thatthe quadrupedal step-trained group is significantly different from the bipedal step-trained and non-trained groups; the second set ofhorizontal lines indicate significant differences between the pre- and each of the post-hemisection groups (P 5 0.05). Similar convention isused in Figs 4 and 6 and in Supplementary Figs 2 and 3.

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In the forelimb, there was a persistent increase in biceps brachii
EMG activity bilaterally at 4, 8 and 21 days and a transient increase in triceps brachii EMG activity bilaterally at 4 and 8 days
post-hemisection in all rats (data shown for non-trained rats in
Fig. 3A), indicating compensatory forelimb activity propelling the
body forward post-hemisection (Lopez-Dolado et al., 2013).
Bilateral biceps brachii activity was decreased at 21 days comparedwith 8 days post-hemisection only in quadrupedal step-trained rats
(Fig. 3B). Interestingly, the lower bilateral EMG activity in thebiceps brachii in quadrupedal step-trained rats coincided with increases in bilateral hindlimb medial gastrocnemius EMG activity at
21 days compared with 8 days (Fig. 3D). These data suggest that
there was greater recruitment of the hindlimb extensor motor
neuronal pools during facilitated hindlimb stepping coincident
with a decrease in forelimb flexor compensation with quadrupedal
step-training. In contrast, tibialis anterior muscle activity remainedlargely unchanged in the right hindlimb at all time points in all
rats post-hemisection, whereas there was an almost 2-fold increase in tibialis anterior activity in the left hindlimb of allgroups at 21 days post-hemisection (Fig. 3D), irrespective of the
training status. This elevated tibialis anterior activity persisted at 21
days post-hemisection most likely as a compensatory mechanism
during the swing phase of the left hindlimb to support longer
stance phase durations of the right hindlimb (Supplementary
Fig. 1).
After quadrupedal step-training, epidurally evoked potential
amplitudes in the tibialis anterior were similar bilaterally at eachstimulation intensity tested 21 days post-hemisection, indicating
similar excitability of the left and right tibialis anterior motor neuronal pools (Fig. 3E–G). In contrast, bipedal step-trained and nontrained rats showed higher amplitude responses in the right (lesioned side) versus left (non-lesioned side) tibialis anterior, indicating asymmetrical excitability. Interestingly, the threshold of
epidural stimulation required to evoke a motor response (bothearly and middle responses) was lowest bilaterally after quadrupedal step-training, indicating overall greater excitability of theP. K. Shah et al.

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Figure 3 EMG recovery patterns post-hemisection and training. (A) Superimposed integrated rectified EMG activity patterns of the left
and right biceps brachii (Bic-Br) and triceps brachii (Tric-Br) during quadrupedal stepping in non-trained (NT) rats (n = 6). This pattern offorelimb EMG activity was similar in all groups. Vertical lines indicate stance and swing phase durations that are normalized to 100% of thestep cycle. (B) Percentage differences between 21 and 8 days (percentage difference 21days–8days) in the peak EMG amplitudes of theleft (L) and right (R) biceps brachii and triceps brachii muscles demonstrate a maximum decrease in the peak amplitude of the biceps
brachii muscle bilaterally in the quadrupedal step-trained rats (QT). (C) Superimposed integrated rectified EMG of the right and left tibialis
anterior and medial gastrocnemius muscles for the same conditions as in (A). (D) Percentage differences between 21 and 8 days in thepeak EMG amplitudes of the tibialis anterior and medial gastrocnemius muscles demonstrate a maximum increase in the peak amplitude of
the medial gastrocnemius muscle bilaterally in the quadrupedal step-trained rats. *Significantly different from quadrupedal step-trained
rats; †Significantly different from bipedal step-trained rats (BT) RMG = right medial gastrocnemius; LMG = left medial gastrocnemius;
RTA = right tibialis anterior; LTA = left tibialis anterior. (E) Representative epidurally evoked responses from the left (black) and right(maroon) tibialis anterior in response to epidural stimulation for one rat per group at 21 days post-hemisection while standing andsupported in a harness (20% body weight support). Vertical dashed lines identify the time periods for the early response (ER) and middle
response (MR) in both the left and right tibialis anterior. The trace starts at the onset of the stimulation pulse. (F) Middle response
amplitudes at various stimulation intensities from the left (black) and right (maroon) tibialis anterior normalized to the maximum early(continued)

3370| Brain 2013: 136; 3362–3377

Intra-hindlimb coordination

Quadrupedal step-training restoressymmetry of gait kinematics
The difference in shape trajectories between the right and left
hindlimbs was 3 pre-hemisection, 9 in the quadrupedal
step-trained rats, and 35 in the bipedal step-trained rats andnon-trained rats, reflecting greater asymmetry in the angle-angle
relationship between the two limbs during stepping in bipedal
step-trained and non-trained rats (Fig. 6D and E). This change inshape trajectory and larger asymmetry in bipedal step-trained andnon-trained rats results from a change in total hip joint excursion
due to greater extension at the hip and lesser extension of theknee. The horizontal distance between the centroids of the shapetrajectory of each hindlimb, an indication of asymmetry between
the left/right hip and knee joint, was larger for bipedal steptrained and non-trained rats than quadrupedal step-trained andpre-hemisection rats (Fig. 6F).

Quadrupedal step-training promotes
plasticity in the thoracic spinal
interneuronal networks
We injected the retrograde tracer rhodamine red into the ipsilesional right L2 spinal segment and quantified the number of retrogradely labelled neurons in the cervical, thoracic, and lumbar
spinal cord segments bilaterally (Fig. 7A). The tracer injection

was confined mainly to the right dorsal and ventral grey matterof the L2 spinal segment (Fig. 7B and C). There were no differences in the dorsoventral and mediolateral spread of the tracer(Fig. 7B and C) or the area of the tracer spread at the site of
injection (Supplementary Fig. 5) between groups.
In uninjured rats, retrogradely labelled neurons were visualizedalong the right and left grey matter of the entire spinal cord,
including an even distribution along the segments of the cervical
cord (Fig. 7D–F and L). In all injured rats, retrogradely labelled
neurons were observed below and above the lesion in the rightand left lumbar (Fig. 7G) and thoracic (Fig. 7H) segments.
Nevertheless, no labelled cells were seen at the cervical segments
in any group post-hemisection (Fig. 7I). Below the injury (T11–L1),
quadrupedal step-trained rats had a greater number of retrogradely labelled propriospinal interneurons bilaterally than the
non-injured, bipedal step-trained and non-trained rats (Fig. 7K).
Above the hemisection (T8–T9), the number of labelled neurons
bilaterally was lower in bipedal step-trained and non-trained rats
than in the non-injured and quadrupedal step-trained rats
(Fig. 7J). Cells were distributed throughout laminae V-VIII and X
and along the medial border of the ventral horn. Few labelledcells also were located in laminae III and IV. The total numbersof neurons were similar in the left and right hemicord for eachgroup with cell counts being highest in the lumbar and lowest in
the thoracic segments (Fig. 7J and K). To examine the sensitivityof the rhodamine dextran-label, we counted the number oflabelled cells using direct fluorescence and in sections processed
for immunohistochemistry with anti-rabbit tetramethylrhodamine
antibody (Invitrogen). There were no differences in the numbers of labelled cells detected by the two methods (data notshown).

Discussion
We hypothesized that actively involving the forelimbs would facilitate the recovery of locomotor function after a spinal cord
injury. We demonstrate three novel findings: (i) actively engaging
both the forelimbs and hindlimbs in a training paradigm (quadrupedal step-training) results in superior locomotor quality and coordination than training only the hindlimbs (bipedal step-training)or not training (non-trained); (ii) quadrupedal step-training is associated with greater excitability of motor neurons as is reflected in
the lower levels of excitation thresholds of epidurally evoked
potentials in the hindlimb muscles in both the lesioned and nonlesioned sides; and (iii) quadrupedal step-trained rats exhibit a significantly greater level of rostrocaudal thoracic interneuron connectivity as the they showed the highest number of labelled

Figure 3 Continued
response for each group. *Significant differences between the right and left hindlimb responses (P 5 0.05). (G) Absolute early response
amplitudes from the left (black) and right (maroon) tibialis anterior for each group. (H) Mean threshold of epidural stimulation to evoke a
middle response and early response for the left and right tibialis anterior muscles for each group. Dotted black horizontal line indicates athreshold of 2.5 mV for the middle responses and early responses. *Significantly different from quadrupedal step-trained rats (P 5 0.05).
Values in B, D, F, G and H are mean  SEM for six rats per group.

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An L-shaped pattern during stepping indicates a strong reciprocal
activation between the tibialis anterior and medial gastrocnemius
muscles (see pre-hemisection patterns in Figs 5 and 6A and B). At
21 days post-hemisection, an L-shaped pattern was most evident
in quadrupedal step-trained rats. The amount of co-activation between the tibialis anterior and medial gastrocnemius muscles washighest in the right hindlimb (lesioned side) of non-trained rats
(Fig. 6B). Importantly, there was no difference between pre-hemisection and quadrupedal step-trained rats bilaterally. But also inthis instance, there was no significant difference between quadrupedal step-trained and bipedal step-trained rats. Qualitatively, theshape of the trajectory created by the joint angle plots was alteredin all groups post-hemisection for hip-knee (Fig. 6D) and ankleknee (Supplementary Fig. 4A and B) angles and maximum rotationof the trajectory for both hindlimbs was observed in bipedal steptrained and non-trained rats (Fig. 6D). Angle-angle plots also indicated that quadrupedal step-trained rats had angle-angle relationships not different from pre-hemisection, whereas bipedal steptrained and non-trained rats showed altered angle-angle relationships (Fig. 6E).

P. K. Shah et al.

Forelimb activity enhances locomotor control

Brain 2013: 136; 3362–3377| 3371Figure 5 Quadrupedal step-training is effective in improving inter-hindlimb coordination. (A) Mean joint probability distributions of the ycoordinates of the left and right metatarsophalangeal (MTP) markers during quadrupedal stepping before, and at 4, 8 and 21 days posthemisection from all animals per group. An L-shaped pattern indicates an alternating motion of the two hindlimbs, whereas a D-shaped
pattern indicates less alternation between the two hindlimbs. The percentage shown on the 21-day plots indicates the average numberof data points that lie outside the 20% margins for an L-shaped pattern. Hx = hemisection; BT = bipedal step-training; QT = quadruped
step-training; NT = non-trained.

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Figure 4 Quadrupedal step-training is effective in improving forelimb-hindlimb coordination. (A) Stick diagrams (30 ms between sticks)
for a representative rat pre-hemisection (Pre-Hx) and from one rat per group at 21 days after a hemisection during the stance phase of asingle step cycle illustrating the coordination patterns between the right hindlimb (RHL) and left forelimb (LFL) during quadrupedal
stepping at 21 cm/s. (B) Bar graphs representing the lag times between the onset of the left hindlimb (LHL) and right forelimb (RFL) andbetween the right hindlimb and left forelimb contact on the treadmill during quadrupedal stepping at each time point. (C) Histograms
depicting the horizontal distance between the left hindlimb and right forelimb and between the right hindlimb and left forelimb during
quadrupedal stepping. After hemisection, the distance between the hindlimb and forelimb increases because of hindlimb drag, decrease in
the knee height, and irregularities in the ratio of the number of forelimb steps for every 10 hindlimb steps (Fig. 2). Values in B and C aremeans  SEM for 10 steps/rat. Note that in B and C the quadrupedal step-trained (QT), bipedal step-trained (BT) and non-trained (NT)groups are significantly different from pre-hemisection at all time points and that the quadrupedal step-trained group is different at 21
days versus 8 days. Additionally, in both B and C, the quadrupedal step-trained group is different than both the bipedal step-trained andnon-trained groups at 21 days.

3372| Brain 2013: 136; 3362–3377

P. K. Shah et al.
propriospinal neurons above and below the hemisection sitebilaterally.

Forelimb training in the recovery ofhindlimb function

Figure 6 Continued

Figure 6 Quadrupedal step-training (QT) improves intralimb
coordination and limb symmetry. (A) Joint probability distributions of rectified EMG for the tibialis anterior (TA) and medial
gastrocnemius (MG) within each hindlimb during quadrupedal
stepping pre-hemisection (Hx) (superimposed black dots) and 21days (coloured dots) post-hemisection. (B) The levels of tibialis
anterior and medial gastrocnemius co-activation within eachlimb are similar after quadrupedal and bipedal step-training (BT)and highest in the right hindlimb of the non-trained (NT) rats.
(C) A triangle is prescribed from the angle-angle plots. Angle Aof the trajectory (i) indicates the relationship between the knee
and hip joint angles during the transition from swing to stance,
segment AC represents this relationship during stance, andsegments AB and BC during swing. Angle C of the trajectory
represents transition from stance to the swing phase. Angle B of
the trajectory represents transition between the initial andmiddle portions of the swing phase. For both stance and swing,

the trajectory is converted into sequential vectors (ii, inset) andthe changes in vector-vector angles (example angle b – angle a)that indicate consistency between joint angles in each phase of
the step cycle are computed algebraically. An example of thechange in angle from the stance to swing phase (rate of changeof angle at point C) is indicated by the angle trace (filled orange)
in inset (iii). (D) Intralimb coordination determined kinematically
via angle-angle plots illustrates the mean right (dark trace) andleft (light trace) hindlimb knee-hip angle trajectories from a
representative animal in each group. The orange triangle is analgebraic representation of the trajectory shapes as explained in
C. The angle of segment AC with the hip joint axis was utilized
to indicate the shape of the trajectory. Maximum anti-clockwise
rotation of the right hindlimb shape trajectory reflects a change
in angular phase relationships between joints within the hindlimb
during stepping. (E) Asymmetry between the two hindlimbs isindicated by the differences in the angle prescribed by segment
AC with the hip angle axis between the right and left hindlimbs
and by (F) greater distances between the centres of trajectories
between the right and left hindlimbs (centroid of triangles,
maroon circles in D). Values in A, B, E and F are mean (  SEM)
for 10 steps from all animals per group and the horizontal lines
above the bars indicate significant differences between andamong groups (P 5 0.05).

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To test the hypothesis that actively engaging the forelimbs canpromote locomotor function, we employed a simple, yet intense
quadrupedal step-training regimen (21 cm/s, 30 min/session for 17
sessions). Training of the forelimbs in this manner served as acondition in which the full set of afferent and efferent inputs/
outputs during quadrupedal locomotion were used. Comparedwith bipedal step-training, quadrupedal step-training includes theuse of the forelimbs during treadmill walking and involves natural
biomechanical adjustments of the entire body for proper control ofposture and locomotion (Perennou, 2012). To isolate the effects
produced by forelimb training, a sub-group of animals underwent
bipedal hindlimb step-training. Assuming that supraspinal influences were similar in all the groups, we attributed differences inmotor recovery between the quadrupedal and bipedal step-trained
rats to the use of the forelimbs.
To our knowledge, no studies have determined the impact offunctionally engaging the forelimbs to improve coordinated quadrupedal locomotion or have emphasized the effects of engaging
the propriospinal pathways connecting cervico-thoraco-lumbar
networks to promote recovery after a spinal cord injury.
Although human studies show that upper extremity activity canacutely influence lower extremity motor function (Visintin andBarbeau, 1994; Huang and Ferris, 2004), none have clearly
demonstrated whether chronically engaging the upper limbs intraining facilitates lower limb function. Studies report that theForelimb activity enhances locomotor control

Brain 2013: 136; 3362–3377| 3373

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Figure 7 Quadrupedal step-training (QT) is associated with greater neuronal reorganization. (A) Schematic illustrating the hemisection at
T10, sites of dye injection at L2, and cross sections where labelled cell counts were made. (B) Localization of rhodamine tracer dye at
injection site in a representative animal shows that the dye was confined to the ipsilateral hemicord. The dorsoventral (yellow dotted)
spread of the dye was normalized to the dorsoventral depth (solid yellow) of the cord (a/b) and the mediolateral spread of the dye (green
dotted) was normalized to the mediolateral width (solid green) of the hemicord (c/d). (C) Both dorsolateral (a/b) and mediolateral (c/d)
spread of the dye were uniform among the groups. Representative transverse micrographs of lumbar (D), thoracic (E), and cervical(F) spinal cord segments show retrograde-labelled neurons in an uninjured rat. Neuronal labelling is seen in all rats 39 days post-hemisection at the lumbar (G) and mid-thoracic (H), but not the cervical (I), spinal cord segments. Mean (  SEM) neuronal counts in the left
and right hemicords above (T8–T9; J) and below (T11-L1; K) the hemisection. (L) Histograms showing the mean (  SEM, n = 6) total
number of labelled cells in select lumbar, thoracic, and cervical spinal segments in uninjured animals. The number of labelled cells was(continued)

3374| Brain 2013: 136; 3362–3377

Role of afferent input from the forelimbs
We propose that the ensemble of afferent input from the forelimbs during quadrupedal stepping strongly influences the neuralsubstrates that impact coordination between the forelimbs andhindlimbs as well as between the two hindlimbs. It is well established that afferent volleys from the forelimbs induced by electricalstimulation of the brachial plexus or mechanical stimulation of theforepaw evokes direct responses of flexor and extensor lumbosacral motor neurons (Aoki and McIntyre, 1975; Schomburg andSteffens, 1986). In humans, interlimb coupling between the arms
and legs is strengthened by adding cutaneous feedback from thehand during a normal locomotor rhythmic activity of all four limbs(Zehr et al., 2007). In the present study, bipedal step-trained andquadrupedal step-trained rats were able to step at similar treadmill
speeds, but the ‘added’ afferent input from the forelimbs andtrunk most likely was associated with the enhanced locomotor
function observed with quadrupedal training.
The additional effects on locomotor recovery observed withquadrupedal step-training are attributable to one or more of atleast three mechanisms [Fig. 7Q(i–iii)]. First, afferent feedback
from the forelimbs can influence hindlimb activity through thedirect activation of the long descending propriospinal pathways(Akay et al., 2006; Gerasimenko et al., 2009). Second, the forelimb to hindlimb interactions could be a consequence of chains ofseveral inter-connecting short propriospinal axons (Aoki and
McIntyre, 1975; Kostyuk and Vasilenko, 1979; Cowley et al.,2008). Third, afferent feedback from the forelimbs could be conveyed by ascending supraspinal pathways to locomotor regions in
the brainstem or cortical structures from which the bulbospinal
pathways descend. This spino-bulbo-spinal loop is activated byafferent input from the forelimbs that ultimately projects onto
the spinal cord (Kausz, 1986) and may contribute to the prominent lumbosacral motor neuron discharge evoked by forelimb
stimulation (Shimamura et al., 1967). Below, we discuss the roleof the propriospinal system as one of the mechanisms of locomotor recovery.

Role of the propriospinal system in therecovery of locomotion
The long propriospinal neurons in the cervical spinal cord of the rat
project ipsilaterally and contralaterally to the lumbar cord with the
majority of the crossings taking place in the cervical or lumbar
enlargement (Reed et al., 2006). Given their extensive remodelling
capacity (Iannotti et al., 2003; Bareyre et al., 2004), robust capability to respond to therapeutic interventions (Houle et al., 2006;
Deng et al., 2013), and their role in interlimb coordination (Zehrand Duysens, 2004) after spinal cord injury, we expected to detect
retrotraced neurons in the cervical spinal cord of quadrupedal
step-trained animals after a unilateral tracer dye injection below
the thoracic lesion site. Surprisingly, we did not observe any propriospinal neurons in the cervical enlargement in all groups posthemisection. A reduction in the amount of dye transport in thelong propriospinal cervical neurons has been reported after thoracic spinal cord contusion injury in rats (Conta and Stelzner, 2004,2010). Thus, the absence of cervical labelled neurons in our study
could be attributed to: (i) the nature of the injury, i.e. comparedwith a lateral hemisection, contusion injuries preserve bilateral
ventral and ventro-lateral white matter through which a majorityof the propriospinal system descends (Reed et al., 2006, 2009); or(ii) the relatively lower volume of tracer dye injected in the present
study. It must be noted, however, that the volume injected wasadequate to observe retrotraced dyes in the cervical spinal cord of
our uninjured animals (Fig. 7L) and low enough to prevent dyeleakage to the contralateral hemicord.

Figure 7 Continued
maximum in the lumbar segment and lowest in the cervical cord. (M–P) Schematic illustrating the likely pathway of dye transport throughpropriospinal interneurons in the thoraco-lumbar and thoracic spinal segments. The number of labelled cells is similar in N and M andsuggests an underlying mechanism of neuronal organization, i.e. axonal sprouting or activation of dormant neurons (dotted coloured lines)that are likely responsible for the higher neuronal count observed after quadrupedal step-training. (Q) Conceptual model for the observed
results: schematic illustrating neuronal pathways that may be modulated by quadrupedal step-training. Afferent input from the forelimbs
activates the lumbosacral locomotor networks (LLN) in the spinal cord through direct activation of (i) long (solid long arrow); and/or(ii) intersegmental short propriospinal neurons (solid short arrows); (iii) a longer neuronal loop (spino-bulbo-spinal pathways) that projects
from the forelimbs to the brain and eventually down to the lumbosacral locomotor network; (iv) a bulbospinal direct relay that activatesthe lumbosacral locomotor network; and/or (v) descending supraspinal pathways that activate the propriospinal system (red dottedarrows).

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magnitude of leg muscle recruitment during recumbent steppingand cycling increases as healthy subjects push against elevatedmechanical resistance with their arms (Huang and Ferris, 2004)
or perform higher frequencies of rhythmic arm cycling (Ferris
et al., 2006; Hundza and Zehr, 2009). Similarly, in individuals
with an incomplete spinal cord injury, there is some facilitationof the lower extremity muscles in spinal cord injury subjects
when walking with, versus without, the arms swinging (Visintin
and Barbeau, 1994; Behrman and Harkema, 2000). This facilitation was phase dependent on the step cycle (Kawashima et al.,2008). Interlimb coupling of upper extremity movements andstimulation of the peripheral nerves in the upper extremities also
can modulate reflexes in the legs (Mezzarane et al., 2011).
Whereas all of the abovementioned reports demonstrate that sensory input from the arms can contribute to the modulation of the
motor output from the legs in humans, we show that actively
engaging the forelimbs by ‘training’ over a period of 2.5 weeks
elevates the EMG activity in the hindlimb muscles and enhances
spinal cord excitability resulting in a significant improvement inlocomotor function after spinal cord injury in rats.

P. K. Shah et al.

Forelimb activity enhances locomotor control| 3375(Giszter et al., 2008) exert greater control over the hindlimb musculature after quadrupedal step-training than bipedal step-training
for effective quadrupedal locomotion. After quadrupedal steptraining, the rats step effectively without tail support unlike bipedal step-trained and non-trained rats (Supplementary Video
1). As such, we suggest that the anatomical reorganization of
the thoracic interneurons contributed to improved postural controlof the trunk, and hence hindlimb locomotion. Interestingly, ourresults show that the vertical distance between the pelvis/kneeto the ground (indicating vertical postural alignment) is similar
between the trained groups and pre-hemisection (Supplementary
Fig. 2C and D). Collectively, these data suggest that improvements
in gross adaptive postural control of the trunk (Giszter et al.,2008) might partially contribute to enhanced locomotor function,
but is not a major contributor in the differential recovery observed‘among’ groups.
In conclusion, hindlimb function and coordination patterns improve after a hemisection when the locomotor circuitry is activated, presumably as a result of neuromodulation via sensory
input from the forelimbs. The propriospinal system strategicallyintegrates signals from supraspinal (and spinal) centres withthose from the periphery for accurate activation of motor neuronalpools (Darlot et al., 2012; van den Brand et al., 2012). Based on
the present data alone, we cannot distinguish to what extent the
improved motor function observed is attributable to the reorganization of supraspinal descending pathways [Fig. 7Q(iv-v)] versus
changes in the processing of sensory input (proprioception andcutaneous) to the propriospinal pathways [Fig. 7Q(i–iii)].

Relevance to clinical rehabilitation
Observations of the impact of upper extremity training on lower
extremity function in persons with spinal cord injury are preliminary and inconclusive. We demonstrate that actively engaging theforelimbs when training the hindlimbs in hemisection rats can enhance hindlimb locomotor function to a much greater extent thanwith hindlimb training alone. Identification of this phenomenon
provides a strong rationale for proceeding toward preclinical studies for determining whether training paradigms involving active
upper arm training in concert with lower extremity training canenhance locomotor recovery after neurological damage. Examples
of such training paradigms might include arm-cycling during functional electrical stimulation leg-cycling exercises, use of an ellipticaltrainer after incomplete paraparesis, and active arm-swinging
during overground locomotion and treadmill training.

Funding
This work is supported by the Craig Neilsen Foundation PostDoctoral Fellowship award granted to PKS (Grant # 82616);
Christopher and Dana Reeve Foundation; the programs Number
5 and 7 of the Presidium of RAN; the Russian Foundation of Basic
Research Grants: 13-04-01091 and 13-04-12030-ofi_m; and the
National Institute of Biomedical Imaging and Bioengineering
R01EB007615.

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In contrast to the absence of labelled cells in the long descending cervical propriospinal neurons, large numbers of short propriospinal neurons were labelled bilaterally in all animals in thethoracic and lumbar segments, above and below the lesion site.
These findings are consistent with a previous report showing retrogradely labelled short propriospinal neurons bilaterally above andbelow a lateral thoracic hemisection lesion in mice allowed to recover spontaneously (Courtine et al., 2009). These short neurons
may create new functional circuits around an incomplete lesion
and provide the substrate for locomotor recovery.
Based on the present results, we propose that training the forelimbs significantly impacted the hindlimbs through a link of intersegmental connecting interneurons. As such, the propriospinal
axons from the cervical cord segmentally activate interneurons in
the thoracic segments that, in turn, activate neurons in the lumbar
segments [Fig. 7Q(ii)], a hypothesis previously suggested in thecat (Aoki and McIntyre, 1975; Shik, 1997; Manjarrez et al., 2003)
and neonatal rat (Juvin et al., 2005; Cowley et al., 2008). Studies
in the neonatal rat demonstrate that the propriospinal relay system
serves as an important and likely sufficient conduit for descending
activation of the locomotor networks (Cowley et al., 2008).
Indeed, a drastic reduction in locomotor cervico-lumbar coordination is observed when the excitability of thoracic neural elements
is decreased or when synaptic transmission in these segments is
suppressed (Juvin et al., 2005, 2012). Additionally, the short propriospinal neurons primarily located in T6–T8 which innervate
lumbar motor neuronal pools (Conta and Stelzner, 2004) may
also relay signals from the long supraspinal descending systems
to the motor neurons in the lumbar cord, as shown in the cat(Vasilenko et al., 1972). In fact, based on the latency of theevoked responses from the triceps brachii muscle in response tomagnetic stimulation of the sciatic nerve (Beaumont et al., 2006),
it was suggested that transmission through this ascending propriospinal pathway likely involves three to five synapses, providing
further evidence of extant polysynaptic connections between theforelimbs and hindlimbs. Reorganization of the thoraco-lumbar
spinal circuitry by sprouting of spared fibres (Siebert et al.,2010; Darlot et al., 2012) and/or alterations in the strength ofexisting neural circuits through, e.g. changes in neurotransmitter
release or postsynaptic receptor density (Flynn et al., 2011), may
account for the increased number of labelled cells observed after
quadrupedal step-training. Alternatively, the possibility exists thatpropriospinal axons engaged in more activity (as a result of forced
forelimb training) took up and transported the retrograde tracer
more efficiently, resulting in the differences in labelling amonggroups.
The greater number of labelled cells in the thoracic segments ofquadrupedal step-trained rats represents increased excitation ofthoracic interneurons through rostrally connecting interneurons.
The remarkable improvements in locomotor function and bilateralsymmetry in the excitability of lumbosacral motor neurons inquadrupedal step-trained rats also suggests that these neurons
were responsive to the training paradigm. Given that the propriospinal system is implicated in the regulation of the axial
musculature and in postural mechanisms (Anderson, 1963;
Vasilenko, 1975), it is very likely that the trunk afferents andmusculature that are rhythmically active during locomotion

Brain 2013: 136; 3362–3377

3376| Brain 2013: 136; 3362–3377

Supplementary material
Supplementary material is available at Brain online.

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