NIH Public Access
Author Manuscript
Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

NIH-PA Author Manuscript

Published in final edited form as:
Nat Neurosci. 2009 October ; 12(10): 1333–1342. doi:10.1038/nn.2401.

Transformation of nonfunctional spinal circuits into functional
states after the loss of brain input
Grégoire Courtine1,2, Yury Gerasimenko3,4, Rubia van den Brand1,2, Aileen Yew5, Pavel
Musienko1,2,4, Hui Zhong3, Bingbing Song6, Yan Ao6, Ronaldo M Ichiyama3, Igor Lavrov3,
Roland R Roy3,6, Michael V Sofroniew5,6, and V Reggie Edgerton3,5,6
1Neurology Department, University of Zurich, Zurich, Switzerland 2Rehabilitation Institute and

NIH-PA Author Manuscript

Technology Center Zurich, Zurich, Switzerland 3Department of Physiological Science, University ofCalifornia Los Angeles, Los Angeles, California, USA 4Motor Physiology Laboratory, Pavlov Instituteof Physiology, St. Petersburg, Russia 5Department of Neurobiology, University of California Los
Angeles, Los Angeles, California, USA 6Brain Research Institute, University of California Los
Angeles, Los Angeles, California, USA

Abstract
After complete spinal cord transections that removed all supraspinal inputs in adult rats, combinationsof serotonergic agonists and epidural electrical stimulation were able to acutely transform spinal
networks from nonfunctional to highly functional and adaptive states as early as 1 week after injury.
Using kinematics, physiological and anatomical analyses, we found that these interventions could
recruit specific populations of spinal circuits, refine their control via sensory input and functionallyremodel these locomotor pathways when combined with training. The emergence of these newfunctional states enabled full weight-bearing treadmill locomotion in paralyzed rats that was almost
indistinguishable from voluntary stepping. We propose that, in the absence of supraspinal input,
spinal locomotion can emerge from a combination of central pattern-generating capability and theability of these spinal circuits to use sensory afferent input to control stepping. These findings provide
a strategy by which individuals with spinal cord injuries could regain substantial levels of motor
control.

NIH-PA Author Manuscript

Severe spinal cord injuries that remove all supraspinal input to lumbosacral spinal circuits leadto permanent paralysis of the legs in adult rodents1–3 and humans. Nevertheless, networks ofneurons in the lumbosacral spinal cord retain an intrinsic capability to oscillate and generate
coordinated rhythmic motor outputs. Circuits underlying such rhythmic and oscillatory outputs
are commonly referred to as central pattern generators (CPGs) and are found in all invertebrate
and vertebrate animals4,5. Although the anatomical architecture of locomotor CPGs remains
poorly understood, especially in mammals5, the functional phenomenon, central patterngeneration, has been documented extensively. Indirect evidence suggests that CPGs are present
in human spinal cord6,7. These observations offer the possibility of directly accessing andactivating spinal cord CPGs to facilitate locomotor recovery after a severe spinal cord injury(SCI).

Correspondence should be addressed to G.C. (gregoire.courtine@bli.uzh.ch).
Note: Supplementary information is available on the Nature Neuroscience website.
Author Contributions: G.C., Y.P.G., I.L., M.V.S. and V.R.E. designed the study. G.C., R.R.R., H.Z. and P.M. performed the surgeries.
G.C., Y.P.G., R.v.d.B., P.M. and A.Y. carried out the experiments. G.C., A.Y., B.S., Y.A., R.I. and M.V.S. conducted the anatomicalassessments. G.C. analyzed the data. G.C., R.R.R., M.V.S. and V.R.E. wrote the manuscript. G.C. supervised the study.

Courtine et al.

Page 2

NIH-PA Author Manuscript

Several experimental strategies have been tested to activate locomotor circuits in mammals
after a complete spinal cord transection, including pharmacological treatments8–10,
epidural2,11,12 or intraspinal13,14 electrical stimulation, and motor training1,2,8,15,16. Serotoninor agonists of 5-HT2A and 5-HT1A/7 receptors can activate the quiescent locomotor circuitry
in neonatal rodent fictive locomotion preparations17,18 and can facilitate treadmill steppingwith limited weight bearing in adult rats9 and mice10 with SCI. Epidural electrical stimulation(EES) applied dorsally at the lumbar (L2)2,11 or sacral (S1)12,19 spinal segments induces
rhythmic hindlimb movements2,12. Locomotor training, notably in conjunction withpharmacological8,9 or electrical stimulation2 interventions, can promote use-dependent plasticchanges in sensorimotor circuits below the injury16,20,21 that lead to specific improvements of
stepping patterns. These interventions, however, have shown limited potential for promoting
weight-bearing capacities and there have been few attempts to correlate the specific functional
states induced pharmacologically9,10, electrically11–13,19,22 or by locomotor training2,20 with
distinct characteristics of stepping motor patterns. When studied in sufficient statistical detail,analyses of kinematics and electromyographic (EMG) features revealed that such induced
spinal locomotion differed from voluntary stepping in many important aspects2,9,13. In
addition, it remains unknown whether lumbosacral neuronal networks in the absence of brain
input could sustain full weight-bearing locomotion that resembles nondisabled stepping.
Considering the diffusely distributed5 and heterogeneous4 character of the spinal locomotor
system, it is likely that multiple complementary approaches, both acute and chronic, would be
required to attain the full possible expression of effective stepping in the absence of supraspinal
input.

NIH-PA Author Manuscript

We tested the hypothesis that combinations of specific pharmacological and electricalstimulation interventions, together with locomotor training, may interact synergistically toactivate and functionally remodel spinal locomotor circuits, possibly enabling a coordinatedand context-dependent function of the paralyzed hindlimbs of adult rats after a complete spinal
cord transection. We tested combinations of 5-HT2A and 5-HT1A/7 serotonin agonists and EES
at two different positions distal to the lesion, each of which individually exerted somefacilitating effects on hindlimb function. Using detailed kinematics, EMG and anatomical
analyses, we found that such combinatorial interventions induced unique functional states that
correlated with distinct patterns of locomotion in paralyzed rats. We demonstrate for the firsttime, to the best of our knowledge, the ability of rats with SCI to generate full weight-bearing
bipedal treadmill locomotion that is almost indistinguishable from voluntary stepping recordedin the same rats prior to injury. We also found that sensory input determines the formation ofadaptive motor patterns in the absence of supraspinal influences.

Results
NIH-PA Author Manuscript

Experiments were conducted on adult rats that received a complete mid-thoracic (∼T7) spinal
cord transection, permanently removing all supraspinal input below the level of the lesion. At
7–8 d after injury, all of the rats showed flaccid paralysis of the hindlimbs; no bursts of EMG
were observed in extensor or flexor muscles (Fig. 1) when a rat was positioned in a bipedal
posture on a moving treadmill belt (9 cm s−1) while secured in a jacket that was attached to arobotic arm used to control and measure the amount of hindlimb body weight support (BWS;
Supplementary Fig. 1)2.
Accessing spinal locomotor circuits
We first examined the ability of combined pharmacological and electrical stimulations totransform spinal circuits from a dormant to a functional state. These experiments wereconducted sequentially (Fig. 1a–i) on the same rats to identify the specificity of and synergy
between each intervention on the modulation of stepping patterns (Supplementary Fig. 2). To

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 3

NIH-PA Author Manuscript

facilitate stepping with electrical stimulation, we applied EES over the dorsal surface of L2and S1 (Supplementary Fig. 1). We previously found that stimulation at either site can facilitate
treadmill locomotion in paralyzed rats with SCI after 5–7 weeks of recovery post-injury11,19.
Applied 7–8 d post-injury, EES (40–50 Hz, 0.2 ms, 1–4 V) at L2 and/or S1 modestly increased
EMG bursting patterns or tonic activity in the hindlimb muscles (P < 0.05; Fig. 2), but failed
to generate any step-like movements (Fig. 1c). To engage locomotor networks
pharmacologically, we administrated quipazine, a predominantly 5-HT2A receptor agonist, and
8-OHDPAT, a 5-HT1A and 5-HT7 receptor agonist. Both serotonin agonists can facilitate
treadmill locomotion in spinal rodents after some weeks of recovery from SCI9,10, but whensystemically administered at 7–8 d post-injury, quipazine (0.3 mg per kg of body weight,
intraperitoneal) and/or 8-OHDPAT (0.1–0.3 mg per kg, subcutaneous) induced only brief
periods of erratic hindlimb movements in response to treadmill motion (Fig. 1f), with limited
weight bearing (Fig. 2c) and small EMG bursts in both extensor and flexor muscles (Fig. 2g,h).
Thus, pharmacological or EES interventions alone, administrated 1 week after a complete
interruption of supraspinal input, could not induce functional states that would enable stepping.
In contrast, several combinations of serotonin agonists and EES were strongly synergistic,
resulting in hindlimb locomotion whose features clearly varied with the procedures used (Fig.
1d–i).

NIH-PA Author Manuscript
NIH-PA Author Manuscript

We extensively quantified the kinematics and EMG characteristics of stepping movements
caused by specific treatment combinations (n = 135; Supplementary Table 1). However,
because a large number of these parameters changed substantially across combinations, weresorted to principal component analysis (PCA) to identify the specific contributions ofindividual interventions to the observed variations in hindlimb stepping. PCA transforms theraw dataset to a new coordinate system such that the explained variance is maximized on eachaxis. In the PCA-based statistical representation, the differences between gait patterns are
related to the distance between the data points. The distinct spatial locations (P < 0.05; Fig.
1k–m) occupied by each combination (Fig. 1j) indicated that each intervention promoted a
specific pattern of hindlimb locomotion. The distinct characteristics captured by eachcomponent can be extracted from the analysis of factor loadings, that is, correlations between
each variable and each component. To visualize factor loadings, we developed a color-coded
representation that clearly highlighted the variables that cluster on each component (Fig. 1n).
For example, principal component 3 (Fig. 1j) differentiated the effects of quipazine from theeffects of the other interventions (P < 0.05; Fig. 1j). Variables associated with joint extension(variables 56, 57 and 76) and EMG activity of extensor muscles (soleus, P < 0.01; Fig. 2g)
clustered on principal component 3 (Fig. 1n), indicating that quipazine primarily facilitatedextension components. 8-OHDPAT was significantly more effective in facilitating rhythmic
movements of the hindlimbs under EES at S1 plus L2 compared with quipazine (principal
component 1, P < 0.05; Figs. 1k and 2b,e). However, stepping patterns were less variable (P< 0.001; Fig. 2e), showing markedly improved interlimb coordination (P < 0.01; Fig. 2b) andhigher levels of weight bearing (P < 0.01; Fig. 2c) when combining quipazine, 8-OHDPAT
and EES at either site. In turn, principal component 2 differentiated gait patterns under EES atthe L2 versus S1 levels (P < 0.001; Fig. 1i). Analysis of the variables that clustered on principal
component 2 (Fig. 1m) showed that EES at L2 facilitated flexion and resulted in an enhanced
swing phase (P < 0.01; Fig. 2f), whereas EES at S1 was more biased toward extension.
To further investigate the modulation of hindlimb movements by site-specific EES, we
compared the changes in limb postures produced by EES at L2 compared with EES at S1.
During standing (20% weight bearing), EES at L2 (Fig. 3) induced a rapid flexion (meanduration to peak flexion, 263 ± 96 ms) at all joints (P < 0.001; Fig. 3c,d) that was maintained
tonically during the duration of the stimulation (3 s). In contrast, EES at S1 promoted a
progressive whole-limb extension (mean duration to peak extension, 1,050 ± 361 ms; P < 0.001;
Fig. 3a,d) that persisted as long as the stimulation was continued (3 s) and was comparatively
Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 4

NIH-PA Author Manuscript

slower (P < 0.001) and less stable than the effects of EES at L2. Next, we tested the effects ofincreasing intensity at L2 versus S1 during locomotion enabled by quipazine, 8-OHDPAT and
dual-site EES. Gradually increasing the intensity of EES at L2 progressively increased hip
flexion (P < 0.05; Fig. 3d), step height (P < 0.05; Fig. 3c) and foot velocity (P < 0.05) duringswing. Opposite effects (P < 0.05; Fig. 3c,d) were obtained when increasing EES at S1 (Fig.
3b). Together, these results demonstrate that upper lumbar stimulation engages the circuits
controlling flexion, whereas upper sacral stimulation primarily recruits the circuits controlling
extension.

NIH-PA Author Manuscript

When EES was simultaneously applied to L2 and S1 in the presence of quipazine and 8OHDPAT (Fig. 1i), the variability of stepping motion was reduced compared with EES
application at either site alone (P < 0.01; Fig. 2e). Weight-bearing ability increased twofold(P < 0.01; Fig. 2c) and EMG activity in extensor and flexor muscles reached levels similar to
those observed during voluntary locomotion recorded in the same rats pre-injury (Fig. 2g,h).
Although some defects persisted, for example, toe dragging at swing onset (P < 0.05; Figs. 1dand 2a) and only partial weight-bearing ability (P < 0.001; Fig. 2c), the resulting locomotor
patterns resembled voluntary stepping (Fig. 1a,i). These results indicate that there is a strong,synergistic potential for combined pharmacological and electrical stimulations to functionallyengage spinal locomotor circuits as early as 1 week after a complete spinal cord transection,
promoting weight-bearing locomotion with plantar placement of the paws in the hindlimbs ofparalyzed adult rats (Supplementary Video 1).
Learning in spinal locomotor circuits
Next, we sought to optimize the use of spinal circuits for recovery of locomotor function in
the absence of supraspinal input. To attain this goal, we subjected rats with SCI to the above
pharmacological and/or EES interventions in combination with well-established rehabilitative
locomotor training procedures. We hypothesized that use-dependent mechanisms would
functionally remodel the pharmaco-electrically activated spinal circuits and further improve
stepping ability.
We exposed the rats to 20-min locomotor training sessions every other day for 8 weeks, starting
7-8 d post-injury. Stepping was enabled by the full combination of serotonergic agonists anddual-site EES. We compared the locomotor performance of these rats (Fig. 4) with theirperformance measured at 1 week post-injury, before any training (Fig. 1i), with that of
nontrained rats that received no pharmacological or electrical stimulation (Fig. 4a) and withthat of rats trained with either the serotonin agonists (Fig. 4b) or dual-site EES (Fig. 4c) alone(Supplementary Fig. 2).

NIH-PA Author Manuscript

PCA (Fig. 4f–j) identified clusters of variables on each component (Fig. 4f) that allowed us todifferentiate the effects of the various conditions on stepping abilities. Principal component 1
identified improved performance in rats that were trained with the full combination ofserotonergic agonists and dual-site EES compared with nontrained rats and rats trained witheither the serotonergic agonists or EES alone (P < 0.01; Fig. 4h). Principal component 2
highlighted the deterioration of stepping ability in nontrained rats after 8 weeks compared withthe gait patterns recorded at 1 week post-injury (P < 0.001; Fig. 4i). Principal component 3
revealed the importance of combining the serotonergic agonists and EES for promoting
stepping improvement with locomotor training (P < 0.01; Fig. 4j). Deterioration of stepping
capacities in chronic nontrained rats (Fig. 4a) was made evident by the substantial increase in
movement variability (P < 0.001; Fig. 2e), the loss of interlimb coordination (P < 0.001; Fig.
2b) and the partial coactivation of normally reciprocally activated flexor and extensor hindlimb
muscles (Fig. 4a and Supplementary Fig. 3). In contrast, rats trained with the full combinationof treatments (Fig. 4d) recovered the ability to sustain full weight-bearing locomotion (Fig. 2c)with kinematic profiles of hindlimb joint angles (P > 0.2; Fig. 2d) and limb endpoint trajectories
Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 5

NIH-PA Author Manuscript(Fig. 4d) that were nearly indistinguishable from those observed during voluntary bipedal
treadmill stepping in the same rats pre-injury (Fig. 1a). Although locomotor training with thefull combination did not completely prevent the hindlimb muscle atrophy that accompanies a
SCI, the amount of muscle weight loss was significantly less (mean for all muscles, 14%) intrained versus nontrained rats (P < 0.01; Supplementary Fig. 4). Deterioration of locomotor
performance was reduced in rats trained with either the pharmacological or EES intervention
compared with nontrained rats (P < 0.01; Fig. 4i). However, locomotor training enabled by theindividual interventions failed to promote substantial improvement in stepping ability.

NIH-PA Author Manuscript

We next investigated use-dependent plastic changes in the spinal circuits that mediate theobserved functional improvements. To assess physiological changes, we chronically measured
the efficacy of monosynaptic inputs to extensor and flexor motoneurons in awake standing(20% weight bearing) rats23. The rats showed a significant decrease in the amplitude ofmonosynaptic responses in extensor motoneuron pools 1 week post-injury (P < 0.05; Fig. 5a),whereas these values were unchanged in flexor motoneuron pools (Fig. 5b) compared to prelesion values. Before the onset of locomotor training, no significant differences could be
detected between the two groups (P > 0.4; Fig. 5a,b). After 9 weeks of the absence of weightbearing in non-trained injured rats, we observed no significant changes in the amplitude of the
responses in flexor motoneuron pools (Fig. 5b), whereas there was a moderate facilitation of
the extensor motoneuron pools (P < 0.05; Fig. 5a). In contrast, after 8 weeks of locomotor
training, all of the rats demonstrated a substantial increase in the efficacy of monosynaptic
inputs to both extensor (P < 0.01; Fig. 5a) and flexor (P < 0.01; Fig. 5b) motoneuron pools.
We further assessed the functional remodeling of lumbosacral locomotor networks
anatomically by examining FOS expression patterns2 induced by 45 min of continuous bipedal
stepping under the full combination of treatments. Although FOS-positive nuclei were foundmainly in laminae I–IV in the vicinity of the site of stimulation when EES was delivered withthe rats in a prone, suspended non–weight-bearing position (Supplementary Fig. 5), FOSpositive nuclei were present in all of the laminae of the examined lumbar and sacral segments
in response to locomotor activity (Fig. 5c,d). However, the number of FOS-positive cells per
segment (L1 to S2) was two- to threefold higher in nontrained than in trained (P < 0.01) andnoninjured (P < 0.001) rats (Fig. 5d and Supplementary Fig. 5). Moreover, the total number
of FOS-positive cells was inversely correlated with the level of locomotor recovery (P < 0.001;
Fig. 5e). These results confirm earlier observations2 that motor learning can occur in adultrodent lumbosacral circuits after a complete spinal cord transection and extend these findings
by showing that, in the absence of any supraspinal input, use-dependent learning mechanisms
can promote the recovery of full weight-bearing treadmill locomotion that is kinematicallyvery similar to pre-injury voluntary stepping in the same rats (Supplementary Video 2).

NIH-PA Author Manuscript

Control of spinal locomotor circuits
We next investigated how successful control of spinal locomotor circuits occurs in the absenceof supraspinal input in adult rats. Cats with SCI can recover hindlimb locomotion when
lumbosacral circuits have access to sensory information8,15. This is usually attributed to thesensory systems providing feedback correction24 and reinforcement25 of motor patterns
generated by the spinal circuitry4,9,26. However, the precise contributions of sensory input
during spinal locomotion have not been defined. To investigate the influence of sensory input
on the initiation and control of standing and stepping in the absence of supraspinal input, we
imposed substantial changes in the patterns of afferent information while the rats' spinal circuits
were engaged by the full combination of interventions. For this purpose, an additional groupof rats (n = 6) was trained to step for 3 weeks before testing (Supplementary Fig. 2).
We first compared the effects of dynamic versus static patterns of sensory input. In the absenceof treadmill motion, but under weight-bearing conditions (37% ± 3% of weight bearing),

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 6

NIH-PA Author Manuscript
NIH-PA Author Manuscript

pharmacological and EES interventions facilitated the tonic recruitment of extensor muscles,
whereas the flexor muscles were quiescent or weakly active, behaviorally apparent as standing(Fig. 6a). When treadmill belt motion (5 cm s−1) was initiated, all hindlimb joints underwent
changes toward extension (limb moving backward), creating dynamic proprioceptive input that
immediately transformed the motor patterns from a tonic to a rhythmic state (Fig. 6a). To further
test the influence of velocity-dependent afferent input on motor pattern formation, we
incrementally increased the treadmill belt speed from 5 cm s−1 (slow walking) to 25 cm s−1(running). Increasing treadmill speed induced a velocity-dependent lengthening of the stride(P < 0.01; Fig. 6a), an increase in stepping frequency (P < 0.001; Fig. 6a,b), a progressive
decrease in the duration of the stance phase (P < 0.001; Fig. 6b) and of extensor EMG bursts(P < 0.001; Fig. 6c), and a progressive adjustment in the relative timing between hindlimb
segment oscillations (P < 0.05; Fig. 6d). In contrast, the duration of the swing phase (Fig. 6b)
and of flexor bursts (Fig. 6c) were unchanged across speeds. Similar adjustments of thekinematics and EMG parameters were observed in noninjured rats (Supplementary Fig. 6). Allof the rats with SCI (n = 6) accommodated displacement of the limbs and recruitment of motor
pools to changing treadmill belt speeds in a single step and were able to locomote for extended
periods of time on the treadmill even at the fastest speeds (25 cm s−1; Supplementary Fig. 7).
When the treadmill belt was stopped at either slow or fast speeds, rhythmic hindlimb
movements arrested instantly (Fig. 6a). Ongoing bursts in extensor muscles persisted assustained tonic activity, whereas flexor muscles became quiescent, similar to what would occur
in noninjured rats.
Next, we assessed the influence of weight-bearing input on the formation of motor patterns inrats with SCI (Fig. 7). When hindlimbs were suspended above the treadmill belt, that is, in theabsence of load-related input, pharmacological and EES interventions induced step-likemovements with alternate recruitment of extensor and flexor motor pools in all of the rats (n= 6; Fig. 7a). In the absence of belt motion, contact of the hindlimbs with the treadmill
immediately arrested the rhythmic movements (Fig. 7a), stopped the recruitment of flexor
muscles (P < 0.001; Fig. 7d), and induced tonic levels of EMG activity in extensor muscles(P < 0.001; Fig. 7d) that resulted in vertical reaction forces (P < 0.001; Fig. 7e), enabling asustained standing posture. Stepping movements continued if the treadmill belt was moving
at the time of hindlimb contact, even with minimal weight bearing (Fig. 7b). Increasing thelevel of weight bearing resulted in a progressive increase in the amplitude of the extensor bursts(P < 0.001; Fig. 7b–d), an increase in the vertical reaction forces during stance (P < 0.001; Fig.
7b,c,e) and adjustments in hindlimb endpoint trajectories (longer stride, lower height) (P <0.01; Fig. 7b).

NIH-PA Author Manuscript

Finally, we assessed the effects of direction-dependent afferent input on locomotor patternformation (Fig. 8). When reversing the direction of the treadmill belt from forward to backward,
all of the rats (n = 6) showed alternate stepping movements with backward-oriented motion of
the foot during swing (P < 0.001; Fig. 8b) and significant adaptations (timing and amplitude,
P < 0.001) in muscle activity (Fig. 8b). Notably, a substantial coactivation of antagonist
muscles was observed systematically during stance (Fig. 8b), as also occurred, although lessfrequently, in noninjured rats (Supplementary Fig. 6). In contrast, reciprocal recruitment ofextensor and flexor motor pools was the only pattern observed during forward locomotion (Fig.
8a). Likewise, rotating the rat perpendicular to the treadmill belt direction did not arrest
stepping movements. Instead, all of the rats (n = 6) showed sideways displacements of the feet(P < 0.001; Fig. 8c) with a reversed pattern of hip abduction and adduction (P < 0.001; Fig.
8c) compared with forward locomotion (Fig. 8a). Although variable among rats, a major
reorganization in the coordination patterns of proximal and distal flexor and extensor motor
pools was observed during sideways locomotion. Well-defined coactivation patterns (Fig. 8c)
between motor pools that were activated reciprocally during forward locomotion (Fig. 8a) also
occurred during sideways stepping. These results reveal a previously unrecognized ability of
Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 7

NIH-PA Author Manuscript

velocity-, load- and direction-dependent sensory inputs to regulate both the control of standing
and the control of adaptive and flexible stepping movements with an exquisite degree ofrefinement and without any influences from supraspinal centers in adult rats (Supplementary
Video 3).

Discussion
Accessing the circuits and receptors in lumbosacral spinal cord

NIH-PA Author Manuscript

Although several studies have documented the potential of pharmacological9,10 and/or
electrical stimulation11–14,19,27, as well as motor training2,8,16, to facilitate stepping in rats
with a complete spinal cord transection, limited weight-bearing capacities have been achieved,
and when investigated in detail, kinematics and EMG patterns differed substantially from thoseunderlying voluntary stepping. A major result of our study is that a combination ofpharmacological and electrical stimulations was able to acutely transform rodent lumbosacral
circuits from nonfunctional to highly functional and adaptive states as early as 1 week postinjury. Furthermore, we found that, in conjunction with rehabilitative motor training, the samecombined stimulations promoted the recovery of full weight-bearing hindlimb locomotion ona treadmill that was nearly indistinguishable from voluntary stepping, although robotic
assistance was necessary for the rats to maintain balance. Together with previous evidence incats24, our results indicate that the mammalian lumbosacral spinal cord contains circuitry thatis sufficient to generate close-to-normal hindlimb locomotor patterns in the absence of any
supraspinal input.
The mechanisms underlying locomotion induced by intraspinal stimulation13,14 or EES11,12,
19,27 in rats and cats remain undetermined. Neurophysiological recordings28 and computer

NIH-PA Author Manuscript

simulations29, however, suggest that the electrical stimulation engages spinal circuits primarily
by recruiting afferent fibers. In fact, dorsal root stimulation can induce stepping similar to
intraspinal stimulation in spinal cord–injured cats14. In humans, leg muscle vibration, which
recruits large-diameter afferent fibers, can evoke locomotor-like movements of the legs6. In
principle, EES applied at discrete lumbosacral locations could recruit both long-range
ascending and descending afferent branches in the segments below the spinal cord transection
and activate profuse intraspinal ramifications that contact subsets of circuits localized aroundthe stimulation site30. Accordingly, although electrical stimulation of virtually any lumbosacral
segment can facilitate step-like movements on a treadmill11, we found clear site-specific effectsof EES on hindlimb locomotion. Consistent with the rostrocaudal anatomical gradient of flexorand extensor motor pools, lumbar stimulation facilitated flexion, whereas stimulation appliedat the sacral level primarily facilitated extension. Distinct locations of serotonin receptors
associated with stepping have also been reported in neonatal rats. A previous study18 foundthat in vitro brainstem-induced fictive locomotion can be blocked by antagonizing 5-HT2A or
5-HT7 receptors and that 5-HT7 receptors are prominent in the upper lumbar segments, whereas
5-HT2A receptors are more localized to the lower lumbar and sacral segments. Further studies
will be required to identify the location and types of neurons, interneurons and receptors thatare engaged by serotonergic agonists and electrical stimulation during treadmill locomotion.
Nevertheless, our statistical analyses indicate that each pharmacological or electricalintervention modulates distinct functions, suggesting the fine-tuning of selective circuits. This
specificity provides the means for the synergy between serotonergic agonist and electricalstimulation interventions, such that only combinations of both were able to functionally engage
spinal locomotor networks 1 week after injury and induce substantial functional improvements
when combined with rehabilitative locomotor training.
These findings support the viewpoint17,31,32 that the spinal motor infrastructure is composedof a widely distributed and heterogeneous, but highly integrated and synergistic, system ofneural circuits and receptors that can generate a range of task-specific movements when
Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 8

NIH-PA Author Manuscript

recruited in different combinations. Evidence for such principles has been extracted statisticallyin humans33,34 and documented directly in anesthetized frogs31, rats32 and cats35. Our findings
are consistent with and extend these organizational principles to the production of adaptive
locomotor movements without any brain input in vivo. Consistent with this, the developmentof high-density electrode arrays that enable detailed, distributed and simultaneous access tothe diffusely located components of the lumbosacral circuits may offer the potential to promotea higher level of motor control than is currently possible in paralyzed subjects.
Functional remodeling of the spinal locomotor circuitry

NIH-PA Author Manuscript

A substantial reorganization of propriospinal circuits and spared descending fibers that leads
to functional recovery occurs spontaneously after an incomplete SCI 3 and can be enhancedwith locomotor training36. However, there has been limited or no evidence for a similar
plasticity of lumbosacral networks when the lesion interrupts all supraspinal inputs in adultrats1,2 and humans37, contrary to functional improvements that have been repeatedly
documented in cats with SCI8,15,16. We found that the combination of locomotor training withpharmacological and electrical stimulation markedly improved the functional capabilities ofthe sensorimotor circuits to sustain locomotion without supraspinal input. Although locomotor
training with either pharmacological or electrical stimulation alone could reduce thedeterioration of stepping ability observed in chronic untrained rats with SCI, only the
combination of all three intervention types could produce a substantial improvement. The
decrease in the number of FOS-positive neurons, together with elevated synaptic efficacy ofmonosynaptic inputs to both flexor and extensor motoneuron pools, suggests that locomotor
training combined with serotonergic and electrical stimulations may reinforce selected spinal
circuits and may suppress other, nonspecific circuitry. Use-dependent selection andstrengthening of neural circuits has been shown neurochemically16 andelectrophysiologically20,21 after motor training in adult cats with SCI and rats with a neonatal
SCI, as well as in the brain in conjunction with the practice of skilled movements38. Our results
suggest that similar mechanisms underlie motor learning in the adult rat spinal cord bereft ofbrain input and that the extent of this functional remodeling is highly correlated with the degreeof improvement of locomotor ability. We also found that the chronic absence of activity leads
to a deterioration in stepping ability, as has been reported in cats15. These results corroborate
clinical insights from individuals with severe SCI39 and emphasize the need to counteract the
rapid deterioration of function that occurs in the absence of weight bearing and motor activity
after a SCI.

NIH-PA Author Manuscript

Combined, these results indicate that the recovery of stepping ability after a spinal cord
transection does not result simply from the activation of an ontogenetically defined hardwired
and unmodified circuitry that persists and recovers post-injury. Instead, specific combinationsof locomotor training and pharmacological and electrical stimulation induce de novo usedependent functional states that enable spinal circuits to learn the motor tasks that are trained
and practiced.
Sensory control of stepping after the loss of brain input
The recovery of hindlimb locomotion in animals with SCI is usually attributed to the neuronal
networks responsible for central pattern generation, that is, oscillatory output without brain or
sensory input4,9,26. However, we found that near normal adaptive stepping and standing
without any brain input emerged in vivo as a result of the capability of spinal neuronal networks
to recognize and use task-specific sensory input. We found that sensory information instantly
transformed motor patterns in response to changing task, load, speed and direction conditions
with a degree of flexibility and precision that has not been previously observed in animals withSCI24.

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 9

NIH-PA Author Manuscript

Although the role of sensory information in feedback modulation of locomotor activity has
been well recognized in cats with SCI15,16,24,25 as well as in humans40,41, our results suggestthat the sensory information is also instructive in a functional, primary feedforward manner.
Indeed, we previously showed that pharmacological and electrical stimulations fail to elicit
rhythmic recruitment of deafferented hindlimb muscles in rats with SCI in vivo42 and a partial
deprivation of cutaneous input strongly reduces the stepping ability of cats with SCI, but not
in intact cats43. Robust changes in the functional properties of spinal locomotor circuits havebeen described in the lamprey when introducing sensory inputs44. However, these interactions
between feedforward and feedback mechanisms have received little attention. Our results
indicate that the ability of afferent information to markedly reorganize functional connections
among spinal sensorimotor pathways, both acutely and chronically, is an important propertyof the spinal motor infrastructure.
Clinical perspectives

NIH-PA Author Manuscript

Our findings have important implications for the understanding of motor pattern formation in
the absence of supraspinal input in vivo and provide a conceptual framework for the design ofstrategies to ameliorate motor function in humans after SCI or in the context of otherneuromotor disorders such as Parkinson's disease45. Preliminary clinical studies have reported
that EES can engage lumbosacral circuits of spinal cord–injured individuals7 and facilitate therecovery of functional walking46. Given the evidence that afferents can serve as a source ofcontrol for stepping and standing and can drive use-dependent plasticity of locomotor networks
in spinal cord–injured individuals37, combinatorial strategies may also promote recovery of
motor function after severe SCI in humans. Such interventions could include neuroprosthetic
spinal cord electrode arrays, customized pharmacological treatments and robotically-assisted
locomotor training procedures. With the development of efficacious neural repair therapies,
these neurorehabilitative strategies may become important for counteracting chronic
deterioration of function39, optimizing the use of intrinsic lumbosacral circuits in regaining
function37 and ensuring functional interactions between spinal mechanisms and regenerative
fibers47,48.

Methods
Methods and any associated references are available in the online version of the paper at
http://www.nature.com/natureneuroscience/.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

NIH-PA Author Manuscript

Acknowledgments
We would like to acknowledge the excellent technical help provided by S. Zdunowski, L. Friedli, J. Heutschi and O.
Märzendorfer for data collection and analysis, as well as M. Herrera for his expert assistance and guidance involving
the care and handling of the animals. This work was supported by the Craig H. Nielsen Foundation (#20062668), theInternational Paraplegic Foundation (P106), the National Center of Competence in Research ‘Neural Plasticity andRepair’ of the Swiss National Science Foundation, the Christopher and Dana Reeve Foundation (VEC-2007), the US
National Institute of Neurological Disorders and Stroke (NS16333), the US Civilian Research and Development
Foundation (RUB1-2872-07), the Roman Reed Spinal Cord Injury Research Fund of California and the RussianFoundation for Basic Research (07-04-00526 and 08-04-00688).

References
1. Kubasak MD, et al. OEG implantation and step training enhance hindlimb-stepping ability in adultspinal transected rats. Brain 2008;131:264–276. [PubMed: 18056162]

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 10

NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

2. Ichiyama RM, et al. Step training reinforces specific spinal locomotor circuitry in adult spinal rats. J
Neurosci 2008;28:7370–7375. [PubMed: 18632941]
3. Courtine G, et al. Recovery of supraspinal control of stepping via indirect propriospinal relay
connections after spinal cord injury. Nat Med 2008;14:69–74. [PubMed: 18157143]
4. Grillner S. Biological pattern generation: the cellular and computational logic of networks in motion.
Neuron 2006;52:751–766. [PubMed: 17145498]
5. Kiehn O. Locomotor circuits in the mammalian spinal cord. Annu Rev Neurosci 2006;29:279–306.
[PubMed: 16776587]
6. Gurfinkel VS, Levik YS, Kazennikov OV, Selionov VA. Locomotor-like movements evoked by leg
muscle vibration in humans. Eur J Neurosci 1998;10:1608–1612. [PubMed: 9751133]
7. Dimitrijevic MR, Gerasimenko Y, Pinter M. Evidence for a spinal central pattern generator in humans.
Ann NY Acad Sci 1998;860:360–376. [PubMed: 9928325]
8. Chau C, Barbeau H, Rossignol S. Early locomotor training with clonidine in spinal cats. J Neurophysiol
1998;79:392–409. [PubMed: 9425208]
9. Antri M, Mouffle C, Orsal D, Barthe JY. 5–HT1A receptors are involved in short- and long-term
processes responsible for 5-HT-induced locomotor function recovery in chronic spinal rat. Eur J
Neurosci 2003;18:1963–1972. [PubMed: 14622228]
10. Landry ES, et al. Contribution of spinal 5–HT1A and 5–HT7 receptors to locomotor-like movement
induced by 8-OHDPAT in spinal cord–transected mice. Eur J Neurosci 2006;24:535–546. [PubMed:
16836640]
11. Ichiyama RM, Gerasimenko YP, Zhong H, Roy RR, Edgerton VR. Hindlimb stepping movements
in complete spinal rats induced by epidural spinal cord stimulation. Neurosci Lett 2005;383:339–
344. [PubMed: 15878636]
12. Gerasimenko YP, et al. Epidural spinal cord stimulation plus quipazine administration enable steppingin complete spinal adult rats. J Neurophysiol 2007;98:2525–2536. [PubMed: 17855582]
13. Guevremont L, et al. Locomotor-related networks in the lumbosacral enlargement of the adult spinal
cat: activation through intraspinal microstimulation. IEEE Trans Neural Syst Rehabil Eng
2006;14:266–272. [PubMed: 17009485]
14. Barthélemy D, Leblond H, Rossignol S. Characteristics and mechanisms of locomotion induced byintraspinal microstimulation and dorsal root stimulation in spinal cats. J Neurophysiol 2007;97:1986–
2000. [PubMed: 17215509]
15. De Leon RD, Hodgson JA, Roy RR, Edgerton VR. Retention of hindlimb stepping ability in adultspinal cats after the cessation of step training. J Neurophysiol 1999;81:85–94. [PubMed: 9914269]
16. Tillakaratne NJ, et al. Use-dependent modulation of inhibitory capacity in the feline lumbar spinal
cord. J Neurosci 2002;22:3130–3143. [PubMed: 11943816]
17. Hochman, S.; Garraway, S.; Machacek, D.; Shay, B. 5-HT receptors and the neuromodulatory controlof spinal cord function. In: Cope, TC., editor. Motor Neurobiology of the Spinal Cord. CRC Press;
Boca Raton, Florida: 2001. p. 47-87.
18. Liu J, Jordan LM. Stimulation of the parapyramidal region of the neonatal rat brain stem produces
locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors. J Neurophysiol
2005;94:1392–1404. [PubMed: 15872068]
19. Lavrov I, et al. Epidural stimulation induced modulation of spinal locomotor networks in adult spinal
rats. J Neurosci 2008;28:6022–6029. [PubMed: 18524907]
20. Petruska JC, et al. Changes in motoneuron properties and synaptic inputs related to step training after
spinal cord transection in rats. J Neurosci 2007;27:4460–4471. [PubMed: 17442831]
21. Côté MP, Gossard JP. Step training–dependent plasticity in spinal cutaneous pathways. J Neurosci
2004;24:11317–11327. [PubMed: 15601938]
22. Barthélemy D, Leblond H, Provencher J, Rossignol S. Nonlocomotor and locomotor hindlimb
responses evoked by electrical microstimulation of the lumbar cord in spinalized cats. J Neurophysiol
2006;96:3273–3292. [PubMed: 16943319]
23. Lavrov I, et al. Plasticity of spinal cord reflexes after a complete transection in adult rats: relationshipto stepping ability. J Neurophysiol 2006;96:1699–1710. [PubMed: 16823028]

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 11

NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

24. Rossignol S, Dubuc R, Gossard JP. Dynamic sensorimotor interactions in locomotion. Physiol Rev
2006;86:89–154. [PubMed: 16371596]
25. Pearson KG. Generating the walking gait: role of sensory feedback. Prog Brain Res 2004;143:123–
129. [PubMed: 14653157]
26. Barrière G, Leblond H, Provencher J, Rossignol S. Prominent role of the spinal central patterngenerator in the recovery of locomotion after partial spinal cord injuries. J Neurosci 2008;28:3976–
3987. [PubMed: 18400897]
27. Iwahara T, Atsuta Y, Garcia-Rill E, Skinner RD. Spinal cord stimulation-induced locomotion in theadult cat. Brain Res Bull 1992;28:99–105. [PubMed: 1540851]
28. Gaunt RA, Prochazka A, Mushahwar VK, Guevremont L, Ellaway PH. Intraspinal microstimulation
excites multisegmental sensory afferents at lower stimulus levels than local alpha-motoneuron
responses. J Neurophysiol 2006;96:2995–3005. [PubMed: 16943320]
29. Rattay F, Minassian K, Dimitrijevic MR. Epidural electrical stimulation of posterior structures of thehuman lumbosacral cord. 2. Quantitative analysis by computer modeling. Spinal Cord 2000;38:473–
489. [PubMed: 10962608]
30. Rudomin P. Central control of information transmission through the intraspinal arborizations of
sensory fibers examined 100 years after Ramon y Cajal. Prog Brain Res 2002;136:409–421.
[PubMed: 12143398]
31. Giszter SF, Mussa-Ivaldi FA, Bizzi E. Convergent force fields organized in the frog's spinal cord. J
Neurosci 1993;13:467–491. [PubMed: 8426224]
32. Tresch MC, Bizzi E. Responses to spinal microstimulation in the chronically spinalized rat and theirrelationship to spinal systems activated by low-threshold cutaneous stimulation. Exp Brain Res
1999;129:401–416. [PubMed: 10591912]
33. Courtine G, Schieppati M. Tuning of a basic coordination pattern constructs straight-ahead and curved
walking in humans. J Neurophysiol 2004;91:1524–1535. [PubMed: 14668296]
34. Ivanenko YP, Cappellini G, Dominici N, Poppele RE, Lacquaniti F. Modular control of limb
movements during human locomotion. J Neurosci 2007;27:11149–11161. [PubMed: 17928457]
35. Lemay MA, Grill WM. Modularity of motor output evoked by intraspinal microstimulation in cats.
J Neurophysiol 2004;91:502–514. [PubMed: 14523079]
36. Engesser-Cesar C, et al. Wheel running following spinal cord injury improves locomotor recovery
and stimulates serotonergic fiber growth. Eur J Neurosci 2007;25:1931–1939. [PubMed: 17439482]
37. Harkema SJ. Plasticity of interneuronal networks of the functionally isolated human spinal cord. Brain
Res Rev 2008;57:255–264. [PubMed: 18042493]
38. Rioult-Pedotti MS, Friedman D, Donoghue JP. Learning-induced LTP in neocortex. Science
2000;290:533–536. [PubMed: 11039938]
39. Dietz V, Muller R. Degradation of neuronal function following a spinal cord injury: mechanisms andcountermeasures. Brain 2004;127:2221–2231. [PubMed: 15269117]
40. Harkema SJ, et al. Human lumbosacral spinal cord interprets loading during stepping. J Neurophysiol
1997;77:797–811. [PubMed: 9065851]
41. Beres-Jones JA, Harkema SJ. The human spinal cord interprets velocity-dependent afferent input
during stepping. Brain 2004;127:2232–2246. [PubMed: 15289272]
42. Lavrov I, et al. Facilitation of stepping with epidural stimulation in spinal rats: role of sensory input.
J Neurosci 2008;28:7774–7780. [PubMed: 18667609]
43. Bouyer LJ, Rossignol S. Contribution of cutaneous inputs from the hindpaw to the control oflocomotion. II. Spinal cats. J Neurophysiol 2003;90:3640–3653. [PubMed: 12944535]
44. Grillner S, Wallen P. Cellular bases of a vertebrate locomotor system-steering, intersegmental andsegmental coordination and sensory control. Brain Res Brain Res Rev 2002;40:92–106. [PubMed:
12589909]
45. Fuentes R, Petersson P, Siesser WB, Caron MG, Nicolelis MA. Spinal cord stimulation restores
locomotion in animal models of Parkinson's disease. Science 2009;323:1578–1582. [PubMed:
19299613]

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 12

NIH-PA Author Manuscript

46. Carhart MR, He J, Herman R, D'Luzansky S, Willis WT. Epidural spinal-cord stimulation facilitates
recovery of functional walking following incomplete spinal-cord injury. IEEE Trans Neural Syst
Rehabil Eng 2004;12:32–42. [PubMed: 15068185]
47. Courtine G, et al. Can experiments in nonhuman primates expedite the translation of treatments forspinal cord injury in humans? Nat Med 2007;13:561–566. [PubMed: 17479102]
48. Maier IC, et al. Differential effects of anti-Nogo-A antibody treatment and treadmill training in rats
with incomplete spinal cord injury. Brain 2009;132:1426–1440. [PubMed: 19372269]

NIH-PA Author Manuscript
NIH-PA Author Manuscript
Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 13

NIH-PA Author Manuscript
Figure 1.

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Accessing spinal locomotor circuits 1 week after the interruption of all supraspinal input. (a–i) EMG and kinematic characteristics underlying locomotion recorded pre-injury (a) and 7–8
d post-injury (b) without any intervention, as well as under various combinations ofserotonergic agonists and/or EES (c–i). The full combination (i) included quipazine, 8OHDPAT and EES at L2 and S1. Recordings were performed sequentially in the same rat.
Horizontal arrows indicate the chronology of the different recordings. A representative stickdiagram decomposition of hindlimb motion during swing is shown for each condition withsuccessive color-coded trajectories of limb endpoint. Vectors represent the direction andintensity of the limb endpoint velocity at swing onset. A sequence of raw EMG activity fromtibialis anterior (TA) and soleus (Sol) muscles is shown below. Grey and red bars indicate theduration of stance and drag phases, respectively. The BWS of the represented rat under eachcondition is shown. Finally, a polar plot representation documents the coordination between
the left and right hindlimbs (thin arrow, single gait cycle; thick red arrow, average of all gait
cycles; 50%, out of phase). (j) Three-dimensional statistical representation of locomotor
patterns. Each small colored label represents the gait pattern from an individual rat under agiven combination of interventions. The area defined by individual points under a given
condition is traced to emphasize the differences between gait patterns under specificcombinations. This analysis revealed that each combination of interventions resulted in distinct,
but reproducible, patterns of locomotion. (k–m) Bar graphs of average scores on principal
components 1–3. (n) Color-coded representation of factor loadings that identify the variables
that contributed most to the differences observed between the experimental conditions. For
example, principal component 2 captured the differences between stepping with EES at L2
versus S1. Variables associated with changes in joint angles toward flexion and limb endpoint
trajectory (left and right step heights, 18–19) clustered on principal component 2, indicatingthat EES at L2 enhanced flexion, whereas EES at S1 enhanced extension. All of the computed
kinematic and EMG variables (n = 135) are reported in Supplementary Table 1. Error bars
represent s.e.m. * P < 0.05, different from all other conditions. ** P < 0.05, significantly
different conditions.

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 14

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 2.

NIH-PA Author Manuscript

Kinematics and EMG features of locomotor patterns. (a–h) Bar graphs of average values (n =7 or 8 rats per group) for locomotor parameters computed under the different experimental
conditions. In each graph, the green horizontal bar represents the pre-lesion baseline recordedin the same rat 1 week pre-injury. The duration of stance (light gray), swing (dark gray) anddrag (red) phases is shown in a. The r values at t = 0 for the cross-correlation function between
oscillations of the left and right hindlimbs computed over a gait sequence of ten steps is shownin b. A r value of −0.5 indicates out of phase coupling between the limbs. The maximum levelof weight bearing (percentage of body weight) at which the rat could perform ten successful
steps is shown in c. Cross-correlation functions were computed between datasets obtained preinjury and under a given experimental condition for the hip, knee, ankle and MTP joint angles,
and associated joint velocity profiles (d). Maximum r values were extracted from each crosscorrelation function and averaged across joint angle and joint angle velocity profiles. The
variability of gait parameters computed as the mean coefficient of variation for all the computed
parameters normalized to the pre-injury baseline are shown in e. Step height, defined as the
maximum vertical distance between the foot (MTP marker) and the stepping surface, is shownin f. The average EMG burst amplitude for left and right soleus (g) and tibialis (h) anterior
muscles normalized to pre-injury values are shown. Error bars represent s.e.m. ** P < 0.05,
significantly different conditions.

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 15

NIH-PA Author Manuscript
Figure 3.

NIH-PA Author Manuscript

Site-specific effects of EES during standing and stepping. (a) Stick diagram decomposition ofhindlimb movements and time course of changes in hindlimb joint angles when delivering EES
at L2 (left) or S1 (right) during standing (20% of weight bearing). Each diagram is separated
by 5 ms (L2) or 50 ms (S1). The dark gray shaded areas indicate the period during which EESinduced changes in hindlimb posture were observed. Light gray shaded areas represent periods
during which EES-induced posture was maintained. (b) Effects of increasing EES by 0.4 and
0.8 V at L2 versus S1 on hindlimb movements during locomotion enabled by the fullcombination of interventions. Data are presented as in Figure 1. Arrows indicate increased
flexion with EES at L2 versus increased extension with EES at S1. (c) Bar graph of averagevalues of maximum foot height during standing and step height during locomotion. Opt,
optimal EES intensity to encourage stepping. (d) Bar graphs of average values of angularchanges during standing and stepping. For standing, values were obtained by measuring, foreach joint angle, the difference between positions at EES onset and at the time of maximum
EES-induced change in hindlimb posture at each joint and then averaging these values acrossjoints. Ext, extension. Fle, flexion. For stepping, the maximum position of the hip joint angle
in flexion during swing was computed. Error bars represent s.e.m. * P < 0.05, different fromall the other conditions. ** P < 0.05, significantly different conditions.

NIH-PA Author Manuscript
Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 16

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 4.

NIH-PA Author Manuscript

Rehabilitation locomotor training enabled by pharmacological and EES interventions improves
stepping ability. (a–d) Representative illustrations of EMG and kinematic characteristics
underlying bipedal hindlimb locomotion recorded at 9 weeks post-injury under the fullcombination of interventions for a nontrained rat with SCI that did not receive pharmacologicalor EES interventions for the entire duration of the post-injury period until the day of testing(a), a rat with SCI that was trained with serotonergic agonists only (b), a rat with SCI that wastrained with EES at L2 and S1 only (c), and a rat with SCI that was trained with the fullcombination of interventions (d). Data for this rat are also shown pre-injury in Figure 1a andat 1 week post-injury in Figure 1i. (e) Successive limb endpoint trajectories from the righthindlimb are shown for all of the other rats from each experimental group. The BWS of each
rat is reported below each limb endpoint trajectory. (f) Color-coded representation of factor
loadings of each variable on principal components 1–3 (as shown in Fig. 1n). Principal
component 1 identified improved gait in rats tested at 1 week post-injury and then trained withthe full combination compared with the other groups. The analysis of variables that clusteredon principal component 1 indicated that reduced variability of gait parameters, improved gaitstability, increased amplitude of EMG activity and recovery of full weight-bearing capacities
were the more salient features for explaining the improved stepping performances of rats
trained with the full combination. (g) Three-dimensional statistical representation of locomotor
patterns. The near absence of spatial interceptions between the different groups indicates thateach group of rats had unique stepping patterns. (h–j) Bar graphs of average scores on principal
components 1–3, which each captured specific effects. Error bars represent s.e.m. * P < 0.05,
different from all the other conditions. ** P < 0.05, significantly different conditions. Data arepresented as in Figure 1.
Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 17

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 5.

NIH-PA Author Manuscript

Functional remodeling of spinal circuits after rehabilitative locomotor training. (a)Representative average (n = 10) traces of monosynaptic motor-evoked potentials recorded fromthe soleus muscle pre-injury and at 1 and 9 weeks post-injury. Data are shown for one
nontrained and one rat trained with the full combination of interventions. Dark shaded areas
indicate the amplitude of pre-injury motor-evoked potentials. Bar graphs report the average
amplitude of motor-evoked potentials recorded in the soleus muscle at the different time points.
(b) Data are presented as in a for the tibialis anterior muscle. (c) Representative example ofcamera lucida drawings of FOS-positive cells in spinal segments L2, L4 and S1 of a noninjured
rat, a nontrained rat with SCI and a rat with SCI trained with the full combination ofinterventions. (d) Average values for the total FOS-positive cell count (all laminae) per spinal
segment. (e) Correlation between the total number of FOS-positive cells (all laminae from L1
to S2) and gait performance measured as individual scores along the principal component 1
axis. PCA was applied on locomotor data (n = 135) recorded from the same rats 3–5 d before
the FOS experiments under the same conditions, that is, no intervention for noninjured rats
and under the full combination for rats with SCI. Error bars represent s.e.m. * P < 0.05, different
from trained group. ** P < 0.05, different from noninjured group.

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 18

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 6.

NIH-PA Author Manuscript

Effects of velocity-dependent afferent input on motor patterns. (a) Representative example ofhindlimb kinematics and EMG activity recorded from a continuous sequence of steps during
which the speed of the treadmill belt was changed gradually (0, 5, 15, 25 and 0 cm s−1). Data
are presented as in Figure 1, except that changes in hindlimb joint angles are also shown. Stickdiagram decomposition of the first step is shown to demonstrate the smooth transition fromstanding to stepping. MG, medial gastrocnemius; St, semitendinosus; VL, vastus lateralis.
(b) The durations of the swing and stance phases are plotted against the cycle duration. Colorcoded labels indicate the measured treadmill belt speed during the performance of therepresented gait cycles. (c) The durations of flexor (TA) and extensor (MG) EMG bursts areplotted against the cycle duration. (d) The temporal lag between oscillations (with respect tothe direction of gravity) of adjacent hindlimb segments is plotted against the cycle duration.
Inter-limb lags were computed by means of cross-correlation functions and expressed as apercent of cycle duration. b–d are shown for a representative rat. Mean ± s.e.m. correlation
values computed by averaging values obtained from linear regressions performed on each rat(n = 6) individually are reported in each plot. All rats were trained with the full combinationof interventions for 3 weeks before the experimental testing.

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 19

NIH-PA Author Manuscript
NIH-PA Author Manuscript
Figure 7.

NIH-PA Author Manuscript

Effects of load-dependent afferent input on motor patterns. (a) Representative example ofhindlimb kinematics and EMG activity induced by the full combination while the rat transitedfrom suspended in the air (0% of body weight) to contact with the immobile treadmill belt
(40% of body weight support). (b) Representative example of limb endpoint trajectories, mean
EMG activity and mean vertical reaction forces during stepping with 0, 20, 60 or 100% weightbearing. (c) The mean vertical reaction force measured during stance is plotted against theamplitude of the medial gastrocnemius EMG burst for a representative rat that demonstrated
full weight-bearing capacities after 3 weeks of training with the full combination ofinterventions. Color-coded labels represent the amount of body weight supported by thehindlimbs of the rat. A strong relationship was observed in all of the rats (n = 6). The mean ±s.e.m. correlation value computed by averaging the values obtained from linear correlation
computed on each rat is reported. (d) Bar graphs of average amplitudes (n = 6) of EMG bursts
in selected hindlimb muscles. Values are normalized to values measured during standing (40%of body weight support). The 100% weight-bearing condition is not represented because only
two rats could step without any support after 3 weeks of training. (e) Bar graphs of averagevalues (n = 6) of vertical reaction forces measured during stance. Error bars represent s.e.m.
** P < 0.05, significantly different conditions.

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.

Courtine et al.

Page 20

NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 8.

NIH-PA Author Manuscript

Effects of direction-dependent afferent input on motor patterns. (a–c) Representative example
of mean (+ s.d.) hindlimb kinematics and raw EMG activity during continuous locomotion in
the forward (a), backward (b) and sideways (c) directions. The same limb from the same rat
is shown for the three directions, which corresponds to the leading (front) limb during sideward
locomotion. Bar graphs show the average (n = 6 rats) linear distance traveled by the foot duringswing with respect to the pelvis orientation for the different directions of stepping. Backward(BW) and forward (FW) motions correspond to displacements in the sagittal plane (defined by
the pelvis orientation), whereas outward (OW) and inward (IW) motions correspond to
displacements in the medio-lateral direction. Probability density distributions of normalized
EMG amplitudes between the semitendinosus and medial gastrocnemius muscles, and thetibialis anterior and vastus lateralis muscles are shown at the bottom. L-shape patterns indicate
reciprocal activation between the pair of muscles, whereas line-shape patterns indicate
coactivation. Abd, abduction (increasing value); Add, adduction. Data are presented as in
Figure 1, except that stick diagrams are represented in three dimensions, with the main planeoriented with the direction of the treadmill belt motion.

Nat Neurosci. Author manuscript; available in PMC 2010 February 25.