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1 Clinical Neurophysiology 124 (2013) Contents lists available at SciVerse ScienceDirect Clinical Neurophysiology journal homepage: Modulation of reciprocal and presynaptic inhibition during robotic-assisted stepping in humans Chaithanya K. Mummidisetty a, Andrew C. Smith a, Maria Knikou a,b,c, a Electrophysiological Analysis of Gait & Posture Laboratory, Sensory Motor Performance Program, Rehabilitation Institute of Chicago, Chicago, IL, USA b Department of PM & R, Northwestern University Feinberg School of Medicine, Chicago, IL, USA c The Graduate Center, City University of New York, College of Staten Island, New York, NY, USA article info highlights Article history: Accepted 13 September 2012 Available online 6 October 2012 Keywords: H-reflex Interneuronal circuits Reflex conditioning Reciprocal inhibition Presynaptic inhibition Robotic gait Spinal circuits Reciprocal inhibition and presynaptic inhibition of soleus Ia afferents are adjusted continuously within a step cycle, and are either increased or decreased based on the phase of the step cycle. Reciprocal inhibition and presynaptic inhibition are modulated in a similar pattern to that reported during human walking without body weight unloading and leg assistance. Activity of spinal interneuronal circuits supports a reciprocal gait pattern during robot-assisted stepping. abstract Objective: To establish the modulation pattern of reciprocal inhibition and presynaptic inhibition of soleus Ia afferents during robot-assisted stepping in healthy subjects. Methods: During stepping, the soleus H-reflex was conditioned by percutaneous stimulation of the ipsilateral common peroneal nerve with a single pulse at stimulation intensities that ranged from 0.9 to 1.2 TA M-wave motor thresholds across subjects. To control for movement of recording and stimulating electrodes, a supramaximal stimulus 80 ms after the conditioned and/or unconditioned H-reflexes was delivered to the posterior tibial nerve. The short (2, 3, 4 ms) and long (60 80 ms) conditioning-test intervals at which the largest amount of reflex depression was observed with the subjects seated were utilized during stepping. Stimuli were randomly dispersed across the step cycle which was divided into 16 equal bins. Results: Reciprocal inhibition exerted from flexor group I afferents onto soleus motoneurons was decreased at mid-stance and increased and late-stance and throughout the swing phase. Presynaptic inhibition of soleus Ia afferents was increased at heel strike and decreased at late-stance and early swing phases. Conclusion: Reciprocal inhibition between ankle antagonistic muscles and presynaptic inhibition of soleus Ia afferents are modulated in a similar pattern to that reported during walking on a treadmill with full weight bearing and without robot-assisted leg movement. Significance: The activity of spinal interneuronal circuits engaged in patterned locomotor activity supports a reciprocal gait pattern during robot-assisted stepping in healthy humans. Ó 2012 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. 1. Introduction The function of the central nervous system during human walking is easily recognized by the phase-dependent modulation of Corresponding author at: Department of Physical Therapy, College of Staten Island, Graduate Center, City University of New York, 2800 Victory Blvd, Bldg. 5 N- 207 Staten Island, NY 10314, USA. Tel.: +1 (718) ; fax: +1 (718) addresses: Maria.Knikou@csi.cuny.edu, m-knikou@northwestern.edu (M. Knikou). URL: (M. Knikou). short-latency spinal reflexes, a phenomenon attributed to segmental spinal inhibitory mechanisms, the central pattern generator (CPG) regulating the segmental spinal reflex circuitry, and descending control of both the CPG and spinal inhibitory mechanisms (Knikou, 2010). The soleus H-reflex increases progressively from mid to late stance in parallel with soleus EMG activity and is significantly depressed or completely abolished during the swing phase of gait, regardless of full or partial weight bearing (Capaday and Stein, 1986; Knikou et al., 2009, 2011). Tonic increase in presynaptic inhibition of group Ia afferent terminals projecting monosynaptically /$36.00 Ó 2012 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

2 558 C.K. Mummidisetty et al. / Clinical Neurophysiology 124 (2013) Fig. 1. Experimental protocol during robot-assisted stepping. Schematic illustration of the relationship between the conditioning stimulus delivered to the common peroneal nerve and the timing of posterior tibial nerve stimulation (conditioned or unconditioned soleus H-reflex) and maximal M-wave at 80 ms after the H-reflex (top panel). to soleus a motoneurons is considered as a key mechanism for the decreased soleus H-reflex amplitude during walking (Capaday and Stein, 1987; Faist et al., 1996; Lavoie et al., 1997; Petersen et al., 1999). This is supported by the fact that the pronounced soleus H-reflex depression at the swing phase remains unaltered upon voluntary contraction of the gastrocnemius or ankle flexor muscles (Yang and Whelan, 1993; Lavoie et al., 1997). The presynaptic inhibition of soleus Ia afferents, estimated based on changes in the amount of soleus H-reflex short-latency monosynaptic facilitation by femoral nerve stimulation at supramaximal intensities, was decreased at mid-stance and increased at late-stance, while the soleus H-reflex depression by common peroneal nerve stimulation was decreased at mid-stance (Faist et al., 1996). Further, the disynaptic reciprocal inhibition between antagonistic ankle muscles increased during the swing phase (when soleus motoneurons are hyperpolarized) but was absent in stance (Petersen et al., 1999), while increased reciprocal inhibition in stance has also been reported (Capaday et al., 1990). Taken together, it is apparent that there is no universal agreement on how reciprocal and/or presynaptic inhibition behaves during human walking. Simultaneous extracellular recordings from Ia inhibitory interneurons and intracellular recordings from lumbar motoneurons during fictive locomotion in cats without brainstem connections revealed that hyperpolarization of soleus a motoneurons coincided with activity of Ia inhibitory interneurons (Degtyarenko et al., 1996; Geertsen et al., 2011; Pratt and Jordan, 1987). Ia inhibitory interneurons are subject to presynaptic inhibition on the basis that stimulation of gastrocnemius-soleus nerve depressed the reciprocal Ia inhibitory postsynaptic potentials evoked by stimulation of the quadriceps nerve for up to 200 ms, while stimulation of flexor, extensor, and cutaneous nerves evoked a long-lasting increase in the excitability of the terminals of the Ia inhibitory interneurons in deeply anaesthetized cats (Enriquez-Denton et al., 2000). We have previously shown that the soleus H-reflex phasedependent modulation pattern remains unaltered during treadmill walking at full or partial body weight bearing (Knikou et al., 2009) and during robot-assisted stepping in healthy subjects (Knikou et al., 2011). These findings suggest that spinal interneuronal circuits, such as reciprocal and presynaptic inhibition, are engaged in a physiologic manner despite being constrained under conditions of body weight unloading and leg assistance. To our knowledge there is yet no study reporting on the excitability changes of reciprocal Ia and presynaptic inhibition across different phases of the step cycle in healthy humans during robot-assisted stepping. Thus, in this study we established changes in reciprocal Ia inhibition exerted from flexor group I afferents onto soleus a motoneurons and presynaptic inhibition of soleus Ia afferent terminals at different phases of the step cycle. We hypothesized that reciprocal and presynaptic inhibition are continuously adjusted within a step cycle, and are modulated in a similar manner to that reported during human walking without body weight support (BWS) and/or legs being assisted by an exoskeleton. 2. Materials and methods All experimental procedures were conducted in compliance with the 1964 Declaration of Helsinki (revised October 2008) after a full Institutional Review Board (IRB) approval was granted by Northwestern University and City University of New York IRB committees. A written consent of all participants was obtained prior to study participation. Fourteen healthy subjects (4 male, 10 female) with an age ranging from 21 to 51 (30.9 ± 8.1; mean ± SD) years participated in 18 experiments (8 subjects in the reciprocal inhibition protocol and 10 subjects in the presynaptic inhibition protocol) conducted on different days. Their daily physical activities ranged from moderate to vigorous. No subject reported low back pain or any type of neuromuscular disorder Robot-assisted stepping A driven gait orthosis (DGO) system (Lokomat, Hocoma, Switzerland) was utilized in this study for robot-assisted stepping. The DGO system was fixed to the treadmill through a parallel frame, while compensation for its weight was also provided. Subjects were fixed into the DGO using straps at the waist, thighs, and shanks, which were adjusted at different body segments, allowing for adaptable fitting. Both feet were enclosed in foot lifters (using elastic straps and springs), but were not tightened in order to allow voluntary ankle flexion and extension. Body weight

3 C.K. Mummidisetty et al. / Clinical Neurophysiology 124 (2013) was removed through an upper body harness that was connected to the orthotic system. Subjects were instructed not to oppose the movement pattern of the robotic device, to promote heel contact and toe off gait events, and actively perform ankle flexion and extension. For the reciprocal inhibition protocol, subjects stepped at an average of 2.81 ± 0.22 km/h treadmill speed and 27 ± 2.58% BWS. For the presynaptic inhibition protocol, subjects stepped at an average of 2.31 ± 0.14 km/h treadmill speed and 28 ± 6.6% BWS. Based on the effects of the walking speed on the reflex gain (Edamura et al., 1991), we tried to match the speed across subjects, but it was adjusted when subjects expressed discomfort during stepping. The average duration of the step cycle for the reciprocal and presynaptic inhibition protocol was 1.4 ± 0.15 s and 1.6 ± 0.2 s, respectively Soleus H-reflex elicitation and recording protocol Following standard skin preparation procedures, bipolar differential surface electrodes of fixed inter-electrode distance (MA , Motion Lab systems Inc., Baton Rouge, LA, USA) were used to record EMG activity from the soleus (SOL), peroneus longus (PL), and tibialis anterior (TA) muscles of the right leg. With subjects seated, the soleus H-reflex was elicited by stimulation of the posterior tibial nerve with single square pulses of 1-ms duration. The most optimal stimulation site was established via a stainless-steel hand-held monopolar electrode (anode) placed on the popliteal fossa. The cathode was a stainless steel of 4 cm in diameter placed proximal to the patella for selective stimulation of the posterior tibial nerve. The stimulation site was defined as the one at which at low stimulation intensities, Ia afferents could be selectively excited without an M-wave being present in the right soleus muscle. When the stimulation site was identified, the monopolar electrode was replaced by a permanent pre-gelled electrode (N- 10-A; Medicotest, Ølstykee, Denmark), and soleus H-reflexes were evoked at low stimulation intensities at 0.2 Hz to ensure a similar reflex expression compared to that observed with the monopolar hand-held electrode. The stimulating electrode was held under constant pressure and maintained in the same position, via athletic wrap, throughout the experiment. The right posterior tibial nerve at the popliteal fossa was stimulated with square pulse stimuli of 1-ms duration delivered by a constant current stimulator (DS7A, Digitimer Ltd., Welwyn Garden City, UK) with subjects seated and by a custom-built constant current stimulator during stepping (see more details on the stimulator below) (Knikou et al., 2009, 2011) Conditioning reflex stimulus The right common peroneal nerve was stimulated according to methods we have previously utilized in human subjects (Knikou, 2011; Knikou and Mummidisetty, 2011; Knikou and Taglianetti, 2006). A single pulse of 1-ms in duration, generated by a constant current stimulator (DS7A, Digitimer Ltd., Welwyn Garden City, UK), was delivered with a bipolar stainless steel electrode placed distal to the head of the fibula. The optimal stimulation site was selected based on the following criteria: the TA motor threshold was lower than that of the peroneal muscles, and at increased levels of stimulation intensities ankle eversion and PL muscle activity were absent. When the stimulation site for the common peroneal nerve was identified, the bipolar electrode was stabilized and secured with athletic wrap. Single pulses were delivered at 0.2 Hz to determine the TA motor threshold and type of contraction (selective dorsiflexion) at increased stimulation intensities. Common peroneal nerve stimulation ranged from 0.9 to 1.2 TA M-wave thresholds across subjects. The TA M-wave was monitored throughout the experiment to ensure consistency of the conditioning stimulation. Fig. 2. Modulation of reciprocal Ia inhibition during robot-assisted stepping. The amplitude of the conditioned and unconditioned soleus H-reflexes (A) and associated M-waves (B) from all subjects as a percentage of the maximal M-wave is indicated for each bin of the step cycle. Asterisks indicate decreased or increased conditioned H-reflexes during stepping compared to the unconditioned soleus H- reflex. Estimated changes of reciprocal inhibition by subtracting the unconditioned soleus H-reflex from the conditioned H-reflex, with both reflexes expressed as a percentage of the maximal M-wave evoked at each bin (C). Positive values suggest decreased reciprocal inhibition and negative values suggest increased reciprocal inhibition. Error bars represent the SEM Reciprocal Ia inhibition and presynaptic inhibition at rest and during stepping Having established the most optimal stimulation sites of the mixed peripheral nerves, the maximal M-wave (M max ) of the soleus was evoked with subjects seated and saved for off-line analysis. The stimulation intensity was adjusted to evoke soleus H-reflexes on the ascending portion of the recruitment curve that ranged from 15% to 30% of the M max, while their corresponding M-waves ranged from 3% to 9% of the M max. The effects of common peroneal nerve stimulation on the soleus H-reflex at the conditioning-test (C-T) intervals of 2, 3, 4, 10, 20, 60, and 80 ms were determined. Forty H-reflexes at 0.2 Hz were recorded randomly with and without conditioning stimulation. The soleus H-reflex depression following

4 560 C.K. Mummidisetty et al. / Clinical Neurophysiology 124 (2013) common peroneal nerve stimulation at short and long C-T intervals is attributed to reciprocal Ia inhibition and presynaptic inhibition of Ia afferent terminals, respectively (Capaday et al., 1995; Crone et al., 1987; Mizuno et al., 1971). The short and long C-T interval, at which the largest amount of depression was observed on the soleus H-reflex with subjects seated, were utilized during assisted stepping. A change in body position constitutes a limitation of the study given the task-dependent modulation of spinal inhibition (Lavoie et al., 1997), but this approach was selected because presynaptic inhibition predominates over other spinal inhibitory mechanisms in standing human subjects (Goulart and Valls-Solé, 2001), and is unknown how reciprocal and presynaptic inhibition behave during standing with partial body loading. Each subject was transferred to the treadmill and while standing at a BWS equivalent to that utilized during stepping, the soleus H-reflex and M-wave recruitment curves were constructed at 0.2 Hz. Approximately responses were recorded at different intensities for each subject. Stimulation intensities and amplitudes corresponding to M-wave/H-reflex thresholds and M max as well as peak-to-peak amplitudes of the M-wave and H-reflex from the constructed recruitment curve were measured, saved and retrieved as reference values during stepping (Knikou et al., 2009, 2011). The orientation of the recording EMG electrode with respect to the underlying muscle fibers may vary due to changes in the muscle architecture during walking (Gerilovsky et al., 1989). Further, the stimulating electrode during flexion of the knee joint (swing) moves away from the tibial nerve while extension of the knee joint (stance) has opposite effects, creating a necessity for appropriate adjustments of stimulation intensities across the step cycle. Lastly, an important criterion in H-reflex studies, especially when conditioning reflex effects are assessed, is the amplitude of the M-wave and H-reflex as a percentage of the M max across subjects (Crone et al., 1990). To meet the aforementioned criteria, during robot-assisted stepping we utilized a custom-built constant current stimulator in which the stimulation intensity is computer controlled and adjusted. During robot-assisted stepping, the stimulation intensity was adjusted by the software based on the parameters measured from the recruitment curve constructed with subjects standing. Most importantly, stimulation intensities were adjusted based on the peak-to-peak amplitude of the maximal M-wave evoked 80 ms after the conditioned or unconditioned soleus H-reflex and the associated soleus M-wave (Fig. 1) (Dyhre-Poulsen and Simonsen, 2002; Knikou et al., 2009). The customized LabView software measured the peak-to-peak amplitude of the un-rectified M-wave and M max at each bin online, and used a self-teaching algorithm to adjust the stimulation intensity as needed for the next bin. The adjustment of intensity was based on the amplitude of the M-wave as a percentage of the M max corresponding to intensities evoking responses on the ascending limb of the recruitment curve, which was set to range between 3% and 9% of the M max (Knikou, 2008; Knikou et al., 2009, 2011). This criterion applied to conditioned and unconditioned H-reflexes, which were randomly recorded during stepping. During stepping, stimulation of peripheral nerves was triggered based on the signal from the ipsilateral foot switch (Noraxon USA Inc., Scottsdale, AZ), and was delivered randomly once every three to five steps. Only one stimulation sequence was delivered per step cycle. Stimuli were randomly dispersed throughout the step cycle, which was divided into 16 equal bins. The step cycle phases of the contralateral leg were identified by a foot switch. A custom made script (Labview, National Instruments, Austin, TX) was used to mark the foot switches and identify the 16 bins of the step cycle for all steps taken. Bin 1 corresponds to heel contact. Bins 8, 9, and 16 correspond approximately to stance-to-swing transition, swing phase initiation, and swing-to-stance transition, respectively. The experiment was concluded when at least five conditioned and unconditioned H-reflexes were accepted at each bin. EMG signals were filtered with a cut-off frequency of Hz and sampled at 2000 Hz using a data acquisition card (NI PCI- 6225, National Instruments, Austin, TX) Data analysis The conditioned and unconditioned soleus H-reflexes, M-waves, and M max evoked at each bin of the step cycle as well as with subjects seated were measured offline as peak-to-peak amplitudes by Fig. 3. The mean normalized soleus (SOL) and tibialis anterior (TA) EMG background activity for the conditioned H-reflex at short C-T intervals and unconditioned H-reflex as a function of the step cycle is indicated in (A, B), respectively. The amplitudes of the conditioned and unconditioned soleus H-reflexes recorded at each bin from all subjects are plotted against the SOL (C, D) and TA (E, F) EMG background activity and equation shows linear relationship between these variables. In these graphs, the 16 points correspond to the 16 bins of the step cycle.

5 C.K. Mummidisetty et al. / Clinical Neurophysiology 124 (2013) customized Labview software. The mean amplitude of the conditioned soleus H-reflex recorded at short and long C-T intervals and expressed as a percentage of the mean amplitude of the unconditioned (or control) H-reflex from all subjects was grouped based on the C-T interval tested, and a one-way ANOVA was applied to the pooled data. The conditioned and unconditioned soleus H-reflex and associated M-waves during robot-assisted stepping were expressed as a percentage of the M max evoked at each bin for each subject. The mean amplitude of the normalized conditioned and unconditioned H-reflexes from all subjects was grouped based on the bin number, and statistically significant differences between them were established with a paired t-test or with a Mann-Whitney rank sum test, depending on whether the data were normally distributed. In order to estimate the relative changes of reciprocal and presynaptic inhibition during assisted stepping, the unconditioned H-reflex at each bin was subtracted from the associated conditioned H-reflex (Mwaves for both reflexes ranged from 3% to 9% of the M max ), both normalized to the M max evoked at each bin. This was done in order the inherent modulation of the maximal M-wave during walking to be counteracted. The mean amplitude of the subtracted conditioned H-reflex was grouped across subjects based on the bin number, and the overall amplitude was estimated. A positive result indicates a condition possibly associated with decreased spinal inhibition, while a negative result indicates increased inhibition at pre- and postsynaptic levels (Knikou and Rymer, 2002). The background activity of the ipsilateral TA and SOL muscles for each bin was estimated from the mean value of the rectified and filtered EMG for 60 ms duration (high-pass filtered at 20 Hz, rectified, and low-pass filtered at 400 Hz), beginning 140 ms before posterior tibial nerve stimulation. The background activity of both muscles was normalized to their maximal locomotor EMG background activity recorded during robot-assisted stepping. The mean amplitude of the conditioned and unconditioned soleus H-reflex (normalized to the M max evoked at each bin) was plotted on the y-axis (dependent variable) versus the associated normalized SOL and/or TA background activity (independent variable) on the x- axis, respectively. A linear regression was then fitted to the data. This analysis was conducted separately for each subject (individual data are not presented) and for the pooled data. increased at late stance and early-mid swing phases (P < 0.05) (Fig. 2C). The overall amplitude of the SOL and TA background activity for the conditioned and unconditioned H-reflex recordings as a function of the step cycle is indicated in Figs. 3A and B, respectively. The SOL and TA background activity recorded during reflex conditioning was not statistically significant different compared to that recorded under control (unconditioned) reflex recordings. A linear relationship between SOL background activity and normalized conditioned soleus H-reflex was found (Fig. 3C, R 2 = 0.63, P < 0.05), which was also the case for the unconditioned soleus H-reflex and normalized SOL background activity (Fig. 3D, R 2 = 0.63, P < 0.05). The conditioned and unconditioned H-reflexes were negatively related in a linear fashion to the normalized TA background activity during assisted stepping (Fig. 3E, R 2 = 0.47, P < 0.05; Fig. 3F, R 2 = 0.58, P < 0.05). 3. Results 3.1. Phase-dependent excitability changes of reciprocal Ia inhibition The conditioned soleus H-reflex from all subjects while seated was significantly depressed at the C-T intervals of 2, 3, and 4 ms reaching overall amplitudes of 77 ± 11.7%, 80 ± 8.1%, and 85 ± 7% of the control H-reflex, respectively (data not shown graphically). The conditioned H-reflexes were not statistically significant different among the short C-T intervals tested (F (2,12) = 0.16, P = 0.85). During stepping, the unconditioned H-reflex reached an overall amplitude of ± 3.37% of M max, which was statistically significantly different from the control H-reflex recorded with subjects seated (25.29 ± 2.67% M max ; P < 0.05). The overall average amplitude of the conditioned and unconditioned soleus H-reflexes as a percentage of the associated M max evoked at each bin from all subjects is depicted in Fig. 2A. A statistically significant difference between the conditioned and unconditioned H-reflexes was found at bins 11 and 12 (P < 0.05) with both reflex recordings conducted at constant M-waves (Fig. 2B). In Fig. 2C, the estimated relative changes of the reciprocal Ia inhibition are indicated as a function of the step cycle. The reciprocal inhibition was decreased at bin 3, increased at bin 5, and remained Fig. 4. Modulation of presynaptic inhibition during robot-assisted stepping. The amplitude of the conditioned and unconditioned soleus H-reflexes (A), and associated M-waves (B) from all subjects as a percentage of the maximal M-wave are indicated for each bin of the step cycle. Asterisks indicate decreased or increased conditioned H-reflexes during stepping compared to the unconditioned soleus H- reflexes. Estimated changes of presynaptic inhibition by subtracting the unconditioned soleus H-reflex from the conditioned H-reflex, with both reflexes expressed as a percentage of the maximal M-wave evoked at each bin (C). Positive values suggest decreased presynaptic inhibition and negative values suggest increased presynaptic inhibition. Error bars represent the SEM.

6 562 C.K. Mummidisetty et al. / Clinical Neurophysiology 124 (2013) Fig. 5. The mean normalized soleus (SOL) and tibialis anterior (TA) EMG background activity for the conditioned H-reflex at long C-T intervals and the unconditioned H-reflex as a function of the step cycle are indicated in (A, B), respectively. The amplitude of the conditioned and unconditioned soleus H-reflex recorded at each bin from all subjects is plotted against the SOL (C, D) and TA (E, F) EMG background activity. In graphs C F, the 16 points correspond to the 16 bins of the step cycle Phase-dependent excitability changes of presynaptic inhibition of soleus Ia afferents The soleus H-reflex, conditioned by common peroneal nerve stimulation with subjects seated, was significantly depressed, reaching overall amplitudes of 81.2 ± 6.2%, 78 ± 5.8%, 75.5 ± 8.6% and 88 ± 5.7% of the control H-reflex at 10, 20, 60, and 80 ms C-T interval, respectively. The conditioned H-reflexes were not statistically significant different among the long C-T intervals tested (F (4, 25) = 0.51, P = 0.72). During stepping, the unconditioned H-reflex reached an overall amplitude of ± 2.46% of M max, which was statistically significantly different from the control H-reflex recorded with the subjects seated at similar M-wave amplitudes (23.23 ± 3.93% M max ; P < 0.05). The overall average amplitudes of the conditioned H-reflex, unconditioned H-reflex and associated M-waves, all expressed as a percentage of the maximal M-wave evoked at each bin, during robot-assisted stepping from all subjects are shown in Figs. 4A and B, respectively. The conditioned soleus H-reflex was significantly decreased at heel contact (bin 1) compared to the unconditioned soleus H-reflex (P < 0.05; Fig. 4A). In Fig. 4C, the estimated relative changes of the conditioned H-reflex from the unconditioned H-reflex are indicated as a function of the step cycle. Presynaptic inhibition was increased at heel contact and was decreased at late-stance (bin 7) and early-swing (bin 10) phases (P < 0.05) (Fig. 4C). The SOL and TA background activity was similar when conditioned and unconditioned H-reflexes were recorded (Fig. 5A and B). A linear relationship between the SOL background activity and the normalized conditioned soleus H-reflex was found (Fig. 5C, R 2 = 0.9, P < 0.05), which was also the case for the unconditioned soleus H-reflex and normalized SOL background activity (Fig. 5D, R 2 = 0.92, P < 0.05). The conditioned and unconditioned H-reflexes were linearly related to the normalized TA background activity during robot-assisted stepping (Fig. 5E, R 2 = 0.47, P < 0.05; Fig. 5F, R 2 = 0.35, P < 0.05). 4. Discussion The principal findings of the study are that the reciprocal inhibition exerted from ankle flexor group I afferents onto soleus motoneurons at a postsynaptic level and presynaptic inhibition of soleus I a afferents are adjusted within a step cycle, are either increased or decreased based on the phase of the step cycle, and are modulated in a similar pattern to that reported during walking in absence of BWS and robot-assisted leg movements. The soleus H-reflex conditioned by stimulation of flexor group I afferents at a short C-T interval was modulated in an identical pattern to the unconditioned soleus H-reflex, being significantly different at bins 11 and 12 (Fig. 2A), suggesting that reciprocal inhibition was increased only at mid-swing phase. However, based on the estimated relative changes, reciprocal inhibition was decreased at early-stance and was increased throughout the swing phase (Fig. 2C). Thus, we can theorize that reciprocal inhibition is continuously adjusted within a step. Our findings are partially in agreement to those reported in healthy humans during treadmill walking without BWS and robot-assisted leg movements. Specifically, reciprocal inhibition has been reported to increase, in a ramp-like fashion in parallel with the soleus EMG, from mid to late stance (Capaday et al., 1990). However, an absent or small reciprocal inhibition in the stance phase has also been reported (Petersen et al., 1999). The most common finding reported across studies is that reciprocal inhibition increases at the swing phase of walking (Lavoie et al., 1997; Petersen et al., 1999), similar to our observations (Fig. 2C). Increased reciprocal inhibition during the swing phase cannot be attributed solely to the relationship of ankle flexors and extensors background excitability (Fig. 3), because soleus H-reflex depression in the swing phase is maintained during tonic contraction of ankle extensors in humans (Yang and Whelan, 1993), is present 50 ms before the onset of TA EMG activity (Crone et al., 1987), and Ia inhibitory interneurons are active in a phasic pattern when limbs were immobilized in the mesencephalic cat (Feldman and Orlovsky, 1975). Based on these observations, one may consider that more powerful inhibitory interneuronal circuits, like the ones that control primary afferent transmission at a premotoneuronal level, mediate the phasic soleus H-reflex excitability during human gait. This notion is supported by the findings that primary afferent depolarization decreases the monosynaptic reflex amplitude in all phases of the fictive step cycle (Menard et al., 1999), muscle afferents induce a phase-dependent presynaptic inhibition of monosynaptic

7 C.K. Mummidisetty et al. / Clinical Neurophysiology 124 (2013) transmission (Menard et al., 2003), and primary afferent depolarization evoked by movement-related sensory feedback can account for changes in the monosynaptic reflex that do not always parallel the recruitment levels of the motor pools as expressed by background EMG amplitude (Duenas et al., 1990; Gossard and Rossignol, 1990). The soleus H-reflex, conditioned at a long C-T interval by common peroneal nerve stimulation, was modulated differently from the unconditioned soleus H-reflex only at bin 1 (Fig. 4A), while both reflexes were linearly related to the SOL background activity (Fig. 5). The estimated relative changes showed that presynaptic inhibition was increased at heel contact (bin 1), and decreased at late-stance and early-swing phases (bins 7 and 10) (Fig. 4C). Our findings are partly in agreement to those reported elsewhere. Specifically, the soleus H-reflex depression by common peroneal nerve stimulation at a long C-T interval was decreased at mid-stance and was increased in 14 out of 21 subjects in early stance phase (Faist et al., 1996; Capaday et al., 1995). Further, it has been shown that the ongoing presynaptic inhibition acting on soleus I a afferent terminals is increased at early stance, decreased at mid-stance, and increased at end-stance and endswing phases (Faist et al., 1996). The small differences between our findings and the published studies may be related to the relative contribution of several segmental neuronal circuits. This is largely based on the fact that Ia afferent terminals exert presynaptic inhibition on Ia inhibitory interneurons, and that interneurons that mediate presynaptic inhibition project to the synapses of Ia afferents on the Ia inhibitory interneurons (Enriquez-Denton et al., 2000). This means that soleus H-reflex depression during gait is the result of changes in the excitability of interneurons mediating reciprocal, presynaptic, and possibly recurrent inhibition (Lavoie et al., 1997; Ethier et al., 2003; Geertsen et al., 2011), synchronized by spinal locomotor neural networks and prone to modulation by descending inputs (Iles and Pisini, 1992). Lastly, comparison across studies becomes problematic when the resolution of the step cycle is different and when a different experimental protocol is utilized to establish conditioning reflex effects. Based on our findings, it is apparent that inhibition exerted at a pre- and post- alpha motoneuronal level contributes to the phasic soleus H-reflex excitability changes during robot-assisted stepping in healthy humans. The regulation of spinal inhibition may rise from movement-related afferent inputs, the CPG, descending pathways affecting CPG expression, and supraspinal control of lumbar propriospinal neurons (Meunier et al., 1990; Meunier, 1999; reviewed in Knikou, 2010; Kiehn, 2006; Rossignol et al., 2006; Pierrot-Deseilligny and Burke, 2005). While we cannot assign the observed effects to a specific group of afferents based on the experimental protocol, it is possible that sensory afferent feedback related to muscle stretch might have contributed to phasic excitability of spinal interneurons (Knikou and Rymer, 2002). In addition, descending neural pathways may have affected the excitability level of Ia inhibitory interneurons and interneurons acting at a premotoneuronal level, because stepping was not passive but rather subjects leg movements were guided in a specific limb trajectory imposed by the Lokomat. It is well established that sensory afferent feedback from receptors registering load and stretch significantly affect the EMG amplitude and force output of ankle extensors during the stance phase (Sinkjær et al., 1996). Body weight unloading during treadmill walking in humans reduces significantly the EMG amplitude of ankle extensors (Sinkjær et al., 2000; Dietz et al., 2002; Finch et al., 1991), but increases the EMG amplitude of the TA muscle (Knikou et al., 2009). Further, EMG activation profiles at 25% and 50% BWS are similar to those observed at 0% BWS (Knikou et al., 2009), while abnormal EMG patterns have been reported for BWS levels that range from 50% to 70% (Finch et al., 1991; van Hedel et al., 2006; Threlkeld et al., 2003). These findings suggest that the amplitude of load affects the behavior of spinal interneuronal circuits and spinal locomotor generating circuitry. Nonetheless, air stepping does not abolish completely the phase-dependent soleus H-reflex modulation (Kamibayashi et al., 2010). Furthermore, during onelegged foot reaching and withdrawal in standing subjects with absent forward body propulsion and limb loading, a motor task that resembles the swing phase of forward and backward walking, presynaptic inhibition of soleus Ia afferent terminals is modulated in a phase-dependent pattern (Knikou, 2011). These findings support the notion that sensory afferent feedback due to rhythmic leg movement patterns when combined with partial body unloading can modulate spinal interneuronal circuits engaged in patterned motor activity in a physiologic manner. However, based on the current experimental protocol, the relative contribution of spinal locomotor neural circuits and supraspinal control of lumbar propriospinal neurons and transcortical pathways cannot be determined and further research is needed to elucidate such contributions. 5. Conclusion The phase-dependent modulation of the soleus H-reflex is tightly coupled to the neural control of human locomotion. Our findings support the notion that reciprocal and presynaptic inhibition contributes to the phasic soleus H-reflex excitability during robot-assisted stepping. The similarities we observed on the modulation pattern of reciprocal inhibition and presynaptic inhibition of soleus Ia afferents to those reported during human walking without body weight unloading and leg assistance, suggest that spinal interneuronal circuits are modulated in a manner that supports a reciprocal gait pattern during robot-assisted stepping in humans. The observed effects are likely due to the central modulation of excitability in motoneurons and interneurons that is known to take place with voluntary and involuntary movements. 6. Clinical perspective Locomotor training on a motorized treadmill with a harness-lift system to provide partial BWS has been utilized for more than 20 years in people with motor incomplete spinal cord injury, with the goal of restoring independent walking (Barbeau et al., 1987). Bilateral leg movements during BWS-assisted stepping are promoted either by manual assistance provided by one or more therapists or by mechanical assistance from a robotic gait orthosis system. Locomotor training with these two therapeutic interventions improves temporal gait parameters, but when compared to conventional therapy, inconclusive and contradicting findings are reported (Mehrholz et al., 2008; Wessels et al., 2010; Field-Fote and Roach, 2011). The findings of this study suggest that activity of spinal interneuronal circuits engaged in reciprocal patterned motor activity is not negatively affected during robot-assisted stepping in healthy humans. It remains to be shown how these spinal interneuronal circuits behave in neurological disorders during robot-assisted stepping and how they change with robotic gait training. Acknowledgements We thank all participants for their voluntary participation to the study, Nupur Hajela for her assistance during the experiments, and Erik B. Simonsen for the prototype stimulator. This work was funded by Grants awarded to MK from the New York State Department of Health (NYSDOH) Spinal Cord Injury Research Trust Fund, Wadsworth Center (Contract C023690) and The Craig H. Neilsen Foundation (83607), and was conducted at the Rehabilitation Institute of Chicago, Chicago, IL, USA.

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