# Horizontal foot orientation affects the distribution of neural drive between gastrocnemii during plantarflexion, without changing neural excitability

Marion Crouzier,¹ Simon Avrillon,² François Hug,³,⁴ and Thomas Cattagni¹

¹Nantes University, Movement - Interactions - Performance, MIP, UR-4334, Nantes, France; ²Department of Bioengineering, Faculty of Engineering, Imperial College London, London, United Kingdom; ³Université Côte d'Azur, LAMHESS, Nice, France; and ⁴School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia

## Abstract

The distribution of activation among muscles from the same anatomical group can be affected by the mechanical constraints of the task, such as limb orientation. For example, the distribution of activation between the gastrocnemius medialis (GM) and lateralis (GL) muscles during submaximal plantarflexion depends on the orientation of the foot in the horizontal plane. The neural mechanisms behind these modulations are not known. The overall aim of this study was to determine whether the excitability of the two gastrocnemius muscles is differentially affected by changes in foot orientation. Nineteen males performed isometric plantarflexions with their foot internally (toes-in) or externally (toes-out) rotated. GM and GL motor unit discharge characteristics were estimated from high-density surface electromyography to estimate neural drive. GM and GL corticospinal excitability and intracortical activity were assessed using transcranial magnetic stimulation through motor-evoked potentials. The efficacy of synaptic transmission between la-afferent fibers and α-motoneurons of the GM and GL was evaluated through the Hoffmann reflex. We observed a differential change in neural drive between GM (toes-out > toes-in) and GL (toes-out < toes-in). However, there was no foot orientation-related modulation in corticospinal excitability of the GM or GL, either at the cortical level or through modulation of the efficacy of la-α-motoneuron transmission. These results demonstrate that change in the motor pathway excitability is not the mechanism controlling the different distribution of neural drive between GM and GL with foot orientation.

**NEW & NOTEWORTHY** Horizontal foot orientation affects the distribution of neural drive between the gastrocnemii during plantarflexion. There is no foot orientation-related modulation in the corticospinal excitability of the gastrocnemii, either at the cortical level or through modulation of the efficacy of la-α-motoneuron transmission. Change in motor pathway excitability is not the mechanism controlling the different distribution of neural drive between gastrocnemius medialis and lateralis with foot orientation.

H reflex; motor unit; motor-evoked potential; presynaptic inhibition; persistent inward current

## INTRODUCTION

It is often assumed that muscles from the same anatomical group share a large proportion of common drive, such that their activation covary during a contraction. Although this is true for some muscles, such as the vastus medialis and lateralis muscles (1), it is not for other anatomically defined synergist muscles, such as the heads of the triceps surae. For example, the gastrocnemius medialis (GM) and lateralis (GL) muscles receive a small proportion of common synaptic inputs (2). This likely gives the central nervous system the ability to independently control these muscles, at least partly. In this way, the distribution of activation between the two gastrocnemii muscles depends on the foot orientation in the horizontal plane. Specifically, when compared with a plantarflexion performed with the foot externally rotated, a plantarflexion with the foot internally rotated generally leads to higher GL activation and lower GM

activation (2–7). The neural mechanisms underlying this task-dependent distribution of activation between anatomically defined synergist muscles are unknown.

Voluntary movements are initiated at the level of the primary motor area by the activation of the cortical motoneurons and the corticospinal tract innervating the muscles selected in the motor-planning phase. The differences in the distribution of activation between the GM and GL observed with different foot orientations may therefore result from two distinct neural processes: a differential change in the excitability of the motor pathway between muscles with the same motor planning or a different motor planning. Evidence from the upper limbs shows that the orientation of the limb alters corticospinal excitability (8–10). It is therefore possible that GM and GL corticospinal excitability are differentially modulated by foot orientation such that the change in the balance of activation between GM and GL would follow the change in the balance of corticospinal excitability between these muscles.
Corticospinal excitability is sensitive to the balance between inhibitory and excitatory processes at the cortical level (11, 12), but is also modulated by mechanisms acting at the spinal level, i.e., α-motoneuron intrinsic excitability. This latter is strongly influenced by the persistent inward currents (PICs), which can amplify the input-output gain of α-motoneurons by at least approximately fivefold (13). It is worth noting that sensory feedback such as Ia afferent inputs coming from muscle spindles also regulates α-motoneuron activity. Furthermore, presynaptic inhibition mechanisms such as primary afferent depolarization also modulate the synaptic transmission between Ia afferents and α-motoneurons. Previous work has demonstrated that the biomechanical configuration of muscles can affect some of these neural mechanisms. For example, muscle length alters both synaptic transmission between Ia afferents and α-motoneurons (14) and primary afferent depolarization (15), while limb orientation alters the PICs (16).

The overall aim of this study was to determine the neural origin of the changes in the relative activation of the GM and GL muscles with changes in horizontal foot orientation. To address this aim, we performed three complementary experiments. The first experiment aimed to verify that the differential changes in GM and GL activation reported in previous work reflect actual changes in neural drive. We used high-density electromyography (HD-sEMG) to decode the spiking activity of motor units (MUs) from the GM and GL muscles and to estimate the MU discharge characteristics together with an estimation of PIC (ePICs) contribution to motoneuron self-sustained firing. In a second experiment, we investigated whether the changes in neural drive between foot orientations were explained by changes in corticospinal excitability. We assessed corticospinal excitability of both GM and GL muscles using transcranial magnetic stimulation (TMS), as well as their inhibitory and excitatory intracortical activity. We finally conducted a third experiment to provide deeper insights into the potential mechanisms underlying modulations in the efficacy of Ia-α-motoneuron spinal transmission with different foot orientation. This was assessed through the Hoffmann (H) reflex, as well as the presynaptic inhibition due to primary afferent depolarization. We hypothesized that the modulation of GM and GL activation with change in foot orientation would be linked to differential changes in neural drive (experiment A) and that this would be due to a differential change in the excitability of the neural pathways controlling the GM and GL at the cortical (experiment B) and spinal (experiment C) levels.

# MATERIALS AND METHODS

## Participants and Study Design

Ethical approval for this study was obtained from the Research Ethics Committee of Nantes Université (#5102020). Participants were recruited by word of mouth. Inclusion criteria for all participants were: 1) 18–45 yr old, 2) no recent lower limb injuries on the right leg; 3) no known neuromuscular disorders; and 4) no contraindications to peripheral electric or magnetic stimulation. Written informed consent was obtained from the participant after eligibility was confirmed. Nineteen participants took part in this study. Of the

participants, 10 were involved in all three experiments. It should be noted that we were not able to identify a sufficient number of MUs from data collected on female participants during our pilot testing (typically < 5 MUs). Therefore, we only recruited males for this study.

In experiment A ($n = 15$; 26 ± 6 yr old, 178 ± 10 cm, 73 ± 7 kg), we investigated muscle activation [estimated from root mean square (RMS) EMG] and MU discharge characteristics (Table 1). ePICs contribution to motoneuron self-sustained firing were investigated with the paired MU analysis technique (17–19). In experiment B ($n = 13$; 25 ± 7 yr old, 179 ± 6 cm, 72 ± 5 kg), we investigated corticospinal excitability through motor-evoked potentials (MEP), and intracortical excitability through the assessment of short interval intracortical inhibition (SICI) and intracortical facilitation (ICF). In experiment C ($n = 13$; 25 ± 6 yr old, 179 ± 7 cm, 74 ± 7 kg), we investigated the efficacy of spinal transmission between Ia afferents and α-motoneurons through an assessment of the H reflex and primary afferent depolarization.

In experiment A, data were collected while participants maintained a constant level of torque (set at 10 and 20% of their maximal voluntary torque; MVT) and while participants performed triangular contractions reaching 30% of MVT. In experiments B and C, experimental measures were performed both at rest and during a low-level isometric contraction, during which participants maintained a constant level of EMG activity level (set at 10% of maximal EMG activity level; EMGmax), for each muscle (GM and GL) and each foot orientation (rotated internally: “toes-in” or externally: “toes-out”). This was done because the amount of descending drive greatly influences corticospinal excitability (20) and spinal transmission efficacy (21). Hence, matching EMG amplitudes were aimed to match the neural drive across tasks, allowing us to consider that any differences in neural excitability between the tasks could be attributed to differences in foot orientation. The neural excitability of the motor system was also assessed at rest because, when an intended movement is not engaged, inputs upstream of the motor cortex involved in the planning of movement are absent, thus providing insights into the cortical map plasticity associated only with the mechanical change in limb orientation (22). Three different conditions (named hereafter muscle states) were thus examined in experiments B and C: when matching 10% of GM EMGmax, when matching 10% of GL EMGmax, and when at rest.

## Experimental Procedures

### Foot position.

In all the experiments, participants were seated with the right leg extended and the foot rigidly strapped on an isokinetic dynamometer (Biodex Medical Systems, Shirley, New York). Hip, knee, and ankle angles were fixed at 70° of flexion (0° being the standard anatomical position), 0° (i.e., knee extended), and 0° (i.e., foot perpendicular to the shank), respectively. The position we refer to “toes-neutral” is defined as when the long axis of the foot is aligned with the sagittal plane of the body. For the testing in toes-in, the foot was internally rotated by ~35° from the long axis of the foot. For the testing in toes-out, the foot was externally rotated by ~50° from the long axis of the foot.
<table><caption>**Table 1.** Content of each experimental procedure</caption><thead><tr><th></th><th>Experiment A</th><th>Experiment B</th><th>Experiment C</th></tr></thead><tbody><tr><th>Method of investigation</th><td>High-density electromyography</td><td>Transcranial magnetic stimulation</td><td>Tibial nerve electric stimulation</td></tr><tr><th>Outcomes</th><td>Muscle activation and motor units discharge characteristics</td><td>Corticospinal and intracortical excitability</td><td>Efficacy of la afferent transmission to α-motoneurons and presynaptic inhibition due to primary afferent depolarization</td></tr><tr><th>Conditions</th><td>Isom. at 10% of MVT<br>Isom. at 20% of MVT<br>Triang. at 30% of MVT</td><td>Isom. at 10% of GM EMG<sub>max</sub><br>Isom. at 10% of GL EMG<sub>max</sub><br>At rest<br>12 single pulses (MEP)<br>12 conditioned pulses (SICI)<br>12 conditioned pulses (ICF)</td><td>Isom. at 10% of GM EMG<sub>max</sub><br>Isom. at 10% of GL EMG<sub>max</sub><br>At rest<br>Recruitment curve<br>20 single pulses (H reflex)<br>20 conditioned pulses (D1 inhibition)</td></tr><tr><th>Protocol (repeated for each condition)</th><td></td><td></td><td></td></tr></tbody></table>

EMG<sub>max</sub>, root mean square electromyography recorded during the maximal contraction; GM, gastrocnemius medialis; GL, gastrocnemius lateralis; H, Hoffman; ICF, intracortical facilitation; isom, isometric contractions; MEP, motor-evoked potential; MVT, maximal voluntary torque; SICI, short intracortical inhibition; triang, triangular contractions.

### Experiment A.

After a familiarization with the isometric plantarflexion tasks and a standardized warm-up, the following procedure was conducted: 1) two maximal voluntary plantarflexions (three if the difference between the first two was > 10%) for 3 s each, with 120 s of rest in between during which MVT was determined; 2) a series of 15-s plateaued contractions at 10 and 20% of MVT (randomized order); and 3) triangular contractions from 0 to 30% of MVT at a rate of 2% of MVT/s. For each submaximal task, two contractions were performed with at least 45 s of rest in between. All tasks were conducted with toes-in and toes-out in a randomized order, except for the MVT, which was additionally performed in toes-neutral. This was done to verify that the maximal RMS EMG<sub>max</sub> of GM and GL would not differ between foot positions. Visual feedback of the torque was displayed on a screen in front of the participants.

### Experiment B.

Participants were seated in the same position as for experiment A, with the right leg extended and the foot rigidly strapped in an isokinetic dynamometer. After a standardized warm-up, participants performed two maximal voluntary plantarflexion in toes-neutral to determine the EMG<sub>max</sub> of GM and GL. This was done in a toes-neutral position because regardless of the muscle, no difference in EMG<sub>max</sub> was observed across foot positions in experiment A (see RESULTS). Visual feedback of GM or GL RMS EMG was displayed in front of the participant. For each muscle state, 36 stimulations were delivered in a semirandomized order (12 MEP, 12 SICI, and 12 ICF; see Table 1), in each foot orientation (toes-in and -out; randomized order). For the active muscle states, isometric contractions of ~40 s were performed while six TMS stimulations were delivered with an interval of 5–6 s between stimulations.

### Experiment C.

Participants were seated in the same position as for the other experiments, with the right leg extended and the foot rigidly strapped in an isokinetic dynamometer. After a standardized warm-up, participants performed two maximal voluntary plantarflexion in toes-neutral to determine the maximal RMS EMG activity (EMG<sub>max</sub>) of the GM and GL. Again, the following measures were taken for each muscle state and each foot

orientation. Recruitment curves were first recorded. Then, for the estimation of primary afferent depolarization activity, 20 test H-reflexes and 20 conditioned H-reflexes were randomly evoked with a 5-s interval. For the active conditions, isometric contractions of ~30 s were performed while five pulses were delivered. Ten seconds of rest was respected between each contraction, but the stimulation continued to be delivered to keep the level of homosynaptic postactivation depression constant throughout the H-reflex procedure.

### Electromyography

For all experiments, the skin was shaved and cleaned with alcohol before electrodes were attached. For experiment A, HDsEMG signals were recorded using a 64-channel electrode grid [13 × 5 electrodes with one electrode absent from a corner, gold-coated, interelectrode distance: 8 mm; (ELSCH064NM2, OT Bioelettronica, Turin, Italy)]. GM and GL grids were placed at the central part of their muscle bellies, with the columns oriented along the orientation of the fibers. The grids were held on the skin using semidisposable biadhesive foam layers. Skin-electrode contact was made by spreading conductive paste (SpesMedica, Battipaglia, Italy) into cavities on top of each electrode. A strap electrode dampened with water was placed around the contralateral ankle (ground electrode). The reference electrode was placed on the ipsilateral patella. The EMG signals were recorded in monopolar mode, bandpass filtered (10–500 Hz), and digitized at a sampling rate of 2,048 Hz using a multichannel acquisition system (EMG-Quattrocento; 400-channel EMG amplifier, OT Bioelettronica, Turin, Italy).

For experiments B and C, myoelectrical activity was recorded with pairs of self-adhesive surface EMG electrodes (Kendall Medi-Trace 100, Covidien, Mansfield, MA; recording zone: 1 cm diameter, Ag-AgCl), in a bipolar configuration with a 20-mm interelectrode distance. GM and GL electrodes were placed on the middle line of the muscle belly. The ground electrodes were placed on the tibia shaft. The EMG signals were digitized at a sampling rate of 2,000 Hz (MP35, BIOPAC, Goleta).

### Transcranial Magnetic Stimulation

A double-cone coil was placed tangential to the scalp with the handle parallel to the interhemispheric fissure.
Stimulations were delivered by a monophasic magnetic stimulator (Magstim 200 stimulator, Magstim, Dyfed, UK). The hotspot for the right GM and GL within the contralateral (left) primary motor cortex was determined as the optimal coil position that elicited maximal MEP responses. Coil position and orientation at the hotspot were carefully drawn on the scalp and used as the target throughout the session. The coil was held manually by an investigator dedicated to this task. Active motor thresholds were defined for each muscle as the lowest intensity at which at least five responses out of 10 stimulations were discerned above the background EMG level while participants were matching 10% of EMGmax (of the tested muscle) with the foot in the neutral orientation. The resting motor threshold was defined similarly when people were at rest. To assess corticospinal excitability, single pulses were delivered while TMS intensity was set at a suprathreshold level (120% of motor threshold). The technique of paired-pulse TMS was used to assess SICI and ICF in the motor cortex. For SICI, pulses were delivered by conditioning a test MEP (120% of motor threshold) with a subthreshold TMS (80% of motor threshold) delivered 2 ms before the test stimulation (23, 24). For ICF, a similar procedure was conducted except with a 13-ms delay between conditioning and test MEP.

## Electrical Stimulation

Percutaneous electrical stimulation (rectangular pulse duration: 1 ms; voltage: 400 mV) was delivered on the tibial nerve by a constant current stimulator (Model DS7-AH, Digitimer, Hertfordshire, UK) to evoke GM and GL electrophysiological responses. A self-adhesive cathode electrode (Kendall Medi-Trace 100) was placed in the popliteal fossa and the anode electrode (5 × 10 cm, Medicompex SA, Ecublens, Switzerland) was placed on the anterior surface of the knee. The best site for cathodal stimulation was determined with the participants in the toes-neutral condition. This corresponded to the site in the popliteal fossa that generated the greatest GM and GL H reflexes. Once the best site was located, the cathodal electrode was firmly fixed with tape to avoid any change of cathode position throughout the experiment.

## Recruitment Curve

To determine the recruitment curves of H reflex and M wave of GM and GL in toes-in and toes-out, the stimulation intensity was increased by a 1 mA increment from the H reflex threshold to Hmax plus 1 mA. Then, the stimulation intensity was increased by a 10 mA increment until reaching Mmax (highest intensity of the two muscles). At rest, 1) the recruitment curve started at a stimulation intensity of the H reflex threshold of the first muscle recruited, 2) the 1 mA increment was used when both the GM and GL had reached their Hmax response, and 3) the stimulation intensity increased until Mmax was obtained in both muscles. For active muscle states, a recruitment curve was performed for each muscle at 10% of EMGmax. Five stimuli were delivered at each intensity, for both the passive and active states. Stimuli were interspaced by 5 s. The maximal intensity of the stimulation was reached when the amplitude of the peak-to-peak amplitude of GM and GL M-wave

plateaued (25). Three Mmax were recorded at 120% of this intensity.

## Presynaptic Inhibition

To investigate the levels of GM and GL presynaptic inhibition due to primary afferent depolarization in toes-in and toes-out, we used the D1 inhibition method (26). This method consists of conditioning (conditioned H reflex) or not (test H reflex) GM and GL H reflexes by a prior stimulation train (3 pulses at 300 Hz) of the fibular nerve with an interstimulus interval of 21 ms. As in previous studies (27, 28), we used a stimulation train rather than a single stimulation as the conditioning stimulus, since the D1 inhibition of the H reflex is more regularly found when the conditioning stimulus is a train of a few shocks (26, 29). H reflexes were evoked at a stimulation intensity eliciting GM and GL H reflex amplitudes of ~50% of Hmax in the first orientation (toes-in or -out, randomized). The stimulation intensity was adjusted so that a similar test H-reflex peak-to-peak amplitude was elicited in both foot orientations. This was important to make sure the same pool of motoneurons was recruited, hence having a similar sensitivity to inhibitory and excitatory sensorimotor inputs. It is noteworthy that if different stimulation intensities were required for the two muscles to generate the targeted reflex responses at rest, GM and GL D1 inhibition were assessed separately. The fibular nerve was stimulated with the cathode (Kendall Medi-Trace 100,) placed next to the fibular head and the anode (Kendall Medi-Trace 100) near the medial part of the tibia. The optimal stimulation site was defined as the site eliciting the greatest M wave in the tibialis anterior for a given stimulus intensity. We further ensured that the stimulation maximized the activation of the tibialis anterior with no activation of peroneal muscles (checked by palpation). The intensity of the conditioning stimulation of the fibular nerve was set at 130% of the tibialis anterior motor threshold obtained in the toes-out orientation (arbitrary choice). The stimulation intensity of the fibular nerve was adjusted so that a similar M wave peak-to-peak amplitude was elicited in both foot orientations.

## Data Processing

### Torque and activation level.

All torque and EMG data were processed offline using Matlab 2020 b (MathWorks Inc., Natick, MA). Torque data were low-pass filtered at 10 Hz. EMG signals were band-pass filtered (20–500 Hz), visually inspected for noise or artifact on each channel, and differentiated (along columns). Maximal torque and maximal EMG amplitude value (RMS EMGmax) were calculated from MVT trials, as the highest torque and highest RMS EMG were calculated from a moving average window of 300 ms with 99% overlap. To obtain RMS EMGmax, we averaged the results across all channels of the grid. For the submaximal tasks, the RMS EMG was calculated over 10 s in the middle of the force plateau, averaged across channels, and normalized to the RMS EMGmax.

### Motor unit decomposition.

The HDsEMG signals were decomposed with the convolutive blind source separation method (30) implemented in a
custom-made Matlab routine to iteratively identify and extract the sources of the HD-sEMG interference signals, i.e., the motor unit spike trains. The following extraction parameters were selected: 150 iterations, maximal coefficient of variation of the interspike intervals set at 50%, and minimal pulse to noise ratio set as 30 dB (31). After the automatic decomposition, all the MU spike trains were visually inspected and manually edited to add missing spikes and remove artifacts selected as spikes, as described previously (32, 33).

**Tracking of the motor units across contractions.**

To ensure the comparison of the same pool of motoneurons between conditions, the best option is to match MUs across tasks. However, when matching MU, we lost 39 and 64% of the identified MU pool from the decomposition of GM and GL, respectively. As this dramatically reduced the dataset, it is the nonmatched data we report in the present paper. Data from matched MUs are reported as supplemental material. Importantly, they always led to similar observations.

**Motor unit discharge characteristics.**

From the plateaued contractions, we calculated the mean instantaneous discharge rate. From the triangular contractions, we determined the time at recruitment as the delay between the beginning of force rise and the first discharge spike.

**Estimates of persistent inward current.**

From the triangular contraction at 30% of MVT, we also measured the mean ΔF value per participant. Mean ΔF is a well-established method to measure ePICs in humans indirectly from the firing pattern of individual MU. ePICs are

responsible for amplifying α-motoneuronal synaptic inputs and thus enhance the excitability of the motoneuron pool (13). The mean ΔF value for each muscle and foot orientation was calculated to determine whether ePICs are modulated by foot orientation. As previously proposed (17), each ΔF value was calculated as the difference in the smoothed firing rate of a control MU (lower threshold) between the times of recruitment/derecruitment of a test MU (higher threshold). In the current study, we used a 2-s Hanning window smoothing of the instantaneous discharge rate of MU (19, 34). In agreement with previous work (19, 34), the criteria for inclusion of ΔF value from MU pairs were as follows: 1) a test MU should discharge for at least 2 s, 2) it should be recruited at least 1 s after the control MU to ensure full activation of ePICs, 3) it should be derecruited at least 1.5 s before the control MU to prevent overestimation of ΔF. For the analysis, we set a minimal number of five pairs of MU for data to be included in the analysis, which led to the consideration of *n* = 14 participants for GM and *n* = 5 participants for GL. For all motor unit properties, motor units were averaged per participant in the analysis.

**Motor-evoked potentials.**

The peak-to-peak amplitude of the conditioned and unconditioned MEPs was computed off-line from the unrectified EMG signal and averaged for each muscle, foot orientation, and muscle state (Fig. 1). The SICI and ICF were expressed as the percentage of inhibition and facilitation by using the following equation: conditioned MEP/test MEP × 100. Thus, a lower calculated percentage for SICI indicates a higher inhibition level, whereas a higher percentage for ICF indicates a higher facilitation level.

<figure><img src="image_1.png" /><figcaption>**Figure 1.** Individual example of data for the motor unit behavior (A; participant #16) and the neurophysiological measures (B, participant #4). A: the smoothed discharge rate is shown for each motor unit with colored lines. The black line shows the torque produced, which was matching 20% of the maximal voluntary torque. B: examples are shown for the motor-evoked potential and Hoffman reflex data. It is important to note that for the recordings in the active muscle state, data are reported for the GM when the EMG level of GM was matched, and data are reported for the GL when the EMG level of GL was matched. GL, gastrocnemius lateralis; GM, gastrocnemius medialis.</figcaption></figure>
## Efficacy of spinal transmission and presynaptic inhibition.

The peak-to-peak amplitudes of the H reflex and M wave were computed off-line from the unrectified EMG signal and averaged for each muscle, foot orientation, muscle state, and stimulation intensity of the recruitment curves (Fig. 1). Each mean H reflex was expressed as a percentage of the mean $M_{max}$ obtained in the same conditions (foot orientation and contraction intensity) ($H/M_{max}$). Peak-to-peak amplitudes of $H_{max}$ and $M_{max}$ were measured ($H_{max}/M_{max}$) to estimate the efficacy of spinal transmission from Ia afferents to α-motoneurons (35). $M_{atHmax}$ was calculated to characterize motor fiber activation when Ia-fiber activation was maximal. For each individual and foot orientation at rest, we also calculated the slope and intercept of the relationship between the GM $H/M_{max}$ and GL $H/M_{max}$ from the first stimulation intensity where both muscles had a motor response to the stimulation intensity at which one of the muscles reached its $H_{max}$. From these relationships, we also calculated the theoretical GM $H/M_{max}$ value that accompanied a theoretical GL $H/M_{max}$ value of 50% of $H_{max}/M_{max}$ obtained in toes-in orientation (arbitrary choice; Fig. 2). The amount of D1 inhibition, i.e., our index of presynaptic inhibition due to primary afferent depolarization, was calculated as: mean conditioned H reflex/mean test H reflex × 100 ($H_{cond}/H_{test}$), so that the lower the calculated value, the higher the D1 inhibition.

## Statistical Analysis

All statistical analyses were performed in R (version 4.1.2). The threshold of significance was set at *P* < 0.05. Data are reported as mean ± standard deviation. Linear mixed models

<figure><img src="image_2.png" /><figcaption>**Figure 2.** Individual example of the relationship between GM and GL $H/M_{max}$ during electrical stimulation at rest (participant #8). GM $H/M_{max}$ is plotted against the corresponding GL $H/M_{max}$ obtained at the same stimulation intensity and foot orientation. Each circle corresponds to the mean of five H reflex responses recorded for a stimulation intensity. Each continuous line represents the best-fit regression line for each foot orientation. The blue and red colors represent data obtained in toes-in and toes-out, respectively. The equation of the linear regression was used to determine the theoretical GM $H/M_{max}$ obtained in the two foot orientations for the same GL $H/M_{max}$ value corresponding to 50% of the $H_{max}$ recorded in toes-in. For this participant, a theoretical GL $H/M_{max}$ value of 12.0% provides theoretical GM $H/M_{max}$ values of 8.3% in toes-in and 9.8% in toes-out. The GM of this participant has therefore a greater spinal reflex excitability in toes-out than in toes-in compared with the GL. GL, gastrocnemius lateralis; GM, gastrocnemius medialis.</figcaption></figure>

were used to compare outcomes across conditions (see factors described below) using the lme4 package (36). Mean estimates are reported on a table in the supplementary data. In the linear mixed models, the interindividual variability is accounted for by including participants as random effects. The model used was adapted to the independent variables being tested, since such variables were tested through different fixed effects. The under mentioned factors [in brackets] included all or some of the following categories, as needed, based on the experimental set-up: toes-in, neutral, and -out [foot orientation]; 10 and 20% of MVT [contraction level]; GM and GL [muscle]; at rest, 10% match GM EMGmax, and 10% match GL EMGmax [muscle state]. We tested the following with linear mixed models: 1) the effect of foot orientation and muscles (fixed effects) on EMGmax (dependent variables); 2) the effect of foot orientation, contraction level, and muscle (fixed effects) on the submaximal activation level, MU discharge rates and time at recruitment (dependent variables); 3) the effect of foot orientation and activation level (fixed effects) on the ΔF values (dependent variables); and 4) the effect of foot orientation, muscle state, and muscle (fixed effects) on MEP, SICI, ICF, $H_{max}/M_{max}$, $H_{atMmax}/M_{max}$, and $H_{cond}/H_{test}$ (dependent variables). The statistical significance of the fixed effect(s) was tested by fitting the model with restricted maximum likelihood and deriving degrees of freedom via Satterthwaite approximation. When significant differences were found, we compared the estimated marginal means of the levels of significant effects with Tukey adjustments for multiple comparisons using the updated estimated marginal means package (37). Finally, a paired two-tailed *t* test was used to compare the slope and intercept of the GM/GL $H/M_{max}$ relationship between toes-in and toes-out.

# RESULTS

## Maximal Torque and Activation Levels

Participants produced maximal plantarflexion torque of 221 ± 47, 227 ± 45, and 198 ± 37 Nm with toes-in, -neutral, and -out, respectively. Activation levels, calculated from the differentiated EMG signals showed that the maximal RMS EMG values of GM and GL were not significantly different between foot orientations (foot orientation effect: *P* = 0.116; foot orientation × muscle interaction: *P* = 0.482). During the submaximal isometric contractions, the balance of normalized EMG amplitudes between GM and GL depended on foot position (Fig. 3). Specifically, there was a significant interaction between foot orientation and muscle (*P* < 0.001), showing that regardless of the contraction level, GM activation was higher in toes-in than toes-out (post hoc: *P* < 0.001) and GL activation was lower in toes-in than toes-out (post hoc: *P* = 0.005).

## Motor Unit Discharge Characteristics

Across intensities, 27 ± 11 (GM) and 7 ± 7 (GL) MU per participant were identified during contractions with toes-out; 25 ± 13 (GM) and 9 ± 7 (GL) MU per participant during contractions with toes-in (Table 2). For the sake of clarity, we only report below the differences between foot orientations, which are related to the main aim of this study. There was a significant interaction between foot orientation and muscle for MU discharge rate (*P* < 0.001, Fig. 3). Regardless of the
<figure><img src="image_3.png" /><figcaption>**Figure 3.** Gastrocnemius RMS EMG and mean discharge rate during plateaued contraction at 10% of MVT (A) at 20% (B), and time at recruitment and ePICs during triangular contractions at 30% of maximal voluntary torque (C). The torque level was matched between foot orientations. Each colored dot represents a participant (motor units were averaged per participants in the analysis). The short full black lines indicate the group average for each condition. A linear mixed models was used to test (i) the effect of foot orientation, contraction level, and muscle (fixed effects) on the submaximal activation level, MU discharge rates and time at recruitment (dependent variables), and (ii) the effect of foot orientation and activation level (fixed effects) on the DF values (dependent variables). Statistics reported indicate post hoc results. \*\*P < 0.010, \*\*\*P < 0.001. ePICs, estimation of PICs; MVT, maximal voluntary torque; RMS, root mean square.</figcaption></figure>

contraction level, GM discharge rate was lower in toes-in than toes-out (post hoc: *P* = 0.013) and no difference was found for GL discharge rate between foot positions (post hoc: *P* = 0.066). When considering the time at recruitment during the triangular contraction at 30% of MVT, there was a significant interaction between foot orientation and muscle (*P* < 0.001, Fig. 3). The time at recruitment for GL MU occurred earlier with toes-in than toes-out (post hoc: *P* = 0.004). For GM MU, the time at recruitment was not significantly different between toes-in and toes-out (post hoc: *P* = 0.438). Overall, the investigation of MU discharge characteristics demonstrated that the balance of neural drive between GM

and GL depends on foot orientation, and is in line with the change of muscle activation distribution (Fig. 3).

## Estimates of Persistent Inward Currents

Setting a minimal number of five pairs of MU for data to be included in the analysis reduced the sample size to *n* = 14 participants for GM and *n* = 5 participants for GL. We considered too small the number of participants for which ΔF could be calculated for GL thus no statistical comparison was performed. It is important to note that in the current protocol, ΔF was recorded during a matched-torque task, leading to differences in the muscle activation between the two foot

<table><caption>**Table 2.** Number of MU in the GM and the GL for the tasks investigated</caption><thead><tr><th rowspan="2">Decomposition Characteristics</th><th colspan="2">GM</th><th colspan="2">GL</th></tr><tr><th>Toes-in</th><th>Toes-out</th><th>Toes-in</th><th>Toes-out</th></tr></thead><tbody><tr><td>Total Number of MU</td><td>1,123</td><td>1,214</td><td>421</td><td>301</td></tr><tr><td rowspan="2">Number of MU per participant</td><td>10% MVT</td><td>26 ± 16<br/>n = 16</td><td>29 ± 13<br/>n = 16</td><td>11 ± 8<br/>n = 13</td><td>4 ± 2<br/>n = 11</td></tr><tr><td>20% MVT</td><td>28 ± 13<br/>n = 16</td><td>28 ± 11<br/>n = 16</td><td>12 ± 6<br/>n = 12</td><td>11 ± 7<br/>n = 13</td></tr><tr><td rowspan="2">30% MVT</td><td>For time at recruitment</td><td>22 ± 10<br/>n = 16</td><td>24 ± 10<br/>n = 16</td><td>10 ± 6<br/>n = 13</td><td>10 ± 6<br/>n = 12</td></tr><tr><td>For ΔF (number of pairs)</td><td>261 ± 238<br/>n = 16</td><td>402 ± 275<br/>n = 16</td><td>62 ± 56<br/>n = 9</td><td>78 ± 124<br/>n = 10</td></tr></tbody></table>

For some participants, no MU could be extracted for GL. In such cases, the number (*n*) of participants included in the reported data are indicated. GL, gastrocnemius lateralis; GM, gastrocnemius medialis; MU, motor units; MVT, maximal voluntary torque.
orientations, i.e., for GM it was 28% (toes-in) and 35% (toes-out) of RMS EMG<sub>max</sub>. A recent study revealed that PIC strength is affected by the muscle activation level (38). This is the reason why muscle activation level has been included in the analysis as a fixed effect. For GM, ΔF was not significantly different between toes-out (3.3 ± 0.7 pps) and toes-in (2.8 ± 0.9 pps) (foot orientation effect: *P* = 0.678). GL ΔF values were 3.4 ± 1.3 with toes-in and 3.4 ± 1.1 pps with toes-out.

## Motor-Evoked Potentials

MEP amplitudes were not affected by foot orientation, regardless of the muscle state, i.e., rest or contraction (foot orientation × muscle interaction: *P* = 0.781; foot orientation × muscle × muscle state interaction: *P* = 0.500; Fig. 4). Similarly, neither SICI nor ICF was affected by foot orientation, regardless of the muscle state (foot orientation × muscle interaction for SICI: *P* = 0.467, for ICF: *P* = 0.829; foot orientation × muscle × muscle state interaction for SICI: *P* = 0.096, for ICF: *P* = 0.345; Fig. 4). Our results indicate that foot orientation did not affect the balance of corticospinal excitability between GM and GL, nor their balance of intracortical activity.

## H Reflex and M Wave

$M_{max}$ was not significantly different between foot positions for GM or GL (all *P* > 0.104; Table 3), which demonstrates that recording conditions were similar between horizontal foot positions.

$H_{max}/M_{max}$ values were not significantly different between foot orientations for any muscle or at any muscle state (foot orientation × muscle interaction: *P* = 0.670; foot orientation × muscle × muscle state interaction: *P* = 0.957; Fig. 5). Similarly, when considering $M_{atHmax}/M_{max}$, interaction between foot orientation × muscle was not significant (*P* = 0.785) and neither was the interaction between foot orientation × muscle × muscle state (*P* = 0.887), demonstrating that at the occurrence of $H_{max}$, the same proportion of efferent fibers was recruited for the two foot orientations. Lastly, from the analysis of the relationship between the GM $H/M_{max}$ and GL $H/M_{max}$ at rest, no significant differences between foot orientations were found for mean slope (toes-in: 1.3 ± 0.9, toes-out: 1.4 ± 0.8; *P* = 0.493), intercept (toes-in: 0.0 ± 1.8, toes-out: 0.7 ± 2.7; *P* = 0.312), or theoretical GM $H/M_{max}$ for a similar GL $H/M_{max}$ (toes-in: 9.3 ± 4.4, toes-out: 10.4 ± 5.3; *P* = 0.279).

### A At rest

<figure><img src="image_4.png" /></figure>

### B Matching EMG background

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Motor-evoked potentials (MEPs; left), intracortical inhibition (SICI; middle), and facilitation (ICF; right) for gastrocnemius medialis (GM) and lateralis (GL) during the different conditions investigated. Each colored dot represents a participant. On A, data were collected at rest. On B, data were collected during matched background EMG. No difference was found between foot orientations, neither for GM or GL, at rest or when comparing GM responses during matched-background EMG of GM, or GL responses during matched-background EMG of GL. A linear mixed models was used to test the effect of foot orientation, muscle state, and muscle (fixed effects) on MEP, SICI, ICF (dependent variables). GL, gastrocnemius lateralis; GM, gastrocnemius medialis; ICF, intracortical facilitation; SICI, short interval intracortical inhibition.</figcaption></figure>
<table><caption>**Table 3.** Electrophysiological responses for GM and GL during the tibial nerve electric stimulation, for the $M_{max}$ and test H reflex wave</caption><thead><tr><th></th><th>Toes-in</th><th>Toes-out</th><th>P Value</th></tr></thead><tbody><tr><td>M<sub>max</sub>, mV</td><td></td><td></td><td></td></tr><tr><td>Rest</td><td></td><td></td><td></td></tr><tr><td>GM</td><td>8.1 ± 4.1</td><td>8.2 ± 3.9</td><td>0.850</td></tr><tr><td>GL</td><td>7.5 ± 2.0</td><td>7.3 ± 1.9</td><td>0.258</td></tr><tr><td>10% background EMG</td><td></td><td></td><td></td></tr><tr><td>GM</td><td>8.9 ± 4.3</td><td>7.5 ± 2.2</td><td>0.629</td></tr><tr><td>GL</td><td>8.8 ± 4.1</td><td>7.9 ± 2.6</td><td>0.104</td></tr><tr><td>Test H reflex, mV</td><td></td><td></td><td></td></tr><tr><td>Rest</td><td></td><td></td><td></td></tr><tr><td>GM</td><td>11.2 ± 7.1</td><td>12.0 ± 8.8</td><td>0.336</td></tr><tr><td>GL</td><td>8.0 ± 4.2</td><td>8.0 ± 4.3</td><td>0.876</td></tr><tr><td>10% background EMG</td><td></td><td></td><td></td></tr><tr><td>GM</td><td>12.7 ± 6.4</td><td>12.6 ± 5.2</td><td>0.886</td></tr><tr><td>GL</td><td>11.3 ± 4.0</td><td>10.2 ± 4.3</td><td>0.544</td></tr></tbody></table>

This analysis was conducted to verify that recording conditions were similar between foot orientations (for Mmax) and that the chosen intensity during the D1 inhibition technique did not influence the Hcond/Htest results (for the test H reflex). GL, gastrocnemius lateralis; GM, gastrocnemius medialis; $M_{max}$, highest intensity of the two muscles.

## D1 Inhibition

Test H reflex did not differ significantly between foot orientations for the GM or GL (all $P > 0.344$; Table 3). This validated the requirement that the amplitude of the test H reflex

was not significantly different between foot positions to enable comparison.

$H_{cond}/H_{test}$ was also not significantly different between foot orientations for any muscle or any muscle state (foot orientation × muscle interaction: $P = 0.810$; foot orientation × muscle × muscle state interaction: $P = 0.370$; Fig. 5). Our results showed that foot orientation did not affect the efficacy of Ia-α-motoneurons transmission for the GM or GL muscles or their primary afferent depolarization activity.

## DISCUSSION

In this study, we aimed to determine whether the differential change in the neural drive sent to two anatomically defined synergist muscles, induced by a change in limb orientation, is explained by a change in their respective neural excitability. We focused on the lateral and medial head of the gastrocnemii as it constitutes a good model to manipulate the distribution of neural drive through a change in foot orientation. Despite a clear differential change in neural drive to the GM, there was no modulation in the corticospinal excitability related to foot orientation, either at the cortical level (intracortical inhibition or facilitation) or the spinal level (ePICs), or through modulating their afferent feedback loops. This suggests that the change in the excitability of motor pathways of the gastrocnemius muscles is not

### A At rest

<figure><img src="image_6.png" /></figure>

### B Matching EMG background

<figure><img src="image_7.png" /></figure>

**Figure 5.** $H_{max}$ normalized to $M_{max}$ (left), $M_{at Hmax}$ normalized to $M_{max}$ (middle), and $H_{cond}/H_{test}$ (right) and for gastrocnemius medialis (GM) and lateralis (GL) during the different conditions investigated. Each colored dot represents a participant. On A, data were collected at rest. On B, data were collected during matched-background EMG. A linear mixed models was used to test the effect of foot orientation, muscle state, and muscle (fixed effects) on $H_{max}/M_{max}$, $M_{at Hmax}/M_{max}$, and $H_{cond}/H_{test}$ (dependent variables). No differences were found for any measure between foot orientations, neither for GM or GL, in the resting or active state.
the underlying mechanism for the differential change in
neural drive between foot orientations.

Differential Changes in Neural Drive

Most previous studies that assessed the change in GM and
GL activation with toes-in and toes-out investigated func-
tional tasks such as heel raises (2–5) or walking (6), and used
the amplitude of surface EMG as a proxy of muscle activa-
tion. In the present study, we focused on a controlled isomet-
ric plantarflexion performed in a seated position to limit the
influence of postural control. In line with the results
reported in the aforementioned studies, we observed a dif-
ferent balance of EMG amplitude between foot orientations;
GM EMG amplitude being 1.4 times higher with toes-out
compared with toes-in, GL EMG amplitude being 1.6 times
lower with toes-out compared with toes-in. Because surface
EMG amplitude provides a crude estimate of the neural drive
that is sent to the muscles (39), it was important to verify
that the difference in EMG amplitude between tasks
reflected actual differences in the neural drive. We found
that the MU discharge rate decreased for GM from toes-out
to toes-in, which confirmed that the differential changes in
EMG amplitude can be interpreted as differential change in
amplitude of the neural drive. Furthermore, we observed dif-
ference in time at recruitment between foot orientations,
with the MU being recruited earlier in toes-in than in toes-
out for GL.

Excitability of the Motor Pathways

A change in the neural drive sent to a muscle associated
with a change in its mechanical configuration has been pre-
viously observed. For example, the neural drive to the first
dorsal interosseus is coupled with its moment arm (40). It is
also the case for the external intercostal muscles, for which
the neural drive mirrors the spatial distribution of inspira-
tory mechanical advantage (41). The origin of this neurome-
chanical coupling remains poorly understood. In the present
study, we hypothesized that the mechanical change induced
by the foot orientation would impact the neural drive to the
gastrocnemius muscles by changing the excitability of their
motor pathways.

Cortic spinal Excitability

GM and GL corticospinal excitability at rest or during EMG-matched plantarflexion, quantified using MEP amplitude, were not affected by foot orientation. Importantly, as the measure of MEP amplitude is the result of activation of both upper and lower motoneurons, it may have revealed changes in the excitability of cortical motoneurons or α-motoneurons, or both (42). At the cortical level, the inhibitory and facilitatory intracortical activities of the gastrocnemius muscles were not affected by foot orientation. The long-latency facilitation is considered to be mainly due to the activation of corticocortical glutamatergic excitatory pathways (43), and the short-latency inhibition is attributed to the activation of intracortical GABAergic inhibitory interneurons (44). Therefore, our results suggest that none of these pathways are sensitive to the change in foot position. This contrasts with previous work showing that the corticospinal pathways to forearm muscles are influenced by

variations in shoulder joint angle due to a change in intra-
cortical facilitation (45). The excitability of α-motoneurons
was not measured by spinal cord stimulation in the current
study to avoid painful measurements [e.g., thoracic electrical
stimulation; (46)]. However, through the ΔF method (17), we
assessed ePICs that affect the intrinsic excitability of α-moto-
neurons. We found that ΔF of GM was not influenced by foot
orientation. It should be noted that GL ΔF results were not
statistically tested due to the limited number of participants
for whom these data were available. Overall, the experimen-
tal approaches used in the present study suggest that there is
no foot orientation-related modulation in corticospinal
excitability of the GM and GL.

Efficacy of Ia-α-Motoneuron Transmission

The impact of the sensory input of the muscles, poten-
tially modifying the afferent feedback to the spinal cord, was
a possible explanation of how the mechanical consequence
of a change in limb orientation could be integrated by the
central nervous system (9). To obtain insights into the spinal
reflex pathway, we measured the efficacy of the Ia-α-moto-
neurons transmission with the H reflex. There exists evi-
dence that a change in the ankle angle in the frontal plane
affects the efficacy of the Ia-α-motoneurons transmission of
the soleus, with a lower H reflex amplitude in dorsiflexion,
compared with neutral position (14). In contrast, the present
study shows that the efficacy of the Ia-α-motoneurons trans-
mission is not affected by foot orientation. This is supported
by 1) similar Hmax/Mmax between toes-in and toes-out, show-
ing that the maximal efficacy of Ia-α-motoneuron transmis-
sion does not change between foot orientations; 2) similar M
wave amplitude that accompanied Hmax between toes-in and
toes-out, showing that, for a given excitation of motor fibers,
the excitability of the monosynaptic Ia-α-motoneuron reflex
is not altered by foot orientation; and 3) similar relationships
of GM H/Mmax and GL H/Mmax during the recruitment
curves, showing that, for a given level of monosynaptic Ia-
α-motoneuron reflex excitation in the GL, the monosynaptic
Ia-α-motoneuron reflex excitation does not differ in the GM
between toes-in and toes-out. To investigate whether the ab-
sence of change in H reflex was hidden by a change in pri-
mary afferent depolarization, we used the D1-inhibition
technique. We kept the test H reflex at a constant size
between foot orientations to ensure a similar sensitivity to
excitatory and inhibitory inputs (47). The similar Hcond/Htest
seen with toes-in and toes-out indicates that the central
nervous system does not adjust the contribution of the mus-
cle afferent inputs onto the α-motoneuron pool of the GM
and GL between foot orientations. Overall, gastrocnemii effi-
cacy of Ia-α-motoneurons transmission and regulation of Ia
afferent inflow by primary afferent depolarization were not
affected by foot orientation. A possible explanation for this is
that the mechanical change is transmitted through other
sensory sources, for example through afferences at the ten-
don level (e.g., Golgi tendon organs), thus not captured by
the neurophysiological measures used in the current study.
Supporting this, foot orientation in the horizontal plane is
known to affect the gastrocnemii subtendon lengths differ-
ently in the two foot positions: GM and GL subtendons are
longer by 4.3 and 2.1 mm, respectively, in toes-in compared
with toes-out (48). Another possibility for the lack of change in neural excitability between foot orientations is that the manipulation of the foot orientation as tested in the current protocol did not generate a sufficient mechanical change in the GM and GL muscles to impact their sensory feedback differently. In this way, it was previously hypothesized that there is a threshold of muscle length change after which increased afferent feedback is sufficient to generate differences in central nervous system excitability in a muscle; for example, a 5 mm fascicle length change in the soleus would be too small (49).

## The Role of Motor Planning in the Differential Change in Neural Drive between GM and GL

Taken together, our results suggest that changes in excitability of the main motor circuits of the GM and GL cannot explain the differential change in the neural drive they receive between plantarflexions performed with different foot orientations in the horizontal plane. This invalidated our main hypothesis. It is therefore possible that motor execution is programmed differently during an isometric plantarflexion with internal versus external foot rotation, and our results suggest that the two foot orientations tested herein are two tasks with different motor planning. The fact that recruitment strategies of the GM and GL are different when foot orientation in the horizontal plane is changed suggests that a functional specialization between gastrocnemius muscles could exist outside the sagittal plane. Gastrocnemii are intensively studied during plantarflexion tasks (i.e., in the sagittal plane), but their possible function in other planes has been suggested (50–53). For example, recent studies on standing showed that multidirectional leaning or unilateral perturbation generated the recruitment of specific MU differing between the GM and GL (52, 53). It was also observed that electrostimulation of the GM produced a lateral moment at the foot and that a medial moment was seen during electrostimulation of the GL, suggesting that the GM contributes to external rotation and GL to internal rotation (54). Even though the estimation of GM and GL muscle actions in the horizontal plane is not straightforward, this element would provide strong explanation for different motor planning. It echoes recent results supporting the distribution of two common inputs during plantarflexion, primarily grouping motoneurons innervating GM-soleus and GL-soleus (55), and could explain the small proportion of common synaptic input sent to the GM and GL (2). The last theories of motor control propose that synergies exist at the motoneuron level, where common inputs do not necessarily project to all motoneurons in a pool but to groups of motoneurons that pertain to different pools (56). In other words, subparts of GM and GL, or functional groups of motoneurons, could be controlled differently as belonging to different synergies, explaining the relative differences of gastrocnemii neural drive in the two tasks investigated herein. Such a motor plan being embedded in the primary motor area would explain why such coordination options can exist without differences in the excitability state of the corticospinal pathway. To test whether motor execution is programmed differently when foot orientation is changed, further studies could assess motor planning with the use of motor imagery, i.e., the

mental simulation of movement without the corresponding muscle contraction. Since MEP amplitude is known to increase proportionally to contraction intensity during motor imagery (57), comparing MEPs of GM and GL during mental simulation of plantarflexions with the foot placed in toes-in or -out orientations could reveal the role of motor planning in such a task.

## Methodological Considerations

It is important to mention that the position tested in the current study is not restricted to the ankle joint, but also involves the knee and hip joints (58). This study investigated the impact at the ankle level but differences may also exist between lower limb positions at other joints. Also, only males were included in the study, hence results may differ in females. We were able to track the same MUs between the two foot orientations for 61 and 36% of the total number of identified MUs for the GM and GL, respectively (data for matched MU are reported in Supplemental data). Matched MU data showed similar trends to the full pool of MU extracted (see Supplemental data). This complementary analysis provides insight into whether similar MUs change their behavior in response to the change in foot orientation, making it the best approach to compare the strength of neural drive between foot orientations (59). There are two possibilities that could explain the significant proportion of unmatched MUs that were left: 1) methodological limitations, in which the algorithm failed to recognize MU shape of similar MU because, for example, of different motions of muscle fibers underneath the electrodes between plantarflexions in each foot orientation, or 2) a physiological phenomenon, in which a subset of different MUs was recruited between tasks. Spatial recruitment, i.e., the number of motor units recruited rather than just their discharge rate, is another potential contributor to the change in activation of the GM and GL seen with foot orientations, which was not assessed in the current study. Regarding the measures of central nervous system and spinal reflex excitability, in the current study, we took care to measure them when background-EMG activity was matched between foot orientations, for each muscle assessed separately. The chosen methodology was the only way to remove the impact of muscle activation level on the measured outcomes, since the amount of descending drive influences corticospinal excitability (20) and spinal transmission efficacy (21). It guaranteed that any differences seen in neural excitability could be attributed to foot orientation.

## Perspectives and Significance

We have demonstrated that there is a different modulation of neural control between the gastrocnemii according to the foot in the horizontal plane during plantar flexion. Our results do not support that this different modulation is due to a change in the excitability of the neural pathways controlling the gastrocnemii. Further work is needed to confirm such results at other anatomical sites. The novelty of this work is to produce knowledge about the origin of motor control for muscles belonging to the same anatomical group.
DATA AVAILABILITY

Individual data that support the findings of this study are available on FigShare at https://doi.org/10.6084/m9.fishare.22692574.

SUPPLEMENTAL DATA

Supplemental data: https://doi.org/10.6084/m9.fishare.22692574.

ACKNOWLEDGMENTS

The authors thank Valentin Goreau for his assistance on statistical analysis.

GRANTS

F.H. was supported by a grant from the French National Research Agency (ANR-19-CE17-002-01, COMMODE project).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.C., F.H., and T.C. conceived and designed research; M.C. and T.C. performed experiments; M.C., S.A., and T.C. analyzed data; M.C., S.A., F.H., and T.C. interpreted results of experiments; M.C. prepared figures; M.C. drafted manuscript; M.C., S.A., F.H., and T.C. edited and revised manuscript; M.C., S.A., F.H., and T.C. approved final version of manuscript.

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