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Brain Res Rev. Author manuscript; available in PMC 2009 January 1.

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Published in final edited form as:
Brain Res Rev. 2008 January ; 57(1): 241–254.

Training Locomotor Networks
V. Reggie Edgerton1,2,3, Grégoire Courtine1,2, Yury P. Gerasimenko1,4, Igor Lavrov1,2,
Ronaldo M. Ichiyama1,2, Andy J. Fong5, Lance L. Cai5, Chad K. Otoshi2, Niranjala J.K.
Tillakaratne1,3, Joel W. Burdick5,6, and Roland R. Roy3
1Department of Physiological Science, University of California, Los Angeles, Los Angeles, CA 90095
2Department of Neurobiology, University of California, Los Angeles, Los Angeles, CA 90095
3Brain Research Institute, University of California, Los Angeles, Los Angeles, CA 90095
4Pavlov Institute of Physiology, St. Petersburg, 199034, Russia
5Division of Engineering, Bioengineering, California Institute of Technology, Pasadena, CA 91125

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6Division of Engineering, Mechanical Engineering Options, California Institute of Technology, Pasadena,
CA 91125

Abstract

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For a complete adult spinal rat to regain some weight-bearing stepping capability, it appears that asequence of specific proprioceptive inputs that are similar, but not identical, from step to step mustbe generated over repetitive step cycles. Furthermore, these cycles must include the activation of
specific neural circuits that are intrinsic to the lumbosacral spinal cord segments. For thesesensorimotor pathways to be effective in generating stepping, the spinal circuitry must be modulated
to an appropriate excitability level. This level of modulation is sustained from supraspinal input inintact, but not spinal, rats. In a series of experiments with complete spinal rats, we have shown thatan appropriate level of excitability of the spinal circuitry can be achieved using widely different
means. For example, this modulation level can be acquired pharmacologically, via epidural electricalstimulation over specific lumbosacral spinal cord segments, and/or by use-dependent mechanisms
such as step or stand training. Evidence as to how each of these treatments can “tune” the spinal
circuitry to a “physiological state” that enables it to respond appropriately to proprioceptive input
will be presented. We have found that each of these interventions can enable the proprioceptive input
to actually control extensive details that define the dynamics of stepping over a range of speeds,
loads, and directions. A series of experiments will be described that illustrate sensory control of
stepping and standing after a spinal cord injury and the necessity for the “physiological state” of the
spinal circuitry to be modulated within a critical window of excitability for this control to be
manifested. The present findings have important consequences not only for our understanding of
how the motor pattern for stepping is formed, but also for the design of rehabilitation intervention
to restore lumbosacral circuit function in humans following a spinal cord injury.

Keywords
spinal cord injury; use-dependent plasticity; epidural stimulation; afferent control

Correspondence should be addressed to: V. Reggie Edgerton, PhD, Department of Physiological Science, University of California, Los
Angeles, Los Angeles, CA 90095 Phone: 310 825-1910; Fax: 310 267-2071; E-mail: vre@ucla.edu.
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Page 2Introduction
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Under very specific conditions the lumbosacral spinal cord circuitry of a chronic low spinal
cat can generate full weight-bearing stepping of the hindlimbs on a treadmill (Edgerton andRoy, 2002; Rossignol et al. 2002). Furthermore, the stepping of the hindlimbs can be modulated
to accommodate different treadmill belt speeds, levels of loading, directions of movement of
the treadmill belt (Edgerton et al. 2004), and spontaneous perturbations of the steps in order
to continue stepping successfully (Rossignol et al., 2006). A key phrase here is “under very
specific conditions”, with these conditions varying from specie to specie. For example, theadult chronic spinal cat can generate full weight-bearing stepping with sufficient step training
without pharmacological intervention (de Leon et al., 1998a, b). In adult spinal mice, rats, andhumans similar success in regaining stepping-like activity can be achieved, but the specificconditions differ from those observed in the cat (Edgerton and Roy, 2002). For example, unlikein the adult spinal cat that have received some step training, the execution of a consistent seriesof full weight-bearing steps without any assistance to guide the kinematics of the lower limbs
has not been demonstrated in the adult complete spinal mouse, rat, or human even following
extensive training. There is one exception to this generalization. Leblond et al. (2003) reported
that adult spinal mice “can express hindlimb locomotion within 14 days of spinalization” andhave “similar angular excursions of the hip, knee and ankle as intact mice”. However, fullweight-bearing on the hindlimbs was not observed in that the authors stated “the animal has
to be supported and a lateral stability has to be provided by the experimenters”. This was also
evident from the more limited ankle excursions in the spinal vs. intact mice. With the use ofadditional state-dependent modulatory techniques besides step training, level of success close
to that observed in cats has been achieved in rat and mice as will be discussed below. All ofthese observations have important applications with respect to the potential mechanisms forthe recovery of locomotion following incomplete and complete spinal cord injuries in humans.
These observations lead to two different types of questions. How does the spinal circuitry
generate full weight-bearing stepping while also modulating speed, load and direction without
any neural input from the brain? Upon the loss of input from the brain, there is an immediateloss of the ability to generate full weight-bearing stepping in all animal models studied. Somestep-like neural and movement patterns, however, can be generated by the motor pools within
hours of a complete spinal cord transection via spinal cord stimulation, sensory stimulation,
and/or pharmacological interventions.

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A general concept that will be presented in this paper is that the spinal circuitry capable ofgenerating full weight-bearing stepping is present within the lumbosacral spinal cord of themouse, rat, and human as well as in the cat and that it persists after a complete spinal cord
transection at a mid-thoracic level. The detailed strategies needed to access this circuitry,
however, differ between species. The second general concept is that successful weight-bearing
stepping can be achieved by using presently available techniques to bring the spinal circuitry
to a physiological state under which the afferent information can drive the locomotor activity.

2. Modulation of the physiological state and afferent control of locomotion
What is the logic and experimental evidence that the generation of successful locomotion is areflection of state-dependent modulation and afferent control (Fig. 1)? The concept of statedependence implies that the spinal circuitry responds to a given stimulus differently under
different conditions or “states”. The classic example is the phase-dependent modulation of the
response to touching the dorsum of the paw, which induces a flexor action in the ipsilaterallimb during the swing phase, but extension during the stance phase of stepping (Forssberg,1979). Examples of a more prolonged modulation of state dependence have been demonstratedusing pharmacological agonists and antagonists of selected neurotransmitter systems known

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to be important in the generation of locomotion (Rossignol and Barbeau, 1993). We also havedemonstrated that the state dependence of the spinal circuitry can be modulated by epiduralelectrical stimulation of the dorsum of the lumbosacral spinal cord of the adult spinal rat, i.e.,
epidural stimulation at specific segments of the spinal cord can elicit and/or enable reflex andlocomotor-like activity in rats having a complete spinal cord transection at a mid-thoracic level(Gerasimenko et al., 2005;Ichiyama et al., 2005;Lavrov et al., 2006). Essentially, the earlyexperiments of Shik and colleagues (1966) demonstrated that tonic stimulation of themesencephalic locomotor region modulates the state dependence of the spinal cord in the sensethat the gait and speed of stepping at a given treadmill speed was dependent on the intensityof the stimulation.

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It appears that the general perception is that the role of afferents in locomotion, and in othermotor tasks, is to modulate and correct movements that are initiated and sustained by themultiple supraspinal motor systems. This is almost certainly true on some occasions, but is notnecessarily the case even when the neuromotor system is intact. For example, there is no clear
evidence to exclude the possibility that the afferent systems play an important, and maybe evena lead role, in controlling movement (Courtine et al., 2007a), while the supraspinal centerscorrect or modulate the details of the motor event (Nielsen and Sinkjaer, 2002). In situations
where there is no supraspinal input to the lumbosacral spinal cord, (Fig. 2B) theoretically thecontrol could come from the specialized spinal networks responsible for generating the rhythm
for locomotion. We suggest, however, that the essential control is provided by the afferents
from the moving lower limbs. We propose that those spinal circuits responsible for central
pattern generation receive the general, tonic control signals, and then carry out the specificdetails of the task with the availability of the continuously projected profiles of dynamic
sensory input (Fig. 2A). This input seems to reflect the kinematics and kinetics of the lower
limbs and subsequently works in concert with the spinal pre-motor circuits capable of central
pattern generation to control and sustain stepping or standing.

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Numerous experiments support our interpretation that the afferents can actually be the sourceof control of locomotion, at least as it occurs in vivo in the spinal animal. A complete spinal
animal readily adjusts the temporal pattern of stepping in accordance with the speed of atreadmill belt (Rossignol et al., 2006). There are multiple examples of the recruitment of motor
pools accommodating the level of loading placed on the hindlimb of cats (Edgerton et al.,1991) and of the lower limbs in humans (Harkema et al., 1997) with complete spinal cord
injuries. Even more impressive evidence that the afferent system can be the controller isdemonstrated by the ability of the hindlimbs of a complete spinal animal to walk backwards
when the treadmill belt is moving in a forward direction (Gerasimenko et al., 2005). Although
locomotor-like rhythms can be induced by the spinal circuitry in the absence of alternating
sensory input, inhibitory or excitatory neurotransmitter agonists/antagonists can be given atlevels that will not induce, but will enable, oscillatory movements of the limbs. However, when
the animal’s limbs are placed on a moving treadmill belt they will begin stepping at a rate andwith kinematics features appropriate for the speed of the belt. A similar effect of the motor
responses to sensory input can be routinely observed during tonic epidural stimulation in acomplete spinal cat or rat (Gerasimenko et al., 2005; Ichiyama et al., 2005).
A level of epidural stimulation that induces no motor response when the hindlimbs are
suspended will readily generate effective, coordinated locomotor movements when the limbs
are placed on the moving treadmill belt, presumably because it provides systematic andcomprehensive afferent information as a source of control. Further evidence for the role ofsensory information in controlling movement is provided by the observation that several weeks
after unilateral deafferentation (intradural dorsal rhizotomy) in spinal rats, epidural stimulation
can elicit hindlimb stepping only on the intact and not the deafferented side. Other striking
evidence that peripheral afferents can initiate locomotor movements is that involuntary step-Brain Res Rev. Author manuscript; available in PMC 2009 January 1.

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like movements can be generated in intact humans when they are placed horizontally on theirside and the quadriceps muscles are stimulated continuously by vibration (Gurfinkel et al.,1998). The type of vibration used has been demonstrated to activate Group Ia muscle spindle
afferents with the probability that some Group Ib fibers also are being activated. All of theseexperimental observations are consistent with our proposal that afferent input to the spinal cord
is capable of initiating and controlling locomotion in the complete spinal mammal.

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Is there a role for state-dependent modulation in the initiation, induction, and correction of
locomotor tasks? Stimulation of the mesencephalic locomotor region can clearly change thestate dependence of the spinal cord, e.g., change its responsiveness to sensory input. Although
the brainstem can be stimulated at an intensity that does not generate locomotor movements
until the limbs are placed on a moving belt, there is evidence that brainstem simulation can
induce a stepping pattern in the limbs even in the absence of a moving treadmill belt (Liu andJordan, 2005). There is little evidence, however, that such stepping-like motions are completely
independent and fully weight bearing in response to stimulation of the mesencephaliclocomotor region. Epidural stimulation of the dorsal region of the lumbosacral spinal cord has
similar effects, except that sub-threshold stimulation rarely generates full weight-bearing
stepping movements until the limbs are placed on a moving belt. Each of these cases are
consistent with the concept that the state-dependence of the spinal circuitry can be modulatedby the mesencephalic locomotor region or epidural stimulation of the lumbosacral spinal cord,
thereby enabling spinal locomotor networks to process the sensory information for generating
effective weight-bearing locomotion.
Pharmacological modulation of the state-dependency of the spinal cord also seems to be aneffective way to enable locomotion in the presence of weight-bearing associated proprioceptive
input. For example, either strychnine (a glycine receptor blocker) or bicuculline (a GABA
receptor agonist) can significantly facilitate and enable locomotion without inducing
locomotor-like movements in the absence of weight-bearing associated proprioception (deLeon et al., 1999). Relatively modest dosages of the serotonergic agonist, quipazine, have asimilar effect. Although higher doses of quipazine and other excitatory drugs can induce
locomotor-like movements, quipazine in itself does not induce and control stepping at lesser
dosages (Fong et al., 2005; Guertin et al., 2004) Thus, it can modulate the inhibitory andexcitatory properties of spinal neural circuits to a net excitable state that step-related
proprioception can generate successful movements.

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This physiological state can be controlled readily via brainstem simulation, epidural
stimulation, pharmacological facilitation, the level of activity of a given synaptic pathway, andto some degree tonic stimulation of afferents (Fig. 3). Indications that these modulatory
procedures are not controlling, but enabling, locomotion is that all of these stimuli are tonic innature and have minimal spatial and temporal meaning, as is essential for controlling the precise
sequence of muscle synergies that underlies locomotion, i.e., there is little detail needed toactivate all of the motor pools in a coordinated fashion as occurs during weight-bearing
locomotion.

3. Sources of control of locomotion before and after a complete spinal cord
injuryAlthough the neural control of locomotion involves multiple supraspinal centers, spinal
networks, and sensory information derived from the periphery (Fig. 1), the specific roles ofeach of these sources are not well defined. As noted earlier, effective weight-bearing stepping
can be generated in the absence of input from supraspinal centers. Often this is presumed to
be possible because of the presence of central pattern generators in the spinal cord (Grillner,2003). Since it is often assumed that without central pattern generation capability humans
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would be unable to generate locomotion without supraspinal input, i.e., following a brain or
spinal cord injury, there has been considerable focus on the issue of whether such central patterngenerators are present in the human spinal cord (Duysens and Van de Crommert, 1998). The
defining feature of central pattern generation considered to be most critical is its ability togenerate repetitive patterns of reciprocal activity between extensor and flexor muscles
consistently in the absence of any supraspinal or oscillating afferent input. We suggest that a
critical feature of central pattern generators is their ability to receive, interpret, and process
sensory information that can in turn generate the appropriate motor output. We propose,
therefore, that rather than focusing on a conceptual dichotomy between central patterngeneration and sensory control of locomotion, the important feature of these two neural systems
is their ability to interact and function as a highly integrative system. The spinal circuitry
associated with locomotion must receive and interpret very complex, highly ordered
continuous proprioceptive information such that the evolving motor output is generated andadjusted accordingly (Fig 2B). Therefore, as illustrated in Figure 2A and B the important pointof focus should be the interaction between the afferents and the spinal circuitry that receives
and processes the proprioceptive and cutaneous inputs linked to weight-bearing stepping morethan the capacity to generate repetitive cycles.

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The ability to carry out this integrative function between central pattern generators and thesensory system becomes essential following a complete spinal cord injury, if sustained stepping
is to occur. Following the loss of supraspinal control, the spinal circuitry will have changed
significantly. The most obvious change will be the withdrawal of synapses associated withsupraspinal input. In addition, even assuming no direct neurotrauma to the neural circuitry thatgenerates stepping, there will be an extensive number of adaptive processes. Following severe,but incomplete, spinal cord injury the ability to selectively activate specific muscle groups
remains but is very limited, as illustrated by the generation of complex and uncoordinatedmovements of multiple joints when the subject is trying to move only one joint. In other words,
the projection of the motor signal to the periphery becomes highly divergent (Maegele et al.,2002). This loss of specificity of projection of the motor commands must reflect changes infunctional connectivity, both supraspinally and spinally (Raineteau and Schwab, 2001). Figure
2B illustrates a conceptual schematic of the changes in the spinal circuitry that has lost its
specificity, order, and directionality. This dysfunctional divergence could be located directly
within the afferent projections and/or within the circuitry responsible for central patterngeneration.

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The expanded divergence of the command signals also is manifested physiologically as poor
coordination among motor pools during locomotion. For example, although flexor and extensor
motor pools could be activated readily in chronic spinal cats that were unable to step
immediately following a period of stand training, there was very poor coordination among
flexor and extensor motor pools (de Leon et al., 1998b). This lack of coordination reflects alow probability of predicting the level of excitation of a given muscle relative to some specificphase of the step cycle. If there is a random chance of a motor pool being activated at any time
during the step cycle, then the probability of excitation would be similar throughout the step
cycle. On the other hand, if there was a highly regular and predictive onset and termination ofactivation of a given motor pool at a specific time during the step cycle, then the motor pools
can be activated in an orderly and coordinated manner. Figure 4 shows a clear example of theunpredictability and lack of order of motor pool activation and complete inability to step in acomplete spinal cat prior to the administration of strychnine compared with the highly
predictable patterns and full weight-bearing stepping within 30 minutes after strychnine
administration. This rapid onset of motor pool coordination following the pharmacologicaldepression of glycinergic inhibition occurred in chronic spinal animals that previously were
not capable of weight-bearing stepping (de Leon et al., 1999).

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4. The enabling and synergistic effects of pharmacological interventions,
epidural stimulation, and training on stepping ability after a spinal cord injury
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4.1 Pharmacology

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Locomotion can be facilitated by enhancing the level of excitation via serotonergic,
noradrenergic, and glutamatergic systems (Rossignol and Barbeau, 1993). Another approachhas been to potentiate disinhibition by blocking the inhibitory neurotransmitters glycine and
GABA, which has been briefly described above. There are numerous examples ofpharmacological manipulations changing the efferent patterns associated with fictive
locomotion using the neonatal isolated spinal cord as an experimental model (Cazalets et al.,
1992; Cowley and Schmidt, 1995; Kiehn, 2006). Fewer studies have examined the role of thesepharmacological manipulations on the locomotor output under in vivo conditions. There arereasonably clear examples that administration of α-2 adrenergic agonists (e.g., clonidine)(Giroux et al., 1998), serotonergic agonists (e.g., quipazine) (Barbeau and Rossignol, 1990;
Fong et al., 2005), glycinergic agonists (e.g., strychnine) de Leon et al., 1999, and GABAergic
agonists (e.g., Robinson and Goldberger, 1986) can improve the locomotion of complete spinal
animals, particularly when the locomotor capability is very poor prior to the pharmacologicaltreatment. In most of these studies, however, a distinction is not made as to whether thepharmacological agent is actually inducing locomotion vs. changing the physiological state of
the spinal cord so that it can generate locomotion when the afferent systems are allowed tocontrol the motor output. In reports of the facilitation of locomotion in response to either
strychnine or quipazine, it was clear that the drug itself, at least for the dosages used, did not
induce stepping (Cai et al., 2006b; de Leon et al., 1999; Fong et al., 2005). When the hindlimbs
were placed on a moving treadmill belt after administration of the drug, however, effective
weight-bearing locomotion could be generated. The observation that the stepping rate readily
accommodated the speed of the treadmill belt provided further evidence that the afferent control
played a crucial role in defining the locomotor output (Fig. 5).
4.2. Epidural stimulation

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Tonically stimulating the dorsal surface of the lumbosacral spinal cord via electrodes placed
on top of the dura can induce locomotor-like movements in vivo in complete spinal rats
(Ichiyama, 2005) and cats Gerasimenko et al., 2003) and in humans that are classified asclinically complete (Dimitrijevic et al., 1998). The intensity, frequency, and site of simulation
are important parameters to control in defining the nature of the locomotor movements that are
generated. In rats and cats, epidural stimulation alone can generate locomotor movements thatcan bear some, although very limited, weight bearing. In humans that receive a similar type ofstimulation, locomotor-like movements can be generated when there is no weight bearing andthere is a recent report that some weight-bearing steps can be generated when some weight bearing is permitted (Huang et al., 2006). Epidural stimulation in combination with step training
also has been reported to improve locomotor ability in clinically incomplete spinal cord
subjects. The data presented to support this conclusion, however, are equivocal andcomplicated by the fact that the subject’s injury was incomplete and had some stepping
capability without the epidural stimulation/step training intervention (Carhart et al., 2004).
On the other hand, epidural stimulation and pharmacological facilitation, each of which
modulates the physiological state of the spinal cord toward one that can more readily generate
locomotion, can act synergistically on locomotor performance. In the presence of both
treatments the quantity and quality of stepping of adult complete spinal rats exceeds that which
is observed when either intervention is used alone (Fig. 6). As noted above, our interpretationof these results is that the two modulatory events do not induce stepping, but enable steppingto occur when the sensory input is used as the source of control. Although we do not know themechanism for the synergistic effect of these two modulatory interventions, it seems reasonably
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apparent that the mechanisms via which they induce their effects differ and, therefore, thebenefits of each approach can be complementary.

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4.3. Use-dependent plasticity

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Repetitive patterns of activation of a synapse changes the properties of that synapse within atimeframe of milliseconds to months. At the systems level it is equally clear that the synaptic
efficacy of sensorimotor pathways can be modulated by repetitive activity. Furthermore, someof the specific pathways that are modified are most likely those that are being activated, but
inhibition can be enhanced as well. For example, when a spinal cat is trained to step it learns
to step, and when the same cat is trained to stand it learns to stand, but steps very poorly (deLeon et al., 1998a). Following a complete spinal cord transection in the adult cat, some weightbearing locomotion generally will begin to occur, particularly with step training, within a matterof a few weeks. Rossignol and colleagues (Chau et al., 1998) demonstrated that administrationof clonidine immediately before training and beginning within 3 days after a complete spinal
transection in adult cats, resulted in the recovery of weight-bearing stepping within 11 days.
Although it was noted that the “spinal locomotor network might be further consolidated or
molded through the peripheral afferent inflow during locomotor training”, there are two
possible explanations for the improved locomotion. One explanation is that the repetitive
administration of clonidine directly induced alterations in the spinal circuitry so that it could
generate stepping. A second possibility is that the clonidine changed the physiological state of
the spinal cord, thus enabling the afferent systems to assume control and activate theappropriate neural pathways, i.e., a use-dependent mechanism, and not a direct
pharmacologically-induced mechanism. In either case, the results are consistent with theinterpretation that neural pathways that can generate stepping persisted after spinal cord
transection, and it was a matter of gaining access to that network to induce stepping.
We hypothesized that it was the repeated use of the sensorimotor pathways associated withweight-bearing stepping, and not the direct pharmacological facilitation, that increased theefficacy of sensorimotor pathways involved in stepping (Edgerton et al., 2004). A similar
concept was proposed by Chau et al. (1998) based the results of repeated injections of clonidine
and step training immediately after a spinal cord transection. It cannot be determined from thisstudy, however, whether the improved stepping was due to the pharmacological effect itself
or by the combined or interactive effect of training and clonidine.

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To address the question of whether there was an important interactive effect of thepharmacological intervention and the training, we administered the 5-HT receptor agonist
quipazine daily to a group of spinal mice that were step trained and to a group that were nottrained (Cai et al., 2006b; Fong et al., 2005). The non-trained mice receiving quipazine showedno sustained improvement in stepping over a period of several weeks, whereas the trained micereceiving quipazine showed a significant improvement in stepping ability. Furthermore,
although there was some improvement in stepping in the trained mice in the absence of
quipazine administration, there was a marked synergistic effect between the two interventions.
These results demonstrate that the improved stepping was a function of the repetitive activationof the sensorimotor pathways that was enabled by quipazine treatment. Further evidence thatthe serotonergic system can be used to facilitate the recovery of locomotion in complete spinal
rats has been demonstrated by Orsal and colleagues (Feraboli-Lohnherr et al., 1997; Ribotta
et al., 2000). They transplanted embryonic raphe cells secreting 5-HT in the spinal cord distal(T11) to a complete lesion at T8. These rats, demonstrated improved locomotion over a periodof 9 weeks compared to non-transplanted and T9-transplanted animals.

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5. Strategies to achieve more optimal physiological states for locomotion
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We have initiated a series of experiments designed to characterize optimal physiological statesof the lumbosacral spinal cord for stepping in chronic spinal rats. Our strategy has been toacutely, i.e., on a daily basis, modulate state dependence pharmacologically combined with
epidural stimulation, in an attempt to achieve a more prolonged or chronic modulation basedon use-dependent plasticity (step training). These three modulatory approaches used in concert
have proven to be highly successful. We have been able to achieve full weight-bearing bipedal
stepping over a wide range of treadmill speeds in rats spinalized as adults that have received
daily step training in the presence of quipazine and epidural stimulation (Gerasimenko,
2006). As emphasized previously, the combination of quipazine and epidural stimulation does
not directly induce weight-bearing stepping. Locomotion, however, can be enabled by each ofthese modulators when administered at levels that do not initiate locomotor-like movementsin the absence of any sensory input associated with weight bearing stepping. When weightbearing, step-related proprioceptive information is provided to the spinal circuitry, however,it can generate full weight-bearing stepping over a range of speeds and loads.

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Although we have achieved significantly improved “physiological states” for generating
stepping, it is unlikely that these states are optimal. For example, our selection of quipazine asa single pharmacological intervention, and the dosages and method of administration(intraperitoneally) are almost certainly not producing an optimal “pharmacological state”.
Rather it seems that some combination of several different pharmacological agonists andantagonists will ultimately prove to be most successful. For example, the combination of 5HT1A, 5-HT7 and 5-HT2 agonists improves stepping ability of spinal rats beyond the action ofeither serotonin agonist (Antri et al., 2003). Furthermore, it is almost certain that the optimum
combination and dosage of the pharmacological agents will change over time following an
injury due to the modulation of chronic levels of activity, diet, psychological state,
immunological status, etc. Although the challenge here is indeed intimidating, it should bepossible to sustain and improved functional state of the spinal circuitry following spinal cord
injury. We are, however, only at the beginning stages of assessing the efficacy of epidural
stimulation in modulating the physiological state of the spinal cord. Electrode design and theability to selectively activate specific regions of the spinal cord should improve with time.
5.1. Biochemical plasticity in response to training

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We have considered multiple experimental approaches in attempting to understand theneurophysiological and biochemical mechanisms involved in spinal learning, one
manifestation of specific use-dependent plasticity. We have attempted to identify biochemical
adaptations that occur in response to learning to step or to stand and have compared these
responses to those associated with learning in the hippocampus. To date, the levels of several
learning-related molecules have been found to change in the spinal cord following step training.
For example, cyclic AMP responsive element binding protein (CREB) has been shown to bephosphorylated (pCREB) during learning. We have observed that the number of motoneurons
expressing pCREB in specific lumbar motor pools is higher in quipazine-treated, step-trained
than non-trained spinal mice. Similarly, only the trained spinal mice contained a comparable
number of immunopositive cells for the phosphorylated GluR1 subunit of the AMPA receptor
in motor pools to levels observed in intact mice, suggesting a facilitation of glutamate receptor
signaling in the trained mice. A significant increase in stepping performance was observed
only in the trained mice. Administration of quipazine alone did not affect stepping ability,
consistent with an absence of cellular changes observed in the non-trained mice. These datasuggest that the repetitive activation of spinal sensorimotor pathways during quipazine-enabledstep training could mediate the facilitation of glutamate receptor function and activation of theBrain Res Rev. Author manuscript; available in PMC 2009 January 1.

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Page 9transcription factor CREB in the lumbar spinal cord, and that these alterations were associatedwith an improvement in stepping ability after a spinal cord injury.

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We also have assessed the roles of supraspinal and afferent input in defining the dynamics of
the 5-HT1A receptor in specific laminae of the spinal cord. We found an increase in 5-HT1A
receptor immunopositive cells and/or axon hillocks following a complete spinal cord
transection in adult rats. However, these increases were no longer apparent when the afferents
were removed via dorsal rhizotomies, suggesting a conditional afferent-related phenomenon
that may or may not be activity dependent. Changes in receptor distribution could be importantdue to the intracellular consequences associated with 5-HT1A receptor activation, i.e., increases
or decreases can be detrimental to the delicate electrophysiological balance in the spinal
locomotor networks. Ultimately, a comprehensive understanding of the mechanisms
underlying the receptor dynamics in the spinal cord may improve the efficacy ofpharmacological interventions following a spinal cord injury.
5.2 Neurophysiological plasticity in response to training

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A range of synaptic changes following a complete transection of several sensorimotor pathways
has been reported. For example, the amplitude of Group Ia fiber-evoked monosynaptic EPSPs(sEPSPs) was higher in trained than non-trained rats that were spinally transected as neonates.
Cote and Gossard (2004) reported a reduction in Ia-evoked EPSPs in trained adult spinal cats,
but enhanced cutaneous reflexes in trained compared to non-trained spinal cats whenstimulating afferents that were thought to be activated during weight-bearing stepping. They
also observed that the Group Ib (Golgi tendon organ) disynaptic input to homonymous
motoneurons was reversed from inhibition to excitation more frequently in trained than in nontrained cats (Cote et al., 2003).

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Synaptic input to motoneurons from propriospinal pathways in step-trained spinal rats has been
examined. Petruska et al. (in press) observed that the efficacy of propriospinal pathways was
closely linked to stepping ability in adult rats that had been spinalized at five days of age. For
example, stimulation of the ventrolateral funiculus (central-EPSP, cEPSP) induced higher
amplitude polysynaptic cEPSPs recorded from motoneurons in trained than non-trained or
poorly stepping spinal rats. Passive exercise (motorized bicycle training) in spinally transected
rats also restored the frequency-dependent depression of the H-reflex to the level of intact
animals (Reese et al., 2006). One of proposed mechanisms of training is a restoration ofpresynaptic inhibition. There seems to be some activity-dependent factor that affects theefficacy of a range of synergistic pathways, i.e., a decrease in presynaptic inhibition after a
spinal cord injury and a concomitant facilitation of the monosynaptic H-reflex and less H-reflex
frequency-dependent depression. The progressive nature of the training effect was evident. For
example, after 15 or 30 days of training the H-reflex inhibition was only partially restored,
whereas at 90 days the H-reflex frequency-dependent depression had normalized (Reese et al.,2006). A similar effect has been described in humans where motorized bicycle training
provided recovery of H-reflex frequency-dependent depression within 10 weeks. The
normalization of the monosynaptic reflex by training returned to pre-training level within a
few weeks after training was stopped (Kiser et al., 2005).
Some changes in the intrinsic electrophysiological properties of motoneurons also occur after
a spinal cord injury with and without step training. Petruska et al. (in press) found a significant
relationship between the locomotor capacity of rats spinalized as neonates and the depth of theafter-hyperpolarization (AHPd) of action potentials generated in the motoneurons. “Passive”
cyclic exercise in rats spinally injured as adults also can return AHPd toward values observedin control rats (Beaumont et al., 2004). The fact that imposing cyclic activity on the hindlimbs
induces considerable activation of the spinal circuitry and the associated muscles suggests thatthe mechanism of this intervention is an activity-related event.
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Given that the morphology of a motoneuron has a predominant effect on its
electrophysiological properties, it is interesting that motoneuron structure is affected bytraining after spinal transection (Gazula et al., 2004). The size of the dendritic tree ofmotoneurons following spinal cord transection decreases, but these morphological features canbe returned to normal values by training on a cycle. This same study, however, showed nomorphological effects of spinal cord transection or training on the size of the soma. Previous
data are consistent with stability of the motoneuron morphology following either spinal cord
transection or spinal cord isolation (Chalmers et al., 1992).

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Although a number of electrophysiological changes of the spinal circuitry have been reported
in in situ preparations and some of these changes are use-dependent, it remains difficult todetermine whether, and which, of these effects might be responsible for improved performancein vivo. The ability to test reflex responses during recovery from a spinal cord injury under invivo conditions may provide a means of determining more clearly any cause-and-effectrelationships. For example, we (Gerasimenko et al., 2006; Lavrov et al., 2006) havedemonstrated that chronic electrodes implanted epidurally over specific spinal cord segments
can be used to evoke and track monosynaptic and polysynaptic responses in multiple flexorand extensor muscles of the hindlimb over a prolonged period of time after a spinal cord injury(Fig. 7A and B). We also have reported that the progressive reappearance of polysynaptic reflex
responses in hindlimb muscles (Fig. 7B) are correlated with the gradual recovery of locomotor
capacity of adult rats with a complete thoracic transection, i.e., about 3 weeks after the injury(Fig. 7A).
Examining the dynamics of these monosynaptic and polysynaptic responses to epidural
stimulation during stepping should be even more useful than stimulation in a static and/or
anesthetized state (Gerasimenko et al., 2006). The amplitude of spinal cord evoked motor
potentials in hindlimb muscles during continuous bipedal stepping in non-disabled rats varies
as a function of the phase of the step cycle (Fig. 8A). In response to L2 stimulation, theamplitude of the epidurally induced monosynaptic motor response in vivo in normal rats wasstrongly facilitated during the stance and swing phases of gait in extensors and flexor muscles,
respectively. In general, there was a strong co-variation between the output of motoneurons,
as seen in the recorded EMG activity (Fig. 8A, left traces), and the modulation of monosynaptic
reflex responses. Courtine et al. (2007b) observed similar modulation pattern of monosynaptic
reflex responses from lower limb muscles during human locomotion (Fig. 8B). These studies
demonstrate the highly dynamic nature of the activity in the spinal neural circuitry during
stepping, i.e., sensory information provided to the spinal cord is interpreted in a state-dependent
manner.

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5.3. Are the variations in the pathways activated from step to step an important feature inspinal learning?
The limitation with the kinds of biochemical and physiological observations noted above isthat they give little insight into how complex networks of neurons learn. Although it is difficult
to address the cellular mechanisms of learning in such a complex spinal neural network, systemlevel mechanisms may provide important insight into how spinal animals relearn to step. There
are many examples of even the simplest task having some intrinsic level of variation in boththe biomechanics and the timing of the neurons recorded during the repetitive performance ofthe task. Stepping is an excellent example of a motor task that is performed routinely andrepetitively, but even under the most controlled conditions on a treadmill, no two steps are
identical (Fig. 9). Regardless of how much even a professional athlete practices a task, there
will be variation in the success of the performance. This intrinsic variation in all movements
is highly suggestive of a fundamental feature of the neural control of movement. We addressed
this issue by studying the ability of spinal mice to learn to step. We hypothesized that controlling

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the kinematics of the lower limbs during bipedal stepping such that there was minimal variation
would result in poorer stepping ability then if some level of variation was allowed during the
stepping (Fong et al., 2005). The variation was permitted by applying an “assist-as-needed”
mode of control of a robotic arm attached to the ankle of each hindlimb during a 10-min training
session, five days a week. Two “assist-as-needed” modes were used on separate groups ofmice, where the robotic device permits the ankle position of the animal to step within apredefined level of variance from the desired training trajectory. This way, only when the
trajectory deviates from the desired training trajectory greatly, does the robotic device assist
the animal back to the target training trajectory. In this experiment (Cai et al., 2006b), one
group was assisted such that each leg could operate independently. A second group was assistedwith the additional criterion being that the two limbs must function in an alternating, out ofphase fashion. An additional group of mice were trained using a rigid training paradigm inwhich no variation was allowed during stepping. Based on several measures of step
performance, the mice trained with the assist-as-needed mode and some degree of alternatinginterlimb coordination performed better than the other two groups. These data suggest thatsome toleration of variance in the stepping trajectory is beneficial, in particular when interlimb
coordination is imposed. This conclusion is consistent with previous evidence suggesting that
one of the more important features of successful stepping in the spinal cats was the ability tosustain the appropriate interlimb coordination (de Leon et al., 1998b).

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These data imply that the most effective learning paradigm for spinal cord injured subjects
would allow some critical level of variation in stepping kinematics and kinetics (Cai et al.,2006a). We propose that this could be an important feature of the software control that is applied
to robotics designed to retrain spinal cord injured subjects to step. While these data raise theissue as to what the ideal pattern of variation might be for a given subject, it is impossible totest every permutation experimentally. Thus we are currently using a computational learningmodel to test our hypothesis that there is an optimal level of variation in the kinematics anddynamics of stepping. We predict that step training related learning will not occur if the pattern
is too fixed or too variable (Cai et al., 2006b).

6. Gaining access to the spinal circuitry that can generate locomotion inhumans: taking advantage of state-dependent modulation of the spinal
circuitry while using afferents as a source of control

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The clinical question that these experimental observations in animal models raise is: How do
we gain access to the circuits that have the ability to generate and control full weight-bearing
stepping? All of the results from the animal models suggest that any strategy for recovering
locomotor ability should include modulation of the physiological state of the spinal cord while
using the sensory system as the “controller” for motor function. In fact, state-dependent
processing of sensory input has been demonstrated in humans as well. Courtine et al.
(2007b) showed that the gain in the monosynaptic neural pathway is modulated in virtually all
lower limb muscles of normal subjects during walking in a manner similar to the patterns
observed in the bipedally walking rat (Fig. 8B) (Gerasimenko et al., 2006).
For the interventions noted above to be practical for a spinal cord injured subject a number offactors must be considered. We have presented an “enabling” concept as opposed to an“inducing” concept to describe the responses to pharmacological agents, to epidural
stimulation, and to some degree use-dependent plasticity such as step training. In other words,
it is technically feasible, at least from an engineering perspective, to administer some“pharmacological cocktail” directly to the spinal cord at a dosage that is needed at any given
time point. It also is technically feasible to apply electrical stimulation to the dorsum of theBrain Res Rev. Author manuscript; available in PMC 2009 January 1.

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spinal cord at specific segmental levels in a manner that can induce step-like patterns(Dimitrijevic et al., 1998).

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These new interventions (Fig. 3) may make it feasible for a patient with a complete lesion of
the spinal cord to stand and to step when the individual chooses. If the spinal cord in the absenceof any input from the brain is “ready”, i.e., the physiological state of the spinal neural circuits
is appropriately tuned, it can initiate standing or stepping by manipulating the position of the
body so that pressure is placed on the bottom of the feet to initiate an extensor thrust. With
appropriate training this extensor thrust can be used to sustain standing in complete spinal cord
injured subjects (Wernig et al., 1998). Once the individual is standing, manipulations of loading
the limbs and joint angles in the legs also can be used to initiate stepping. This approach ismore feasible for paraplegics than quadriplegics, because they can use their upper limb andtrunk muscles to aid in balance and support as is necessary when using a walker or cane.

7. Conclusions

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In the present chapter we have shown that electrical stimulation of the spinal cord and/or
pharmacological administration such as serotonin agonists can enable stepping in adultmammals with complete spinal cord transection. We propose that these stepping-enablingstrategies can bring the spinal cord to a physiological state at which the motor circuits canprocess the sensory information in a manner sufficient for generating coordinated, weightbearing stepping. We emphasize, however, that sensory information can be used by the spinal
circuitry to actually control, and not just modulate, locomotion. A combination of steppingenabling agents and taking advantage of the capacity of afferent input to drive stepping activity
can be used to improve locomotor function in humans with a spinal cord injury.
Acknowledgments
The work presented in this paper was supported by NIH NS16333, NIH NS42291, RFBR-CRDF 07-04-91106, theChristopher and Diana Reeve Foundation, and the California Roman Reed Bill.

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Figure 1.

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This illustration provides a global perspective on the sources of neural control of posture andlocomotion which normally includes the exchange of information between the brain and thespinal cord and between the sensory receptors within the muscles, joints, and skin and the spinal
cord. This chapter emphasizes the importance of the afferent information from the periphery
as a source of control of posture or locomotor tasks, in conjunction with the spinal circuitry towhich central pattern generation is routinely attributed, when there can be no exchange of
information between the brain and spinal cord, i.e., following a complete spinal cord injury.
Given that it is possible to generate locomotor movements with a tonic stimulation of themesencephalic locomotor region (MLR), details of the control of posture and locomotion are
not likely to be derived from a tonic signal from the brainstem. However, this tonic stimulation
can be an important source of modulatory control of the spinal circuitry, as can be epidural
stimulation, afferent stimulation, and pharmacological modulation. Factors listed on the rightside of the Figure point out details of the physical environment that can be detected by the
spinal cord and used to instruct the spinal circuitry to activate the appropriate motor pools at
the appropriate time. The sensory perception of the factors listed in the box on the right sidecan play an important role in shaping the physiology of the spinal circuitry through repetitive
activity, i.e., training. Further evidence of the importance of the sensory information isdemonstrated by the acute effects of unilateral deafferentation after which epidural stimulation
will induce locomotor movements only on the intact side.

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Figure 2.

This illustration emphasizes several important features of the spinal circuitry in addition its
ability to generate repetitive rhythmic cycles of flexion and extension. We propose that an
important feature of the spinal circuitry is its intrinsic order of activation of selected groups ofneurons and that this order is reinforced by the expectation of the synchronization of order
being derived from the sensory fibers from the periphery as well as being synchronized withthe supraspinal input. In other words, the supraspinal input, the intrinsic circuitry of the spinal
cord, and the peripheral information projecting to the spinal circuitry occur in an orderly fashion
normally (A). On the other hand, when there is loss of input from the brain, the disorder evolves
in the spinal circuitry and there is a loss of the routine orderly sensory information projecting
to the spinal cord due to the lack of routine weight-bearing and postural tasks. All of these
events contribute to a loss of order and specificity of the activation patterns to specific groupsof neurons in a timely fashion (B).

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Figure 3.

This chapter centers around the concept that the spinal cord circuitry changes its functional
properties readily and routinely and that some of these functional properties can be modulated,with the principal modulators being related to how the spinal circuitry is being used, how it
responds to a given pharmacological agonist or antagonist, and how it responds to tonic epidural
stimulation. Each of these factors can readily modulate the state-dependence of the spinal cord.
Modulation of this circuitry in combination with “control” from the sensory input can resultin effective weight-bearing stepping.

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Figure 4.

EMG recordings from the hindlimbs of an adult spinal cat that had been trained to stand for
12 weeks after a complete spinal cord injury. The animal was incapable of generating any
weight-supporting stepping following this stand training. The cat was then given a small dosageof strychnine (STR), intraperitoneally, a glycinergic blocker, and 30 minutes later could walkon the treadmill with full weight-bearing at speeds of up to 0.6 m/sec. The Figure shows theerratic bursting of activity of flexors and extensors prior to receiving strychnine, but a highlyordered and predictable bursting pattern of flexor and extensor muscles following its
administration. St, semitendinosus; VL, vastus lateralis; IP, iliopsoas; Sol, soleus; MG, medial

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gastrocnemius; and TA, tibialis anterior. Horizontal calibration, 1 s; vertical calibration is 1
mV for all muscles except for the Sol, which is 2 mV. (Taken from de Leon et al., 1999).

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Figure 5.

Modulation of the kinematic features with changes in treadmill belt speed in a complete spinal
rat walking bipedally under epidural stimulation and quipazine administration. The stickdecompositions of the hindlimb movements during the swing phase of gait as well as successive(n = 10 steps) trajectories of the hindlimb endpoint during both the stance and swing phase of
gait show the progressive adaptation of hindlimb kinematics with increasing treadmill belt
speed. Likewise, increasing the treadmill belt speed resulted in a decrease in stance durationand increase in step length while interlimb coordination remains preserved. The boxes at thebottom of the Figure indicate the period of stance.

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Figure 6.

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Average (n = 15 steps) trajectory of hindlimb joint angles (±SD) and associated patterns of
EMG activity during bipedal stepping enabled by quipazine, epidural stimulation, and the
combination of both. The gait diagram of the left and right hindlimb movements is also
displayed for each condition. Dark (red) and light (gray) boxes represent the duration of the
stance and drag phases. Quipazine facilitates the flexion phase, whereas epidural stimulation
applied over the S1 spinal segment promotes the extension phase. Applying both quipazine
and epidural stimulation results in improved locomotion whose characteristics combine theindividual effect of either treatment. Muscle abbreviations, same as in Fig. 4; marker
positioning, same as Fig. 5. The mean duration (+ SD) of the stance and dragging phases isreported at the bottom of each panel.

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Figure 7.

A: EMG patterns of the MG, TA, VL, and St muscles at 3, 4 and 6 weeks post-surgery during
stepping induced by epidural stimulation (40 Hz) at S1. Note the more robust and consistent
EMG burst patterns at 6 compared to 3 and 4 weeks. B: Representative ER (early response,
direct activation), MR (middle response, a monosynaptic response), and LR (late response, apolysynaptic response) from the TA muscle at 4.5 V stimulation 3, 4, and 6 weeks after ST.
Note that the LR is minimal at 3 weeks and progressively increases from 4 to 6 weeks. Muscle
abbreviations are the same as in Fig. 4. (Taken from Lavrov et al., 2006).

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Figure 8.

Monosynaptic reflex responses were elicited by epidural stimulation at L2 in rats (A) and bytranscutaneous electrical stimulation between the T12-L1 spinal processes in humans (B).
Stimulations were delivered at different times of the stride period while both non-disabled rat
and human subjects were stepping bipedally on a treadmill. The EMG activity recorded during
all the steps preceding stimulation (n > 100 steps) were averaged and are displayed in left
panels. The mean amplitude (±SD) of reflex responses is shown in right panels. There was astrong correlation between the ongoing motor output and the size of the reflex responses, bothin rats and humans. BF, biceps femoris, all other muscle abbreviations, same as in Fig. 4. (A,modified from Gerasimenko et al., 2006: B, modified from Courtine et al., 2007b).

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Figure 9.

Trajectory plot of the ankle of an adult transected mouse successfully stepping on a moving
treadmill for 10 s at a rate of 3 cm/s after 4 weeks of quipazine-enabled step training. Arrows
are showing the direction of ankle movements. Note the variability in the ankle trajectory inspite of the successful plantar stepping, weight-bearing execution of locomotion.

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